Handbook of Cryo-Preparation Methods for Electron Microscopy (Methods in Visualization)

Author: Annie Cavalier | Daniele Spehner | Bruno M. Humbel

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HANDBOOK OF

Cryo-Preparation Methods for Electron Microscopy

© 2009 by Taylor & Francis Group, LLC

Methods in Visualization Series Editor: Gérard Morel

Genome Visualization by Classic Methods in Light Microscopy Jean-Marie Exbrayat Handbook of Cryo-Preparation Methods for Electron Microscopy Annie Cavalier, Danièle Spehner, and Bruno M. Humbel Imaging of Nucleic Acids and Quantitation in Photonic Microscopy Xavier Ronot and Yves Usson In Situ Hybridization in Electron Microscopy Gérard Morel, Annie Cavalier, and Lynda Williams In Situ Hybridization in Light Microscopy Gérard Morel and Annie Cavalier PCR/RT -PCR In Situ Light and Electron Microscopy Gérard Morel and Mireille Raccurt Visualization of Receptors: In Situ Applications of Radioligand Binding Emmanuel Moyse and Slavica M. Krantic

© 2009 by Taylor & Francis Group, LLC

Methods in Visualization Series

HANDBOOK OF

Cryo-Preparation Methods for Electron Microscopy

Edited by

Annie Cavalier Danièle Spehner Bruno M. Humbel

© 2009 by Taylor & Francis Group, LLC

Painting on the cover by Annie Cavalier

CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2009 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-13: 978-0-8493-7227-8 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright material has not been acknowledged please write and let us know so we may rectify in any future reprint. Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Handbook of cryo-preparation methods for electron microscopy / editor, Annie Cavalier, Daniele Spehner, and Bruno M. Humbel. p. ; cm. -- (Methods in visualization) Includes bibliographical references and index. ISBN 978-0-8493-7227-8 (hardcover : alk. paper) 1. Cryobiology--Methodology. 2. Electron microscopy--Methodology. I. Cavalier, Annie. II. Spehner, Daniele. III. Humbel, Bruno M. IV. Title. V. Series. [DNLM: 1. Cryopreservation--methods. 2. Microscopy, Electron--methods. QY 95 H236 2008] QH324.9.C7H36 2008 570.28’25--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

© 2009 by Taylor & Francis Group, LLC

2008016244

V

SERIES PREFACE The second half of the 20th century was a period of intense exploration into the anatomy of the cell. After the ground-breaking discovery of a whole new world of structural complexity, the period between about 1950 and 1970 was characterized by the further development of the electron microscope, and specimen preparation methods were redefined so that cells could be studied at the organelle level, then that of single molecular units. During the 1970s, correlating structure and function was the most productive area of cell biology. Electron microscopy was applied to cytochemical reactions developed for use with light microscopy, but also to histochemical reactions, immunolabeling and autoradiography. The pioneers of the 1960s were already studying individual molecules within cells, and by the 1970s these methods had gained wide acceptance. Since the 1960s, the most effective method for exploring submicroscopic structures has been ultramicrotomy. But the preparation steps carried out in conventional ultramicrotomy, i.e., chemical fixation using osmium tetroxide, chemical solvents and resin embedding, are harmful to structural conformation. In order to preserve the integrity of molecules in sections, researchers such as Leduc,1,2 Pease3,4 and their co-workers introduced water-miscible macromolecules as embedding material. This technique proved effective in enzyme digestion5,6 and the immunochemical labeling of a small number of antigens,7 and Bernhard’s group adapted it for use with sections of frozen aldehyde-fixed tissue.8-10 Finally, Tokuyasu introduced inert cryoprotectants, such as sucrose, into specimens in infusions before freezing so as to improve their plasticity, and the dry, frozen sections were collected on droplets of saturated sucrose.11,12 For x-ray analytical studies, no suitable flotation fluid has yet been found onto which frozen sections can be cut. Dry sectioning systems13,14 are therefore used, without chemical fixation. Electron microscopy, while presenting the advantages associated with frozen sections of unfixed and unembedded material, provides little ultrastructural detail, due to the damage caused by the freezing process. “A very serious problem in any morphological study of frozen tissue is the ice crystal damage that occurs during the initial freezing and subsequent recrystallization.”13 Christensen14 found that rapid freezing minimized the size of the ice crystals. A number of other ways of minimizing these artifacts have been proposed. Chemical fixation or cryoprotection, for example, can reduce them. But only very rapid freezing, using specifically designed equipment, will result in satisfactory frozen preparations. In 1981, Allakhverdov and Kuzminykh15 wrote: “Well-known advantages of specimen preparation by cryomethods are accompanied by some disadvantages, resulting mainly from the inadequate level of presently existing laboratories and instruments.” These days, there are laboratories that specialize in cryomethods, and state-of-the-art equipment is now available for the preparation and observation of frozen specimens. Thin-film vitrification is a practical freezing technique that does not need very specialized equipment, but its application is limited by the fact that the material to be observed has to be thin enough for direct observation by transmission electron microscopy. Cryoultramicrotomes and electron microscopes are now capable of dealing with larger and/or thicker frozen samples, resulting in ever better observations and a growing range of applications.

© 2009 by Taylor & Francis Group, LLC

VI For the moment, cryomicroscopy using water vitrification is the method of choice for research in the field of structural biology, combined with x-ray analysis and particle visualization (e.g., nanoparticles, macroparticles, viruses, filamentary structures). Vitrification in cells and tissue can attain a depth of 10 µm, and it is now possible to observe intact cells and tissue in their natural state. In the words of Dubochet16 — though indeed Fernandez-Moran, in the early 1950s,17 might have said much the same thing — “That is what cryo-EM of vitreous sections (CEMOVIS) does.” Gérard Morel Series Editor 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Leduc, E.H. et al. The use of water-soluble glycol-methacrylate in ultrastructural cytochemistry, J. Roy. Microscop. Soc., 81, 119, 1963. Leduc, E.H. and Holt, S.J. Hydroxy-propyl methacrylate, a new water-miscible embedding medium for electron microscopy, J. Cell Biol., 26, 137, 1965. Pease, D.L. Eutectic ethylene glycol and pure propylene glycol as substitution media for dehydratation of frozen tissue, J. Ultrastructural. Res., 21, 75, 1967. Pease, D.L. and Peterson, R.G. Polymerisable glutharaldehyde-urea mixture as polar, water-containing embedding media, J. Ultrastructural. Res., 41, 133, 1972. Bernhard, W. and Granboulan, N. The fine structure of the cancer cell nucleus, Exp. Cell Res. Suppl, 9, (19), 19, 1963. Granboulan, N. and Granboulan, P. Ultrastructure cytochemistry of the nucleolus. Demonstration of the chromatin inside the nucleolus, Exp. Cell Res., 34, 71, 1964. Kawarai, Y. and Nakane, P.K. Localization of tissue antigens on the ultrathin sections with peroxidase-labeled antibody method, J. Histochem. Cytochem., 18, 161, 1970. Bernhard, W. and Leduc, E.H. Ultrathin frozen sections. I. Methods and ultrastructural preservation, J. Cell Biol., 34, 757, 1967. Bernhard, W. and Viron, A. Improved techniques for the preparation of ultrathin frozen sections, J. Cell Biol., 49, 731, 1971. Leduc, E.H. et al. Ultrathin frozen sections. II. Demonstration of enzymatic activity, J. Cell Biol., 34, 773, 1967. Painter, R.G. et al. Immunoferritin localization of intracellular antigens: The use of ultracryotomy to obtain ultrathin sections suitable for direct immunoferritin staining, Proc. Nat. Acad. Sci., USA, 70, 1649, 1973. Tokuyasu, K.T. A technique for ultracryotomy of cell suspensions and tissues, J. Cell Biol., 57, 551, 1973. Appleton, T.C. A cryostat approach to ultrathin "dry" frozen sections for electron microscopy: A morphological and x-ray analytical study, J. Microsc., 100, 49, 1974. Christensen, A.K. Frozen thin sections of fresh tissue for electron microscopy, with a description of pancreas and liver, J. Cell Biol., 51, 772, 1971. Allakhverdov, B.L.and Kuzminykh, S.B. Some aspects of developing instruments for cryomethods, Acta Histochem., 23, 75, 1981. Al-Amoudi, A. et al. Cryo-electron microscopy of vitreous sections, EMBO J., 23, 3583, 2004. Fernandez-Moran, H. Application of the freezing sectioning technique to the study of cell structure with the electron microscope, Ark. Fys., 4, 471, 1952.

© 2009 by Taylor & Francis Group, LLC

VII

ACKNOWLEDGMENTS

This handbook was initiated by Annie Cavalier and Danièle Spehner as part of a Centre National de la Recherche Scientifique (CNRS) effort in France (GDR 2368) to promote cryo-electron microscopy. Bruno Humbel, in Holland, enthusiastically joined the team. We are indebted to the contributors who have made it come true. It was not such an easy task explaining tricks of the trade, routinely carried out by each of them, but not so readily put into words. Dr. Gerard Morel provided his guidance throughout and we are very grateful to him. We also thank Profs. Daniel Boujard and Gilles Salbert who have encouraged the project and ensured a propitious environment. Our thankfulness extends to Dr. Patrick Schultz and Prof. Arie Verkleij who believed in the usefulness of such a handbook and supported us during its elaboration. Bruno Humbel would also like to thank the European Network of Excellence (NoE), FP6: “Three-Dimensional Electron Microscopy” and the Dutch Cyttron Consortium, “A Window on the Molecular Machinery of Life,” for support. We express our gratitude to Dr. Robert Drillien who not only overviewed the use of the English language, but also shared his insight on the contents of the book. This project has also benefited from the generous help of Emmanuelle Guiot who kindly performed the layout of the entire handbook; her participation was most valuable. Sandrine Pawlicki kindly constructed the book’s temporary Web site for which we are thankful. Last but not least, we would like to thank Christian Cavalier who offered us his home and his hospitality for a few hectic but very enjoyable weeks during finalization of the manuscripts. Annie Cavalier Danièle Spehner Bruno M. Humbel

© 2009 by Taylor & Francis Group, LLC

IX

THE AUTHORS Annie Cavalier, graduate of the University Claude Bernard-Lyon 1, is currently an engineer in Biology in a CNRS laboratory at the University of Rennes I, France. Annie Cavalier is a member of the French Society of Microscopy (SFµ). She was actively involved in the organization of the 13th International Congress of Electron Microscopy (Paris, July 1994), and the first Congress of the French Society of Microscopy (Rennes, June 1996). She is secretary of the GUMP (Groupement des utilisateurs des microscopes Philips-FEI; a unique association that brings together users of FEI-made electron microscopes). Annie Cavalier has regularly given lectures and practical courses for training in the field of cytology at the ultrastructural level. She has edited two technical books and contributed to numerous scientific papers. Until recently, her research interests have focused on the structural and functional characterization of water channels (aquaporins) and glycerol facilitators. Her current research concerns the assembly and dynamics of large macromolecular complexes by means of electron tomography. Danièle Spehner obtained her Ph.D in Biology from the University Louis Pasteur in Strasbourg, France, while working at the Virology Laboratory of the Medical School in Strasbourg. After a few years at Transgene, a biotechnology company, where she developed new vaccines based on poxvirus recombinants, she returned to fundamental research at the French Institute of Health and Medical Research (INSERM). She is currently an engineer at the Institute of Genetics and Molecular and Cellular Biology (IGBMC) in Illkirch, France. Early in her career, she became interested in electron microscopy to study poxviruses as such or recombinant poxviruses expressing foreign proteins. She acquired a passion for cryo-electron microscopy in biology after meeting Professor Hellmut Sitte at a workshop in Seefeld (Austria) and, later on, Bruno Humbel. She transmitted her passion and her know-how through cryo or immuno workshops to the Alsatian community and throughout France. Danièle’s current research focuses on the use of electron tomography and cryo-electron tomography of vitreous cryo-sections (Cemovis) to analyze poxvirus morphogenesis and the cell nucleus. Bruno M. Humbel graduated in Biochemistry at the Federal Institute of Technology (ETH), Zürich, Switzerland. He was privileged to do his Ph.D with Dr. Martin Müller in the group of Prof. Dr. Hans Moor at the Institute of Cell Biology, ETH. After a postdoc period of four years at the Max-Planck-Institute for Biochemistry in Munich, he joined the lab for Electron Microscopy and Structure Analysis at Utrecht University in the group of Prof. Dr. Arie J. Verkleij, where he currently holds the position of an associate professor. Bruno Humbel’s main interest is to develop preparation methods for (electron) microscopy, which not only allow a glimpse into life at high resolution, but also enable identification and localization of the machinery of life within cells. The aim is to visualize the living cell at low resolution and to zoom in to analyze its ultrastructure at high resolution: The gateway to in situ biological nanostructures. Recently, his research focuses on correlative microscopy to introduce the FIB/SEM technology into life sciences and the application of a newly developed integrated laser electron microscope (ILEM). He is teaching cryo-techniques and immunolabeling at Utrecht University and in different workshops in Europe and Asia.

© 2009 by Taylor & Francis Group, LLC

X

CONTRIBUTORS AL-AMOUDI Ashraf EMBL HEIDELBERG Germany

CHRÉTIEN Denis UMR CNRS 6026, Equipe TIPs Campus de Beaulieu Université de Rennes 1 RENNES France

ARNAL Isabelle UMR CNRS 6026, Equipe TIPs Campus de Beaulieu Université de Rennes 1 RENNES France

CRUCIFIX Corinne Institut de Génétique et de Biologie Moléculaire et Cellulaire Inserm U596, CNRS UMR7104 Université Louis Pasteur de Strasbourg ILLKIRCH France

BAUMEISTER Wolfgang Max-Planck-Institut für Biochemie Abteilung Strukturbiologie MARTINSRIED bei München Germany

DE CARLO Sacha MCDB 347 University of Colorado, BOULDER Colorado USA

BORDAT Christian INRA-CRJ NURELICE, Bât. 230 Domaine de Vilvert JOUY EN JOSAS France

DE HAAS Felix FEI Company EINDHOVEN The Netherlands

BOUCHER-MARQUIS Cédric University of Colorado Dept. of Molecular Cellular and Developemental Biology, Porter Science, BOULDER Colorado USA

DUBOCHET Jacques Centre de Microscopie Laboratoire d’Analyses Ultrastructurales Université de Lausanne LAUSANNE Switzerland

BRON Patrick UMR CNRS 6026, Equipe SDM Campus de Beaulieu Université de Rennes 1 RENNES France

EDELMANN Ludwig Anatomie und Zellbiologie, Universität des Saarlandes, HOMBURG/Saar Germany

CAVALIER Annie UMR CNRS 6026, Equipe SDM Campus de Beaulieu Université de Rennes 1 RENNES France

ELTSOV Mikhail EMBL HEIDELBERG Germany

© 2009 by Taylor & Francis Group, LLC

XI FREDERIK Peter M. Maastricht University EM Unit / Pathology MAASTRICHT The Netherlands

KAECH Andres Center for Microscopy and Image Analysis at the University of Zurich ZÜRICH Switzerland

FUKAZAWA Yugo Division of Cerebral Structure National Institute for Physiological Sciences, Myodaiji OKAZAKI Japan

LEIS Andrew Max-Planck-Institut für Biochemie Abteilung Strukturbiologie MARTINSRIED bei München Germany

GEERTS Willie J. C. Electron Microscopy & Structure Analysis Cellular Architecture  Dynamics Utrecht University UTRECHT The Netherlands

MALLETER Marine Laboratoire de pharmacologie marine Faculté des Sciences de Nantes NANTES France

GUERQUIN-KERN Jean Luc Institut Curie Recherche/INSERM U 759 Laboratoire Microscopie Ionique Bat.112 Centre Universitaire ORSAY France

MASUGI-TOKITA Miwako Division of Cerebral Structure National Institute for Physiological Sciences, Myodaiji OKAZAKI Japan

GRUSKA Manuela Max-Planck-Institut für Biochemie Abteilung Strukturbiologie MARTINSRIED bei München Germany

MÉSINI Philippe J. Institut Charles Sadron-CNRS-ULP STRASBOURG France

HAGIWARA Akari Division of Cerebral Structure National Institute for Physiological Sciences, Myodaiji OKAZAKI 444 Japan

MOREL Gérard UMR 5123 CNRSUniversité Claude Bernard-Lyon 1 VILLEURBANNE France

HUMBEL Bruno M. Electron Microscopy & Structure Analysis Cellular Architecture  Dynamics Utrecht University UTRECHT The Netherlands

PAPAI Gabor Institut de Génétique et de Biologie Moléculaire et Cellulaire Inserm U596, CNRS UMR7104 Université Louis Pasteur de Strasbourg ILLKIRCH France

© 2009 by Taylor & Francis Group, LLC

XII SCHMUTZ Marc Institut Charles Sadron-CNRS-ULP STRASBOURG France

STUDER Daniel Institute für Anatomy University of Bern BERN Switzerland

SCHULTZ Patrick Institut de Génétique et de Biologie Moléculaire et Cellulaire Inserm U596, CNRS UMR7104 Université Louis Pasteur de Strasbourg ILLKIRCH France

TARUSAWA Etsuko Division of Cerebral Structure National Institute for Physiological Sciences, Myodaiji OKAZAKI Japan

SCHWARZ Heinz Max-Planck-Institut fuer Entwicklungsbiologie TÜBINGEN Germany

THOMAS Daniel UMR CNRS 6026, Equipe SDM Campus de Beaulieu Université de Rennes 1 RENNES France

SHIGEMOTO Ryuichi Division of Cerebral Structure National Institute for Physiological Sciences, Myodaiji OKAZAKI Japan

VAN DONSELAAR Elly Electron Microscopy & Structure Analysis Cellular Architecture  Dynamics Utrecht University UTRECHT The Netherlands

SITTE Hellmuth HOMBURG-SAAR Germany or SEEFELD in Tyrol Austria

VANHECKE Dimitri Institute für Anatomy University of Bern BERN Switzerland

SPEHNER Danièle Institut de Génétique et de Biologie Moléculaire et Cellulaire Inserm U596, CNRS UMR7104 Université Louis Pasteur de Strasbourg ILLKIRCH France

VERKLEIJ Arie J. Electron Microscopy & Structure Analysis Cellular Architecture  Dynamics Utrecht University UTRECHT The Netherlands

STIERHOF YorkDieter Zentrum für Molekularbiologie der Pflanzen (ZMBP). Elektronenmikroskopie, Universität Tübingen TÜBINGEN Germany

VONCK Janet Max-Planck-Institute of Biophysics Department of Structural Biology FRANKFURT am Main Germany

STORMS Marc M.H. FEI Company EINDHOVEN The Netherlands

ZUBER Benoît MRC Laboratory of Molecular Biology CAMBRIDGE United Kingdom

© 2009 by Taylor & Francis Group, LLC

XIII

TABLE OF CONTENTS Introduction

XVII

Abbreviations

XIX

Chapter 1 Vitreous Water Jacques Dubochet

1

Part I  Cryo-Fixation Methods Chapter 2 Slam-Freezing, Metal-Mirror Freezing Danièle Spehner and Ludwig Edelmann  Chapter 3 Plunge-Freezing (Holey Carbon Method) Sacha De Carlo

17

Chapter 4 Controlled Vitrification Peter M. Frederik, Felix de Haas and Marc M.H. Storms

69

Chapter 5 BAL-TEC HPM 010 High-Pressure Freezing Machine Andres Kaech

101

Chapter 6 High-Pressure Freezing LEICA EMPACT Dimitri Vanhecke and Daniel Studer

129

© 2009 by Taylor & Francis Group, LLC

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XIV

Part II  Cryo-Electron Microscopy Chapter 7 Frozen-Hydrated Macromolecules for Structural Analysis Corinne Crucifix, Gabor Papai and Patrick Schultz

159

Chapter 8 Two-Dimensional Crystals Patrick Bron and Janet Vonck

191

Chapter 9 Cryo-Negative Staining Sacha De Carlo

219

Chapter 10 Vitrification of Dynamic Microtubules Isabelle Arnal, Marine Malleter and Denis Chrétien

237

Chapter 11 CEMOVIS: Cryo-Electron Microscopy of Vitreous Sections Jacques Dubochet, Ashraf Al-Amoudi, Cédric Bouchet-Marquis, Mikhail Eltsov and Benoît Zuber

259

Chapter 12 Cryo-Electron Tomography Andrew Leis, Manuela Gruska and Wolfgang Baumeister

291

Part III  Low-Temperature Embedding Chapter 13 Freeze-Substitution Bruno M. Humbel

319

Chapter 14 Cryo-Fixation, Freeze-Substitution, Rehydration and Tokuyasu CryoSectioning YorkDieter Stierhof, Elly van Donselaar, Heinz Schwarz, Bruno M. Humbel

343

Chapter 15 Freeze-Drying and Embedding of Biological Material Ludwig Edelmann

367

© 2009 by Taylor & Francis Group, LLC

XV

Part IV  Freeze-Fracture and Metal Shadowing Chapter 16 The Shadow of Hydrated Biological Specimens Daniel Thomas

391

Chapter 17 Cryo-Fracture of Self-Assembled Systems in Organic Solvent Marc Schmutz and Philippe J. Mésini

411

Part V  Analysis Chapter 18 Progressive Lowering of Temperature for Immunolabeling and in situ Hybridization Annie Cavalier and Danièle Spehner

433

Chapter 19 Cryo-Sectioning According to Tokuyasu Bruno M. Humbel and YorkDieter Stierhof

467

Chapter 20 Cryo-Preparation Procedures for Elemental Imaging by SIMS and EFTEM Christian Bordat and Jean-Luc Guerquin-Kern

499

Chapter 21 Correlative Light and Electron Microscopy Heinz Schwarz and Bruno M. Humbel

537

Chapter 22 SDS-digested Freeze-Fracture Replica Labeling (SDS-FRL) Yugo Fukazawa, Miwako Masugi-Tokita, Etsuko Tarusawa, Akari Hagiwara and Ryuichi Shigemoto

567

Chapter 23 Immunolabeling of Ultrathin Sections with Enlarged 1 nm Gold or QDots YorkDieter Stierhof

587

Chapter 24 3-D Electron Tomography of Cells and Organelles Willie J. C. Geerts, Bruno M. Humbel and Arie J. Verkleij

617

Final Considerations Hellmuth Sitte

651

Glossary

657

© 2009 by Taylor & Francis Group, LLC

XVII

INTRODUCTION The purpose of this handbook is to provide guidance to newcomers to the field who wish to learn and possibly apply the methods it describes. Of course, no biologist knows every technique in his specialty and this also pertains to those using electron microscopy, so we hope that our fellow colleagues will also find this book useful for one application or another. The chapters have been written by experts in the field, in some instances the very inventors of the method, who have taken care to explain the history behind the techniques they describe and how they are most readily carried out today. Whenever possible, clear step-by-step recipes are presented and the tools and ingredients of the methods, as well as where they can be purchased, are listed. After a quick glance at the contents one will realize that this handbook goes well beyond the preparation methods the title suggests and into the realm of cryo-electron microscopy analysis itself, hence the numerous illustrations from the contributors’ own laboratories. Most biologists have their own idea of what cryo-electron microscopy is all about and this notion may be strongly influenced by one’s own experience as well as the fashion of the times. It has long been realized that freezing specimens is a nearly perfect way to maintain biological samples in a state as close as possible to their native state. Over the years many different methods have been devised to freeze samples and each of them has been presented with advice on how they should be applied and to what kind of material. The ultimate achievement in this field is certainly the ability to embed samples into a somewhat mysterious form of ice known as vitreous water or crystal-free ice. Today, there are numerous ways to take advantage of this technological feat. Whereas some methods aim at perfect preservation for fine structural analysis, others use vitreous water as an intermediate step for in situ localization of biological molecules and ions. Sophisticated methods of resin embedding subsequent to removal of vitreous water, as well as freeze-fracture, have become valuable tools for numerous applications as illustrated in this handbook. Ice also turned out to be a convenient medium for embedding and sectioning, as has been so exquisitely exemplified by the so-called Tokuyasu method, and dealt with in several chapters herein. This and the so-called hybrid methods, freezesubstitution and freeze-drying, have been found to be ideal intermediates to achieve highly efficient immunolabeling, so critical for determining the molecular geography inside tissues and cells. On the other hand, such methods have gained considerably from parallel developments including new ways to stain samples and label them. This opus would have been deficient if it had not also dealt with one of today’s most promising developments in cryo-electron microscopy, namely cryo-electron tomography. Although such tools would deserve an entire book in their own right, the chapters herein should provide a useful overview of the topic. The pace of cryo-electron microscopy has been accelerating in recent years and the methods available are constantly evolving. It is our wish that this handbook be a useful aid to the scientific community for current and future endeavors, and especially for novices to show the beauty of cryo and encourage them to take up the challenge.

© 2009 by Taylor & Francis Group, LLC

XIX

ABBREVIATIONS 2-D 3-D ART CCD CEMOVIS CET CFD CIA CMC Cryo-ET Cryo-EM CS CTF EELS EF EFTEM ESI ET FD FEG FF FRL FS HAADF HC-PRO HPF LR LTE MCS MES MMF MS medium PLT Pt/C QD RTS SIMS SIRT WBP θ

© 2009 by Taylor & Francis Group, LLC

 Two-Dimension/two-Dimensional  Three-Dimension/three-Dimensional  Algebraic Reconstruction Technique  Charge Coupled Device (camera)  Cryo-Electron Microscopy Of Vitreous Sections  Computerized electron tomography  Cryo-sorption Freeze-Drying system  Cutting-Induced Amorphous water  Critical Micelle Concentration  Cryo-Electron Tomography  Cryo-Electron Microscopy (of vitrified specimens)  Cryo-sectioning according to Tokuyasu  Contrast Transfer Function  Electron Energy-Loss Spectrum  Energy Filtering  Energy Filtered Transmission Electron Microscope  Electron Spectroscopic Imaging  Electron Tomography  Freeze-Drying  Field Emission Gun  Freeze-Fracturing/Freeze-Fracture  Freeze-fracture Replica Labeling  Freeze-Substitution  High-Angle Annular Dark-Field  Helper Component PROteinase  High-Pressure Freezer, High-Pressure Freezing  London Resin  Low-Temperature Embedding  Membrane Contact Side  Morpholino Ethane Sulfonic acid  Metal Mirror Freezing  Murashige and Skoog medium  Progressing Lowering of Temperature  Platinum used in conjunction with carbon  Quantum dot  Rapid transfer system  Secondary-Ion Mass Spectrometry  Simultaneous Iterative Reconstruction Technique  Weighted Back-Projection  Angle of shadowing

© 2009 by Taylor & Francis Group, LLC

3

Vitreous Water

CONTENTS

1.

INTRODUCTION ................................................................................................. 5

2.

WATER IS REMARKABLE................................................................................ 5 2.1. 2.2.

Ice floats ....................................................................................................... 5 Water has a high specific heat ...................................................................... 6

3.

VITREOUS WATER IS STRANGE.................................................................... 7

4.

VITREOUS WATER MADE SIMPLE ............................................................... 9 4.1. 4.2. 4.3.

Vitrification is a good preservation method.................................................. 9 What is vitrification and devitrification? ...................................................... 9 How can vitrification be achieved? ............................................................ 10

5.

OBSERVED RESULTS ...................................................................................... 12

6.

REFERENCES .................................................................................................... 14

© 2009 by Taylor & Francis Group, LLC

Vitreous Water

5

1. INTRODUCTION NASA would like to extend biological studies to other worlds and consequently makes every effort to look for water on other planets. For us, on earth, aiming at a better understanding of our earthly life, water is also of primary concern. It is the major constituent of all living organisms; it is the medium in which life takes place and it directly participates in most actions that make life work. Nevertheless, water is frequently neglected and it remains poorly understood in several essential aspects. The systematic exclusion of water as an object of investigation during the first 50 years of electron microscopy certainly contributed to the bias against it. Times have changed, however; for two decades now water has regained the central role it always had in nature. This book tells the story of its changed fortunes.  Many electron microscopists know little about water and even less about the strange properties of vitreous water. The present chapter aims at familiarizing them with the substance with which they are working. The literature on vitreous water is dangerously abundant and it is rather difficult for those who want to gain an understanding of it from first principles.1-3 Most of our review of 1988 still holds.4 For pleasure, we recommend the Bibliography of Water by Philip Ball.5 A recent review by Angell6 sums up the present situation, gives abundant references to the literature and, for the first time, incorporates the observations of cryo-electron microscopy with the bulk of data from the water field. In the following presentation, we aim to examine some fundamental questions about water and vitreous water. This simple presentation — hopefully not simplistic — will probably raise some specialists’ eyebrows. My excuse is my own incompetence. As a long-time tourist of “Waterland” I hope that it may offer a useful understanding for practitioners.

2. WATER IS REMARKABLE Chapters on water traditionally start with a list of the unusual properties that make water remarkable. We will consider only two of them: ice floats and liquid water has a high specific heat. These two properties are easy to grasp and they are convenient proxies for all the others.

2.1. Ice floats When a room is full, a good way to gain space is to make order. The contrary holds for water for which the disordered liquid form is 8 percent more dense than hexagonal ice, the ordered form we are used to. How can this be? Very simply, it all depends on how order is made. For example, in your home, you will use up a lot of space if you place only one book per shelf. This is similar to what happens with water.

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In a plane, one circle can touch up to six identical nonoverlapping neighbours (see Figure 1.1A). In space, identical spheres can accommodate up to twelve nearest neighbours. In hexagonal ice or cubic ice, each water molecule has only four next neighbours. Why so few? Because of hydrogen bonds! Each water molecule offers two and accepts two in a tetragonal symmetry (see Figure 1.1B). The rule of four can be extended to the neighbours and to the neighbours of the neighbours without limit. The result is ice. Two forms are possible: cubic ice (a form with the same density frequently encountered in cryo-electron microscopy) when the bonds of neighbouring tetrahedron are coplanar, or hexagonal ice in which tetrahedrons are rotated by 60° with respect to each other, as is the case in Figure 1.1C. With only four instead of twefve next neighbours, ice is full of holes. Hydrogen bonds can be seen as long sticks holding the molecules at a distance rather than glue holding them tightly together. Because of the large amount of free space between water molecules, other arrangements involving bending and stretching of hydrogen bonds are possible. The result is several other forms of crystalline ice. In most cases, they are only stable under pressure. The hydrogen bond is not very strong. It tends to break when temperature increases. Above 100°C at 1 atm, it cannot even hold the water molecules together, and water vapour becomes the most stable form. At intermediate temperatures, hydrogen bonds constantly break and reform, thus enabling any molecule to fall down for an instant, into the hole, closer to its neighbour. The structure is disordered, the density is higher; this is liquid water.

2.2. Water has a high specific heat Everything that can shake or move does so at the molecular level. This is temperature. Each of these things that can shake or move is called a degree of freedom. It is remarkable that each degree of freedom contains on average the same amount of energy  proportional to the temperature T:  = ½ kT, where the proportionality factor k is the Boltzmann’s constant, 1.38.10-23 J/oC. Everything else remaining equal, warming means increasing the amount of energy contained in each degree of freedom. The specific heat — the amount of heat required to warm one gram by one degree Celsius — is therefore directly proportional to the number of degrees of freedom n. n is a remarkable number. It tells us how complicated a system is. It tells us how many parameters must be introduced in any realistic model of the system. It tells us the magnitude of the task for those who have the ambition to understand it. We shall try an exercise. n is measured to be nine for water vapour. For each molecule, three degrees of freedom are required to specify its location in space (X, Y, Z) and three others for its orientation (3 Euler angles, three are left for internal vibrations of the molecule. This makes sense! What about ice, which has n = 8? As compared to water vapour, we expect the internal vibrations to be the same — that accounts for three degrees of freedom — but the six involved in location and orientation are lost because they are now nearly fixed in a crystal. So, where are the other five? Solid-state physics gives us the answer. These five are the collective vibrations of the crystal lattice; the molecules are allowed to move, but only in restricted cooperative movements. We come now to liquid water. What

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do we expect? Internal vibrations remain; those are still three. Location and orientation: Molecules are quite free in liquid water, but not so much as in gas; it will be some number smaller than six, say, four. We are left with possible cooperative vibrations as in the crystal, but water is not known for its crystalline sound; it goes “splash” not “cliiing”; so instead of five, we can perhaps save three. And thus we have: n = 3 + 4 + 3 =10. But no, this cannot be! The specific heat of liquid water is more than twice that of vapour or of ice; n = 18. That is eight degrees of freedom more than our guess. We can add one or two more degrees for positional freedom and better collective vibrations, but how to reach eight? Where are the missing degrees of freedom? Apparently, nobody knows for sure. They will probably have to be found in some more cooperative interactions resulting in a much richer conformational space. Certainly, the years to come will bring a solution to the problem of the missing degrees of freedom. I like to guess that the solution will have interesting consequences in various fields where water is important — probably in biology also. Certainly, it is wise to keep an eye on water. 

3. VITREOUS WATER IS STRANGE Vitrification of water is an old idea, loaded with hopes and dreams. Imagine cooling water — or an organism — in such a way that it becomes immediately immobile. It is frozen time, suspended life. A science was born out of these ideas and the state of suspended animation got its name: the vitreous state.7 It turned out, however, that vitrification was terribly difficult to achieve except in the case of concentrated solutions. Even worse, the argument was made that vitrification of pure water is fundamentally impossible. The discovery, at the beginning of the 1980s, that the vitreous state can be obtained from liquid water8,9 was first greeted with scepticism, but because the procedure is simple and reproducible, some way must be found to reconcile theory and practice. We will explain why it is still a difficult task. The phase diagram of water sets the scene (see Figure 1.2). There is one domain for the liquid phase at the upper right, one domain for the vapour at the bottom, and one for ice on the left. We note that the vapour and liquid domains are not completely separated. The line goes to the so-called critical point and then stops. This is because there is not always a difference between these two phases. Above the pressure and temperature of the critical point, gas and liquid are all the same. On the contrary, ice is always separated from the domains of liquid and vapour. There is a good reason for the strict separation of the solid domain: The existence of a critical point between two phases means that it is possible to go continuously from one to the other. This cannot exist between the liquid, which has no symmetry, and hexagonal ice, which has hexagonal symmetry. An intermediate state with “just a bit of symmetry” makes no sense. At normal pressure, the most stable state is ice below 0°C, it is liquid between 0°C and 100°C and vapour is the normal state above 100°C. These are the conditions at which the free energy G is minimal as illustrated in Figure 1.3 for the liquid (A) and for the vapour region (B). This does not mean, however, that the liquid does not exist above 100°C; one can still warm it above this temperature provided it does not cross the potential barrier separating it from the vapour phase (see Figure 1.3 C). It is said to be in a metastable state.

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Upon further warming, the advantage of the vapour phase over the liquid phase becomes greater, and the potential barrier separating them becomes smaller. The higher the temperature, the more difficult it is to keep the superheated liquid from escaping as vapour. Upon further warming, there is a point where the potential barrier becomes zero. Above this temperature, the liquid state just does not exist anymore. The line depicting this limit on the phase diagram is called the spinodal line (see Figure 1.2). An analogy may help to clarify the concept. My pen on the desk is in a metastable state; the stable state is when it lies on the floor. If the table is smaller, the pen can still be on the table though it will fall more frequently. If the table is removed, the question whether the pen is on the table or not ceases to make sense. Similarly, there is no question anymore about the liquid state after the spinodal line. However, the region just before the spinodal line is particularly interesting. Of course, it is difficult to explore it because the liquid takes every opportunity to vaporize. In this region, all the properties of the liquid become strange. A graph presenting the variation of any property of the superheated liquid always shows that something special is happening when the temperature approaches the critical line. The signature of the approaching catastrophe is characteristic. What is the freezing temperature of water? This trap question calls for the wrong answer. 0°C is not the freezing temperature of water at normal pressure; it is the melting temperature of ice! The freezing temperature of liquid water is somewhere below 0°C, whenever the liquid finds its way toward the more stable state of ice. This is an important difference because, in nature, it frequently happens that the temperature fluctuates around 0°C. It makes a lot of difference as to whether water freezes or not. The matter has been studied in detail and one basic fact stands out: Water resists undercooling at low temperatures pointing toward a spinodal line at around 45°C. The signature is the same as for superheated water. It explains why attempts to vitrify pure water were unsuccessful for so long, just like keeping the pen on the table does not work when there is no table. But this does not explain the finding in 1980 that ultrarapid cooling can vitrify liquid water! Perhaps, after all, there is no spinodal at 45°C and the strange behaviour of undercooled water must be explained differently? Soon after, another finding was made, complicating the situation even further. Under very high pressure (10,000 bar), and cooled at liquid nitrogen temperature, hexagonal ice crumbled into a High-Density Amorphous state (1.15 g/cm3 after restoration of normal pressure) poetically called HDA, which is clearly different from LDA, the Low Density Amorphous state (0.94 g/cm3) obtained by rapid cooling of liquid water.10 This could be explained by considering that disorder can take many forms from low to high density amorphous, just as the disorder in a room can correspond to any level of heaping. But as usual, water surprises us. When HDA is rewarmed slowly, it suddenly changes into something resembling LDA. The transformation cannot be ignored because the abrupt change of density results in large internal tensions, which make the block fly apart in small fragments. It was also observed by electron microscopy.11 Thus, it appears that amorphous water is disordered in at least two mutually exclusive states. In HDA, the disorder is organised in some way and it becomes unstable above 160°C, at which temperature the disorder must be organized in a different way. The big trouble is that nobody knows what makes the difference. Keep an eye on vitreous water, it could tell us important things about water!

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4. VITREOUS WATER MADE SIMPLE What are the consequences of the above consideration for us, cryo-electron microscopists? For one thing, and as always in science, it tells us that we should remain vigilant because we do not know what we are observing. It could be that our vitrified specimens are fundamentally different from the original material. In them, water is perhaps organised differently. The future will tell.

4.1. Vitrification is a good preservation method The other thing we know is that vitrification is an excellent method for preserving biological material. It makes it possible to observe samples without going through the major transformations of chemical fixation, dehydration and staining. Even more important, vitrification is, by itself, a remarkably conservative procedure. This is demonstrated by a large body of evidence extending from cryo-preservation of embryos for in vitro fertilization, to cryo-x-ray diffraction for best preservation of atomic structures in delicate crystals. The quality and the coherence of the results obtained by cryo-electron microscopy over the last 20 years also contribute to the credibility of the method. 

4.2. What is vitrification and devitrification? In everyday life, and until we know more about vitreous water, cryo-electron microscopists can resist becoming depressed by following Occam’s recipe, adopting the simplest possible view of vitrification and of the vitreous state. In this view, vitrification is the process by which the viscosity of the sample is increased to such a high value that molecular movements become negligible before ice crystal formation has time to start. Any subsequent change is blocked unless considerable forces are applied. Typically, such forces may arise during cutting thin sections and during observation in the electron microscope. For a normal experimental time scale, the vitreous state holds as long as the temperature is well below 135°C. At this temperature, the movement of the water molecules becomes significant enough so that crystallisation takes place within minutes. This is the process of devitrification. It can take place at a slightly lower temperature if one waits long enough or if it is accelerated by electron irradiation. We note that ice formation by crystallisation from the cooling liquid is very different from ice formation by devitrification. In the first case, it takes place in the liquid medium where everything is mobile. As a consequence, the growing ice crystal, which only incorporates water molecules, rejects all the other constituents of the solution, such as salts, other solutes and macromolecules. Segregation takes place between pure water in the ice crystals and all the dehydrated remains of the biological material. Ice formation by devitrification, on the other hand, takes place under conditions of severely restricted mobility — just enough to allow water molecules to rearrange into ice crystals. Under these conditions, no large ramified hexagonal ice crystal is formed, but instead many compact, sub-µm cubic crystals appear.

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They are easily recognized from the characteristic circles of the powder diffraction spectra. In spite of the fact that little is known about the devitrification process, it probably takes place with minimum rearrangement of the biological material. One can infer that the fine structure of a frozen biological specimen in which ice is formed by devitrification is better preserved than when ice grows from the liquid phase.12 These considerations are important for freeze-substitution (see Chapter 13), which is always performed well above the devitrification temperature. Substitution, therefore, deals with water in the form of crystalline ice. It probably makes a lot of difference whether this ice was formed by devitrification of a vitrified specimen or whether it was crystallized during cooling from the liquid phase. The best route is certainly to start with a vitrified sample.

4.3. How can vitrification be achieved? In principle, the method is simple. It suffices to cool the specimen so rapidly that water molecules are practically immobilized before an ice crystal starts to form. By chance, nucleation, the beginning of ice crystal formation, is not an easy task. Even billions of molecules take a while until, by chance, a group of them happens to be in the right conformation. Once a crystal has started, the road is clear; it produces its own heat, further amplifying its growth until all the available water is sequestrated. The question we may address is, therefore: What is the cooling speed required for vitrification?  The answer is: Nobody knows! Fortunately we know quite well what are the important parameters, and experience tells us how they can be combined to reach vitrification. The easiest way is to increase cooling speed by reducing the size of the specimen. Physics has found that cooling speed increases with the inverse square of the size of an object immersed in a good cryogen. Calculation13 tells us that, one cm deep in a piece of meat suddenly placed at liquid nitrogen temperature, the temperature drop is expected to be in the oC/sec range; at 0.1 mm from the surface, it will reach ca. 104 oC /sec and for a 200 nm thin layer, it is deduced to reach the staggering value of 1011 oC /sec. It is a big advantage for electron microscopists, and the green light towards cryo-electron microscopy, that vitrification is relatively easy when the specimens are of a size that is generally observed in a transmission electron microscope (TEM). Chapters 5 and 6 describe how it can be done. In general, it is considered that vitrification of pure water is not possible for dimensions in excess of some µm. Some µm are more than enough if the specimen is to be directly observed in a transmission electron microscope. Macromolecules, viruses, many isolated organelles and even whole cells, if they can be squeezed onto a thin layer, can be directly vitrified in that way.14 Most eukaryotic cells, however, and all tissues are in a larger range of dimensions. Vitrification at least in the 100 µm scale is needed.

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The cooling speeds calculated above are theoretical maxima. They cannot be significantly increased because the limiting factor is the thermal conductivity of water. There are, however, two other parameters that can favour vitrification: the addition of a cryoprotectant and high pressure. The former acts by reducing the capability of water molecules to participate in crystal formation. For example, in a saturated sugar solution, water molecules are so involved in their interaction with the sugar that they cannot crystallize. Most soluble substances act as a cryoprotectant. The material inside a cell, the cytoplasm, which typically corresponds to 15 to 30 percent dry weight, is a cryoprotectant that nature provides freely. We take advantage of it. High pressure is not so easily offered and its advantage must be paid for.15,16 The method stems from the fact that water increases its volume upon freezing; therefore, great pressure applied during cooling makes ice formation more difficult. The trouble is that pressure has only a minor influence on the volume of water, and considerable pressure must be applied in order to induce a significant effect. 2000 atmosphere is the optimal pressure; it is more than that found in the deepest recesses of the ocean. Applying such a pressure in milliseconds while the specimen is cooled at maximal speed is an engineering challenge that may have some similarity with gun technology. Furthermore, it raises the question as to whether this sudden high pressure may induce structural changes. Certainly it does. The very small compressibility of biological matter suggests, however, that the changes are small; this has been confirmed experimentally. For example, it is surprisingly difficult to kill cells by applying high pressure for a short period of time,17 and reports on structural changes produced rapidly by high-pressure freezing concern quite special systems or very minor transformations.18 Certainly, one should pay careful attention to this problem; however, my personal guess is that the effect of cooling has more important structural consequences than high pressure. There are perhaps other means of helping to achieve vitrification. There are substances that are said to be especially efficient cryoprotectants. Trehalose is one of them. Nature has invented surprising ways to avoid — or to help — ice crystal formation. There are efficient antifreeze proteins in plants and animals,5 and it has been claimed that some other common proteins have similar properties (G. Prulière, personal communication). People have also considered using high frequency electromagnetic waves to prevent ice nucleation. These possibilities are certainly worth creative and careful investigation. Summarizing, we note that vitrification of pure water or of any diluted aqueous solution at normal pressure is easy for thicknesses compatible with direct observation in the electron microscope (µm range). This can be increased by a factor of ten by taking advantage of the natural cryoprotective capabilities of most biological specimens. Another factor of ten is gained by resorting to high pressure.19 The 100 µm range for practical bulk vitrification thus is within reach. Practicalities are described in Chapters 5 and 6.

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5. OBSERVED RESULTS  Figure on the Chapter’s title page General view: The three forms of ice.

 The three forms of solid water frequently encountered in electron microscopy and their electron diffractogram. At the left is a single, large, ramified hexagonal ice crystal grown from a thin layer of water on a supporting film. At upper right is a layer of small cubic ice crystals grown from a thin vitreous layer rewarmed at 135°C. The electron diffractogram formed of concentric rings is characteristic of the large number of small crystals in the observed field. Note also the dark regions in the two forms of ice; they are Bragg reflexions taking place where the crystal has exactly the correct orientation. At the bottom is a layer of amorphous water formed by low pressure vapour deposition on a cold supporting film. (From Dubochet et al. (1988),4 with permission.) 

 Figure 1.1 The water molecule.

 Water molecules and their arrangement in space. Centred on the oxygen atom, with tetragonal symmetry, two strong covalent bonds attach hydrogen atoms at short distance (0.1 nm), whereas two weaker hydrogen bonds are stretched at longer distance (0.2 nm) toward other oxygen atoms. (A) In a plane, a circle can have up to 6 adjacent neighbours. (B) A water molecule and its 4 bonds. (C) A pair of water molecules attached as in hexagonal ice. The shadowed plane marks 3 coplanar bonds.

 Figure 1.2 Phase diagram of water.

 Phase diagram of water. For an explanation, see Section 3.

 Figure 1.3 Free energy and spinodal.

 Stability of liquid (L) and vapour (V) water close to the boiling point. (A) The liquid is the most stable state (below 100°C). (B) The vapour is the most stable state (above 100°C). (C) After the spinodal line, the liquid state exists no longer.



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Figure 1.1

Figure 1.2

Figure 1.3

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6. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Eisenberg, D. and Kauzmann, W. The Structure and Properties of Water, Oxford University Press, Oxford, 1969. Franks, F. Properties of aqueous solutions at subzero temperatures, in Water: A comprehensive treatise. Water and Aqueous Solutions at Subzero Temperatures, Franks, F. ed., Plenum Press, New York, 1982, 215. Debenedetti, P.G. Metastable Liquids, Princeton University Press, Princeton, 1996. Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens, Q. Rev. Biophys., 21, 129, 1988. Ball, P. H2O; A Biography of Water, Weidenfeld & Nicolson, London, 1999. Angell, C. A. Amorphous water, Ann. Rev. Phys. Chem., 55, 559, 2004. Luyet, B.J. and Gehenio, P.M. Life and Death at Low Temperature, Biodynamica, Normandy, MO, 1940. Mayer, E. and Brüggeller, P. Complete vitrification in pure liquid water and dilute aqueous solutions, Nature, 288, 569, 1980. Dubochet, J. and McDowall, A.W. Vitrification of pure water for electron microscopy, J. Microsc., 124, RP3-RP4, 1981. Mishima, O., Calvert, L.D., and Whalley, E. An apparently first-order transition between two amorphous phases of ice induced by pressure, Nature, 314, 76, 1985. Al-Amoudi, A., Dubochet, J., and Studer, D. Amorphous solid water produced by cryosectioning of crystalline ice at 113K, J. Microsc., 207, 146, 2002. Dubochet, J. et al., Freezing; facts and hypothesis, Scanning Microsc. Suppl., 5, S11— S16, 1991. Studer, D. et al. Vitrification of articular cartilage by high pressure freezing, J. Microsc., 179, 321, 1995. Garvalov, B.K. et al. Luminal particles within cellular microtubules, J. Cell Biol., 174 (6), 759, 2006. Moor, H. Theory and practice of high pressure freezing, Cryotechniques in Biological Electron Microscopy, Steinbrecht, R.A. and Zierold, K. Springer, eds., Heidelberg, Germany, 175, 1987. Studer, D., Michel, M., and Müller, M., High pressure freezing comes of age, Scanning Microsc. 3, 253, 1989. Sato, M. et al. Schizosaccharomyces pombe is more sensitive to pressure stress than Saccharomyces cerevisiae, Cell Struct. Funct., 21, 167, 1996. Leforestier, A. and Livolant, F. Cholesteric liquid crystalline DNA: A comparative analysis of cryofixation methods, Biol. Cell, 71, 115, 1991. Sartori, N., Richter, K., and Dubochet, J. Vitrification depth can be increased more than 10 fold by high pressure freezing, J. Microsc., 172, 61, 1993.

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Part I Cryo-Fixation Methods

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Slam-Freezing, Metal-Mirror Freezing

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CONTENTS

GENERAL INTRODUCTION ...................................................................................... 21 1.

PRINCIPLES OF SLAM-FREEZING .............................................................. 22

2.

SUMMARY OF THE DIFFERENT STEPS ..................................................... 23

3.

MATERIALS/PRODUCTS/SOLUTIONS ........................................................ 23 3.1. 3.2. 3.3.

4.

PROTOCOLS ...................................................................................................... 26 4.1. 4.2. 4.3. 4.4. 4.5. 4.6.

5.

Preparation of the Copper Block ................................................................ 26 Preparation of the Slammer ........................................................................ 28 Sample Preparation..................................................................................... 29 Cryo-Immobilization Procedure ................................................................. 30 Checking the Quality of Freezing............................................................... 34 Further Processing ...................................................................................... 35

ADVANTAGES/DISADVANTAGES................................................................ 39 5.1. 5.2.

6.

Materials ..................................................................................................... 23 Products ...................................................................................................... 26 Solutions ..................................................................................................... 26

Advantages ................................................................................................. 39 Disadvantages............................................................................................. 40

WHY AND WHEN TO USE A SPECIFIC METHOD .................................... 41 6.1. 6.2. 6.3.

For Ultrastructural and Immunolabeling Experiments ............................... 41 To Study Dynamic Events .......................................................................... 41 At Liquid Helium Temperature .................................................................. 41

7.

OBSERVED RESULTS ...................................................................................... 42

8.

REFERENCES .................................................................................................... 46



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GENERAL INTRODUCTION Visualization inside a cell, a tissue or a whole organism is a fascinating challenge that has exploded in the last century with the development of new microscopes at the light microscopy or electron microscopy level. Light microscopy and, in particular, confocal microscopy has its own limits due to the resolution (see Chapter 21). Electron microscopy aims at acquiring ultrastructural information, which also implies ultraresolution, and therefore, requires the best preservation of samples. This goal was first achieved by molecular structural biologists who preserve their samples in the native state and keep them at low temperature in the cryo-electron microscope. However, only a few organisms, such as viruses or bacteria, can be observed directly in their native state using the bare grids method developed by J. Dubochet and M. Adrian (see Chapters 3, 7 and 9). It has been noted by Bellare et al.3 that the environment (temperature, humidity and chemical atmosphere) is essential for arresting the sample in its native state, and a method was devised to achieve the appropriate conditions. This method was improved for biological applications by P. Frederik,9 who has constructed a robot to plunge samples in a humidity, and temperature-controlled atmosphere (see Chapter 4). Despite the fact that electron microscopes are increasingly more powerful and that, for example, in theory, the electron beam of a FEG 300 kV can go through a 1 µm thick specimen, it is not possible to observe an entire cell or tissue. The largest organism that can be observed in a frozen hydrated state are human platelets.10 Therefore, most biological samples that one wants to observe in an electron microscope have to be sectioned after cryoimmobilization. Sections can be cut directly after cryo-immobilization (see Chapter 11) or after freeze-substitution or freeze-drying and resin embedding (see Chapters 13 and 15). Each method has its advantages and drawbacks, which are described in the chapters mentioned. The initial step of rapidly cooling to cryo-immobilize a sample is probably the most critical one It is amazing to read that freezing techniques appeared very early, just before the 20th century, as the best methods for structural preservation.1 However, “conventional microscopy” involving chemical fixatives and dehydration of the specimen, thus inducing various artifacts in samples, had more success than cryo-techniques. This is perhaps due to the fact that cryo-techniques are difficult to work with and require specific skills, experience and expensive apparatuses. Today, despite the fact that the apparatuses are very expensive, cryo-techniques have gained in popularity due to the development of new techniques, such as cryo-electron tomography (see Chapters 12 and 24). Cryo-immobilization can be carried out on a simple impact freezing machine (a very powerful machine unfortunately no longer on the market is Escaig’s slammer7), on a double-jet freezing machine18 or by high-pressure freezing (see Chapters 5 and 6). This chapter will concentrate on the slamming method also referred to as impact or metal mirror freezing. For electron microscopy this method, which was originally described by van Harreveld and Crowell,27 has been modified and improved by Heuser et al.,12 Escaig,8 Heath,11 and Phillips and Boyne.19 Despite the fact that the method is very easy to perform and the apparatus is inexpensive, very few scientists use it. “The easiest way is from time to time the most difficult way.” So, some tips and tricks included in this chapter can make the slamming method an attractive technique for many applications.

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1. PRINCIPLES OF SLAM-FREEZING Slamming, the process of rapidly projecting cells or tissue onto a cooled metal block is one of the fastest heat transfer methods. One can expect to get several micrometers in depth of well-frozen material.

 Structural preservation of a 1030 µm thick region of the cell pellet or tissue.7

Principle of the method:23 A solid copper block is cooled in a Dewar flask with liquid nitrogen (LN2). Then, using an insulated manipulator, the copper block is lifted just far enough above the LN2 level that it still remains in the cold atmosphere of evaporated LN2, thus avoiding frosting.

 The metal mirror (MM80 from Leica) is described in this chapter.

 It is possible to slam large tissue fragments of up to 2 cm in diameter (see Chapter 20).

 Other apparatuses exist, however most of them are no longer on the market.

Cells or tissue have to be prepared so that  This step influences the quality of they remain in the best physiological state ultrastructural preservation before slamming:  Cells have to be carefully pelleted as is commonly carried out when harvesting cultured cells.  Tissue has to be excised and kept in an appropriate physiological buffer. 

 It is recommended to cut 0.5 to 2.0 mm thick slices using the tissue slicer developed by Sitte et al.23 and previously described by Stadie and Riggs25 included in the MM80 and CPC package (Leica).

Cells or tissue are rapidly frozen on the  metal mirror block.  Further procedures:  Freeze-substitution (FS)

 See Chapter 13

 Freeze-drying (FD)

 See Chapter 15

 Cryo-sectioning (CEMOVIS)

 See Chapter 11

 Room temperature sectioning after FS or FD

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Slam-Freezing, Metal-Mirror Freezing

2. SUMMARY OF THE DIFFERENT STEPS 1. Preparation of the copper block 2. Preparation of the apparatus 3. Sample preparation

 LN2 is required.  For safety procedures using liquid nitrogen, see Sitte et al.24

4. Slamming procedure 5. Checking vitrification 6. Further processing

 Cryo-ultramicrotomy: CEMOVIS (see Chapter 11).  Freeze-substitution (see Chapter 13).  Freeze-drying (see Chapter 15).  Room temperature sectioning after freeze-substitution or freeze-drying.

3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Impact freezer

 Homemade.23  Some are “precious oldies” like the helium Cryovacublock (Escaig) previously sold by Reichert-Jung, Vienna, Austria, and still used in some laboratories.15  CPC (with an automatic LN2 refilling system and microprocessor controlled) from Leica, Microsystems, Vienna.  This chapter will focus on use of the MM80 from Leica (Figure 2.1).

Figure 2.1 MM80 apparatus

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Handbook of Cryo-Preparation Methods for Electron Microscopy

 Plastic sheets  Plastic cups

  Very dense polystyrene sheet





 Spacer rings

 Tweezers

 Insulated cryo-tweezers

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 Orange plastic spacer of a Leitz binder 0.3 mm thick. Small squares of about 3 mm × 3 mm are cut.  Homemade. A 2.4 mm diameter indentation can be made with a punching device, which was formerly used to make EM grids.  The borders of the depression have to be sharp (see Figure 2.2).  Be careful: A belt punching device is not applicable because the rim (A) will be rounded. Consequently, the liquid droplet does not form a meniscus, but flows over and will not be frozen properly.

Figure 2.2 Image and sketch of the plastic cup. The rim (A) has to be very sharp. The liquid droplet (1.5 µL or less) stays in the depression and forms a meniscus.   2 mm thick.  Readily available in any laboratory or can be purchased in a hobby shop.   #16860180; furnished with the impact freezer machine.  For better results, fix the rings on double-sided tape attached on a piece of polystyrene. The best results are obtained with 0.3 mm thick rings.  Those commonly used for EM work, e.g., flat, short and antimagnetic (#5 Dumont & Fils, Switzerland).   The orange ones furnished with the machine. (#16701955) Leica Microsystems, Vienna, Austria.

Slam-Freezing, Metal-Mirror Freezing

 Heating plate or oven at 50°C

25  Useful to rapidly dry the tweezers, copper block and plastic cups before each slam.

Figure 2.3 A metal block (arrow) on filter paper placed on a heating plate, after slamming. Ice from condensed water vapor is melted and evaporated. The two holes visible on the side of the metal block are useful to insert cryo-tweezers and carry the metal mirror.  Precision micropipette with very thin  Preferably the white Eppendorf 110 µL, #0030 073.363, Eppendorf AG, adapted tips 2233 Hamburg, Germany.  Binocular microscope  Cold light source.  Specific material supplied with any of  Refer to the different supplier’s manuals. the apparatus available will not be described here:  Spacer rings  #16860180 Leica.  Specimen holder  #16701887 Leica.  Liquid nitrogen Dewar

 Styrofoam boxes  Cryo-ultramicrotome  Freeze-substitution apparatus  Freeze-drying apparatus

 Room temperature ultramicrotome

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 To refill the slamming apparatus.  It is furnished with the CPC from Leica. In the CPC, the temperature is microprocessor controlled and the LN2 refills automatically.  To transfer the specimen after slamming to one of the additional processing machines.  For cryo-sectioning with an adapted holder that clamps flat specimens, # 16701880 Leica.  For freeze-substitution and lowtemperature resin embedding.  For freeze-drying and low-temperature resin embedding.  Cryosorption freeze-drying system (CFD), Leica.

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Handbook of Cryo-Preparation Methods for Electron Microscopy

3.2. Products  Venol™

    

Liquid nitrogen Cotton hydrophilic wad For cryo-ultramicrotomy For freeze-substitution For freeze-drying

 To clean and polish the copper block before use.  Any other copper cleaning product can be tested and used.   See Chapter 11 and A. Leforestier et al.15  See Chapter 13.  See Chapter 15.

3.3. Solutions  Fetal calf serum  Water-free acetone    Methanol  Epon

 Or any other concentrated serum protein.  Any supplier, e.g., #00570; Fluka, Sigma-Aldrich Chemie GmbH, CH-9471 Buchs SG, Switzerland.  To rinse the copper block after polishing.  M1775GA, Sigma-Aldrich: Fluka, Buchs, Switzerland.  LX112 Embedding kit, Ref. 5.21210, Inland Europe.

4. PROTOCOLS 4.1. Preparation of the Copper Block  Polish the copper block with Venol Use a cotton hydrophilic wad (no paper napkins or other products, such as Kimwipes)

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 This step is absolutely necessary.  Paper napkins and Kimwipes slightly scratch the copper block. The striations will interfere with the slamming process after a while.  Make sure that all the Venol is removed before using the copper block. Any trace of the cleaning agent can interfere with the specimen.  It is not necessary to polish the copper block after each freezing run, but only when the sample has scarred the copper block. In real life, this often happens and the copper block has to be cleaned when this occurs.

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 An alternative to the polishing step is to use gold-covered copper blocks that do not need to be polished after each slam. It just needs washing with acetone or ethanol before each slamming. Figure 2.4 Copper block.  Rinse the block with acetone or ethanol.

Figure 2.5 Copper block held with the orange tweezers.

 Place the copper block in the apparatus and let it cool.

Figure 2.6 The copper block is placed in the apparatus. The two holes on the side are made to pick up the copper block with the orange tweezers to avoid scratching the surface of the metal mirror.  To test if the block has reached the working temperature and is ready for slamming, fill the chamber with liquid nitrogen up to the brass plate (see Figure 2.6, arrow). Cool down the orange tweezers by dipping them in the chamber and remove a drop of liquid nitrogen (only one drop!) and place it rapidly on the copper block.

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 If the copper block is cold enough, the drop stays on the copper for a few moments before evaporating.  Has to be done just before slamming.  Wipe all traces of liquid nitrogen off the copper block (when it is cold!) using a precooled, dry cotton wad.  When using the CPC (Leica) instead of the MM80, the temperature can be set and controlled automatically.

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4.2. Preparation of the Slammer  Fill the MM80 with liquid nitrogen:

 10 to 15 minutes are needed for the apparatus to sufficiently cool down.  When the system has stabilized, care must be taken to avoid pouring liquid nitrogen on the copper or gold-plated copper block.

 Here the metal mirror freezer MM80 from Leica will be described.  In this case, the whole procedure has to be started again: to heat, polish, pour liquid nitrogen, etc.  A beep signal is emitted when the apparatus needs to be filled with liquid nitrogen. When the system has stabilized, i.e., beeping does not occur every couple of minutes, the apparatus is ready to start.  If this happens, cryo-immobilization is not efficient. Under these circumstances, the warm specimen is projected toward the liquid nitrogen covering the copper block. The liquid nitrogen evaporates and produces an insulated gas layer, which prevents cryo-fixation.11  After the light has been switched on, the TF (TransFer of nitrogen vapor) process is activated and will automatically start when the slider is opened. When the TF process is on, LN2 is evaporated and the cold nitrogen vapor fills the chamber to keep the interior dry and at 196°C. This process is essential to prevent the sample from warming up and subsequent ice crystal formation.  Pressing the TF button will cause LN2 vapor formation even if the slider is closed and the lights are off. Figure 2.7 Control buttons of the MM80 (in color on the apparatus)  On the right:  Mains on button: green lamp  Mains off button: red lamp  On the left:  White button corresponds to the light and the TF  Orange button corresponds to the heating mode.

 Settings for the plastic cups:  Thickness = 0  Force = 6  Speed = 6

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 Other sample holders can be used and are referred to in the material section.  Thickness: Has to be adapted depending on the specimen holder; a simple way to adapt it is to measure the overall distance of the mounted specimen holder squeezed between the thumb and the index.

Slam-Freezing, Metal-Mirror Freezing

29  Force: Choose the same value as speed.  Speed: The best results are obtained with short specimen contact (<5 ms-1) if the specimen is mounted on a support of low mass.  Force and speed have to be adapted to the specimen. It is preferable to try different slamming conditions during an experiment because the results can only be observed under an EM. For example, keep the thickness constant and make a series of slamming from force speed 1 to 9. Figure 2.8 Top view of the apparatus: Buttons for force and speed are visible as well as thickness.

4.3. Sample Preparation  For freeze-substitution, isolated cells are pelleted and the supernatant is removed. The cells are then resuspended in a protein-rich medium, such as undiluted fetal calf serum.

 Or the same serum that was used for the cell culture.  Serum protein serves as an extracellular matrix for the entire procedure (cryoimmobilization and freeze-substitution). This extracellular matrix helps to keep the cells together and acts as a cryoprotectant.  Some cells should not be maintained at a high density or pelleted in pure serum protein because their physiological state can change (e.g., human platelets are activated).

Figure 2.9 Activation of human platelets pelleted in pure serum.  For lyophilization, isolated cells are  They do not need an extracellular matrix pelleted, the supernatant is removed because they stick together and are not lost and the pellet is washed with growth during the freeze-drying procedure. medium.

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 Isolated cells to be further cryo-  The cells are maintained together with a cryoglue20 after slamming, especially when sectioned. using the Escaig’s or Heuser’s machines.  The flattened cell pellet can be directly mounted in the cryo-holder of the cryoultramicrotome.  The use of the tissue slicer especially  Tissue developed by Sitte’s group is recommended.  Any other tissue chopper also can be used.  Slicing has to be rapidly done and the tissue has to remain wet and preferably in its physiological medium.

4.4. Cryo-Immobilization Procedure  Place a thin but very dense polystyrene layer on the supplied foam support using double-sided tape as represented in Figure 2.10. Figure 2.10  A: Represents the metal support (3 cm in diameter) covered with 7 mm thick foam (Leica ref 16701888).  B: The very dense 2 mm thick polystyrene is taped on the foam.  C: Sample holder.  D: Plastic cups. This makes a three-layer sandwich.  The plastic cups (up to four) are fixed  The Leica rings are fixed the same way. on the polystyrene layer with doublesided tape.

Figure 2.11 Four plastic carriers are taped on the specimen holder.

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Slam-Freezing, Metal-Mirror Freezing

 Fill the plastic cup. The total volume makes a positive meniscus and the liquid stays in the cup. In general, the volume is less then 1.5 µL.

31  Check how much liquid the plastic cup can contain before starting.  The sample should make a small meniscus for good cryo-immobilization.  Take care: No overflowing, otherwise the sample will not be frozen properly.  The rings provided with the apparatus can also be used as described before. Figure 2.12 Four homemade specimen carriers are taped onto the polystyrene that is fixed onto the sample holder and filled with the cell suspension.

 The sample holder is placed upside down onto the magnetic ejector. Wait approximately one minute before slamming to allow the cells to move into the meniscus and to avoid freezing of only ice or medium.

 The use of a very thin tip on a 10 µL pipette is recommended.  For a mixed population of small and big cells, the big cells will move to the meniscus more rapidly then the small ones. If small cells are the more interesting ones, they will never be cryo-immobilized properly with this method (they will stay in the not well-frozen part of the sample). High pressure freezing is the best solution in this case.

Figure 2.13 The sample is fixed upsidedown (arrow) on the “air-cushioned injection system” developed by Sitte23 and stays for one to two minutes.

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Figure 2.14 By pressing the red button (arrow) the sample is slammed onto the metal mirror surface with the spring-loaded injector rod.

Figure 2.15 The sample remains pressed against the copper block for one minute.  Very important to prevent heat transfer to the frozen specimen and recrystallization.  Again, wait for one minute.

 The sample is not deformed because in the initial phases of contact and rapid cooling, pressure is built up slowly as the 23  Maintain the sample holder with pre- damping air bag slowly empties. cooled orange tweezers on the cooper  This is crucial to avoid recrystallization block (with the left hand in Figure due to the bouncing phenomenon. 2.16) and detach it from the ejector by simultaneously pushing on the tip of the ejector and pulling out the ejector (with the right hand in Figure 2.16).

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Figure 2.16 Unloading the sample holder from the ejector.  The sample and the holder remain on the copper block. With the precooled orange tweezers, turn the entire holder and place it on the copper block.

Figure 2.17 The sample transport unit (arrow) and the orange tweezers are precooled.

Figure 2.18 The entire holder is placed on the brass plate and the impact imprints are checked.

© 2009 by Taylor & Francis Group, LLC

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Figure 2.19 The three well cryoimmobilized samples are transferred to the precooled transport unit using a precooled tweezers.

Figure 2.20 The samples are then transported in a Styrofoam box containing LN2 where they are further checked. Arrow: Plastic carrier to be checked.  The sample can be ejected from the  This step is done under a binocular cup using two clamped tweezers by microscope with a cold light source and in pressing on both sides of the plastic LN2. cup.  For freeze-substitution or freeze-drying, the sample can be cut in four pieces.  This can be done easily by pressing once with a fine tweezers in the center of the sample.

4.5. Checking the Quality of Freezing  On the copper block: In general, the impact of the plastic cup is seen on the copper block as a white imprint of frozen water vapor. The area surrounding the cup is white, but the inner part has to be colorless with no trace of condensation.

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 Slightly white and round traces indicate that the sample contains some ice and is not well frozen: DISCARD IT.  It is possible that only one or two samples are not well-frozen: Discard those only and not all four.  The square, white imprints on the copper block are made by the plastic cups; the round imprints are from the sample droplet.

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Slam-Freezing, Metal-Mirror Freezing

Figure 2.21 In the ideal configuration, all the samples are “well-frozen.” One can only see the imprints left by the carrier itself on the metal block.  The quality of the well-frozen samples is verified under a binocular microscope (see Figure 2.20). The frozen sample is removed from the cup, positioned at an oblique angle on a black background and viewed from the side. The part that was in direct contact with the metal mirror should be colorless, whereas the opposite side is milky due to light scattering in ice.  Checking under microscope.

a

 Since only 10 to 30 μm in thickness of the specimen can be cryo-fixed the whole sample will never be well-frozen with this technique. The two phases are clearly visible: the well preserved one (the smallest in general) and the ice phase.  If only one milky phase is visible, discard the sample; it is not well-frozen.

cryo-electron  The ideal method!  Using electron diffraction within the cryo-electron microscope is the only method to see if the sample is really vitrified.

4.6. Further Processing Cryo-ultramicrotomy (CEMOVIS)

 See Chapter 11.

Freeze-substitution

 See also Chapter 13.

The sample may be substituted as follows: 1. In methanol with 0.5% uranyl acetate     

4872 hours at 90°C. Go to 60°C: 1°C/hour. Remain at 60°C for 812 hours. Go to 45°C: 1°C/hour. Remain at 45°C for 812 hours.

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 12 to 24 hours are sufficient if uranyl actetate is added to the substitution medium

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Handbook of Cryo-Preparation Methods for Electron Microscopy

Embedding in Lowicryl HM20 45°C:  Care has to be taken using these very toxic and allergenic resins. Please follow (~ 2 hours for each step): the manufacturer’s recommendations.  Methanol/Lowicryl:  2+1  1+1  1+2  Pure Lowicryl  Pure Lowicryl: overnight Polymerization: 2448 hours with UV light. For embedding in Lowicryl K4M at 30°C, follow the same procedure as for HM20. 2. In acetone with 2% osmium tetroxide  72 hours or more at 90°C.  12 to 24 hours are sufficient if osmium is  Go to 60°C: 1°C/hour. added to the substitution medium.  Remain at 60°C for 812 hours.  Go to 45°C : 1°C/hour.  Remain at 45°C for 812 hours.  Wash in pure acetone: 3 times over one hour.  Raise the temperature to 0°C in one hour. Embedding in Epon:  30% Epon in acetone for 3 hours at 0°C.  70% Epon in acetone for 3 hours while raising the temperature to 10°C.  100% Epon in acetone for 3 hours while raising the temperature to 25°C.  100% Epon in acetone for 1 hour. Polymerization:  Increase the temperature from  Alternative: Place the specimen directly 25°C to 60°C over one hour. in the oven at 60°C.  Heat polymerize at 60°C for 24 hours.

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3. Membrane visualization28  2% osmium tetroxide in pure acetone for 24 hours.and linear temperature increase from -90 to – 80°C.  2% osmium tetroxide in pure acetone and 2% H20 for 18 hours and linear temperature increase from 80°C to 10°C.  Pure acetone: 3 washes over one hour while increasing the temperature to 0°C.

 Modification from Ludwig Edelmann.  Starting at -90°C.

 During this step the container with the specimen should be closed or covered to prevent contamination of the freezesubstitution chamber with osmium vapors.

Embedding in Epon  30% Epon in acetone for 1 hour at 0°C.  70% Epon in acetone for 1 hour while raising the temperature to 10°C.  100% Epon in acetone for 1 hour while raising the temperature to 25°C.  100% Epon in acetone for 1 hour. Polymerization:  Increase the temperature from 25°C to 60°C over one hour.  Heat polymerize at 60°C for 24 hours. Freeze-drying

 See Chapter 15.

Room temperature sectioning Fix the block on the ultramicrotome and check it. Turn the block in a manner to cut  Except for SIMS (see Chapter 20) where perpendicular to the slamming the sections are made parallel to the direction. slamming surface.  The two layers, seen in the frozen state, are also clearly visible when checking the block under the binocular of the ultramicrotome. The one that is well preserved has a brownish color, the other is yellowish.  Advantage: One can follow the gradient from well-frozen to the ice-damaged phase in the electron microscope.

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Figure 2.22 Under the electron microscope, the ice gradient ghost is clearly visible in this section of a cryo-fixed/freeze-dried sample (the arrow indicates the direction of the gradient from the point of impact toward the less well preserved area of the sample).  If the resin is too soft, several solutions  Polymerization can be continued with exist: natural UV light; let the block stay on a windowsill or in a homemade box for UV polymerization (Carlemalm, Villiger, Garavito and Acetarin in Lowicryl Letters n°1 or in the Lowicryl user’s manual).  It is recommended to cut Lowicryl with a 35° knife.14  If the blocks are still soft like the consistency of chewing gum (due to the high level of humidity in summer, for example, ) they can be sectioned with the oscillating diamond knife.26 Stabilization of trimmed Lowicryl blocks by osmium vapours:6  0.25 g osmium tetroxide or less is deposited in an Eppendorf tube. The Lowicryl block is fixed with Parafilm on the top of the Eppendorf (the trimmed pyramid facing the osmium vapors).  Wait for 2030 minutes and then section.

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 If the contrast obtained is not satisfactory, K4M sections can be embedded like Tokuyasu cryo-sections in:  0.4 mL 2% uranyl acetate.  1.8 mL 2% methylcellulose.

 This method of Roth et al.21 provides excellent results.  Contrast can also be increased in the electron microscope by reducing the acceleration voltage and by choosing a smaller objective diaphragm.

Other tips and tricks  The specimen must not be rewarmed.  If the specimen is warmed above 140°C, ice crystals will appear and grow. This ice crystal damage can be avoided when:  The cover of the apparatus is always closed when not in use.  Tweezers are dry and precooled.  Once prepared, the specimen is maintained in liquid nitrogen.

5. ADVANTAGES/DISADVANTAGES 5.1. Advantages     

Cryo-immobilization: Quicker than chemical fixation. Ion gradients are maintained. Antigenicity of epitopes is not altered.  As it is the case with chemical fixatives. 16 Retains more lipids than chemical  Maneta-Peyret et al. fixation if followed by freezesubstitution.  No external mass is added. As happens with chemical crosslinkers.17  The process can be reversed with the  Useful for time-resolved slam freezing, cryo-block falling onto the specimen. e.g., electrically stimulated muscle.5  Less traumatic then high-pressure  After slamming cell suspensions, cells freezing. do not float out of the carriers during substitution as is commonly the case after high-pressure freezing.13  Material may be checked for proper  Poorly frozen samples are discarded.  Gain in time. freezing before further processing.  Compared to the high-pressure freezing  The apparatus is inexpensive. machine.

© 2009 by Taylor & Francis Group, LLC

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Handbook of Cryo-Preparation Methods for Electron Microscopy

 The unit is easily transportable.

 From one lab to another.

 Impact freezing devices are available  A more sophisticated apparatus in which commercially. the LN2 is automatically refilled and temperature controlled is also available (CPC from Leica).  The only way to cryofix surface  Nearly as large as the cooling block regions of quite large samples. (3 cm in diameter).  However, the depth will never exceed 30 μm.  Copper has a much higher thermal  The same amount of heat is transferred conductivity than organic liquids. 10,000 times faster through copper than through liquid propane.  Physics related to the various freezing  See article by W. B. Bald.2 methods.

5.2. Disadvantages  Major disadvantage: Only a small portion of the sample will be properly cryo-immobilized.  Cell mixtures (i.e., blood cells): Only the heaviest (largest) cells, which are first in the meniscus, will be properly frozen.  Intracellular ice crystal formation may occur and damage cells.

 Approximately 10 μm (30 μm in the best case): 10 times less then by high pressure freezing (see Chapters 5, 6).  They may not be the cells of interest.  For this type of cell suspension, it is preferable to use the high-pressure freezing method.  The ice crystals grow and cellular organelles are displaced.  Utmost care has to be taken to keep the specimen at the LN2 temperature.  Bouncing phenomenon: If the  The quality of metal mirror freezing is slamming apparatus is not well drastically reduced. adjusted, the thermal contact between  Ice crystal formation. the specimen and the metal block is interrupted.  There are significant limitations for  Only the superficial layer of the organ is freezing organs and tissues. frozen and ice crystals disrupt the fine structure of the cells underneath.  Distortion and structural changes can  There is only one paper about structural occur. changes after MM freezing in the literature, but the authors concluded that freezing was not optimal in their experiments.4

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6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. For Ultrastructural and Immunolabeling Experiments  This method can be used for ultrastructural experiments. At low magnification, a gradient of ice crystal formation can be observed. Only the best preserved part has to be investigated.

 Only 10 to 30 µm are well preserved.  Sufficient for samples containing small isolated cells or bacteria, limited for dissected organs.

Figure 2.23 The cell nucleus is the most sensitive organelle for ice crystal damage. Here, the reticulate in the nucleus, indicates insufficient cryo-fixation. 

6.2. To Study Dynamic Events  The method can be used for time lapse experiments.  For special applications, the metal mirror can be employed so as to catapult onto tissue.

 Vesicle exocytosis.12  For a review see Ryan and Knoll.22  Edelmann made a reverse slammer apparatus to slam the copper block onto the heart or the muscle or any organ in situ.5

6.3. At Liquid Helium Temperature  A heated debate is in the literature  The consensus appears to be that if a concerning the most appropriate polished copper block is used, there is little cryogen for best freezing of samples.2 or no gain to use liquid helium instead of liquid nitrogen.  The situation is different in the “Escaig” machine.15 In this case, slamming is done under vacuum to avoid any moisture contamination and, for this application, liquid helium appears to be the best cryogen. However, this machine is no longer on the market and only a few machines are available throughout the world.

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Handbook of Cryo-Preparation Methods for Electron Microscopy

7. OBSERVED RESULTS  Figure on the Chapter’s title page

 HeLa cells were cryofixed using the MM80.  Freeze-substitution was done in the AFS using the protocol 3 for membrane visualization described in this chapter.  The sample was then embedded in Epon.  Observed results: Numerous nuclear pores are visible around the nucleus. The contrast of the membranes is enhanced.

 Figure 2.24

 Human dendritic cells were cryofixed using the MM80.  Freeze-substitution was carried out in the AFS using the protocol 1.  The samples were then embedded in Lowicryl K4M.  Observed results: The nuclei, as well as the nucleoli, are well preserved.

 Figure 2.25

 Human dendritic cells were cryofixed using the MM80.  Freeze-drying was done in the CFD from Leica, according to Ludwig Edelmann (see Chapter 15).  The sample was then embedded in Spurr’s resin.  Observed results: The nucleus is well preserved as are the cellular elements in the cytoplasm.  NOTE: The cytoplasm, as well as the nucleus, is very dense indicating that more cellular elements are preserved with this method as compared with the freezesubstitution method.  N = Nucleus  R = Endoplasmic reticulum

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Slam-Freezing, Metal-Mirror Freezing

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Handbook of Cryo-Preparation Methods for Electron Microscopy

 Figure 2.26

 HeLa cells were cryo-fixed using the MM80.  Freeze-substitution was carried out in the AFS using the protocol C for membrane visualization described in this chapter.  The sample was then embedded in Epon.  Observed results: Two cells are observed. The membranes are so enhanced that the mitochondria appear as if they only contain membranes. This suggests that the “amplification of membrane contrast” may have induced different artifacts.  M = Mitochondria  N = Nucleus

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Handbook of Cryo-Preparation Methods for Electron Microscopy

8. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Altmann, R. Die Elementarorganismen und ihre Beziehungen zu den Zellen. Veit und Co., Leipzig, 1890. Bald, W.B. The relative merits of various cooling methods J. Microsc., 140, 17, 1985. Bellare, J.R. et al. Controlled environment vitrification system: An improved sample preparation technique, J. Electron Microsc. Tech., 10, 87, 1988. Bennett, P.M. Structural changes in samples cryofixed by contact with a cold metal block, J. Microsc., 192, 259, 1998. Edelmann, L. The contracting muscle: A challenge for freeze-substitution and low temperature embedding, Scanning Microsc. Suppl., 3, 241, 1989. Edelmann, L. and Ruf, A. Freeze-dried human leukocytes stabilized with uranyl acetate during low temperature embedding or with OsO4 vapor after embedding, Scanning Microsc. Suppl., 10, 295, 1996. Escaig, J. New instruments which facilitate rapid freezing at 83K and 6K, J. Microsc., 126, 221, 1982. Escaig, J. Control of different parameters for optimal freezing conditions, in The Science of Biological Specimen Preparation, Revel, J.-P., Bernard, T., and Haggis, G.H., eds., SEM Inc, AMF O'Hare, IL, 1984, 117. Frederik, P.M. and Hubert, D.H. Cryoelectron microscopy of liposomes, Methods Enzymol., 391, 431, 2005. Frederik, P.M. et al. Perspective and limitations of cryo-electron microscopy. From model systems to biological specimens, J. Microsc., 161, 253, 1991. Heath, I.B. A simple and inexpensive liquid helium cooled "slam freezing" device, J. Microsc., 135, 75, 1984. Heuser, J.E. et al. Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release, J. Cell Biol., 81, 275, 1979. Hohenberg, H., Mannweiler, K., and Müller, M. High-pressure freezing of cell suspensions in cellulose capillary tubes, J. Microsc., 175, 34, 1994. Jesior, J.C. Use of low-angle diamond knives leads to improved ultrastructural preservation of ultrathin sections, Scanning Microsc. Suppl., 3, 147, 1989. Leforestier, A., Dubochet, J., and Livolant, F. Bilayers of nucleosome core particles, Biophys. J., 81, 2001, 2001. Maneta-Peyret, L. et al. Immunocytochemistry of lipids: Chemical fixatives have dramatic effects on the preservation of tissue lipids, Histochem. J., 31, 541, 1999. Morgenstern, E. and Edelmann, L. Analysis of dynamic cell processes by rapid freezing and freeze substitution, in Electron Microscopy of Subcellular Dynamics, H, P., ed., CRC, Boca Raton, FL, 1989, 119. Müller, M., Meister, N., and Moor, H. Freezing in a propane jet and its application in freeze-fracturing, Mikroskopie, 36, 129, 1980. Phillips, T.E. and Boyne, A.F. Liquid nitrogen-based quick freezing: Experiences with bounce-free delivery of cholinergic nerve terminals to a metal surface, J. Electron Microsc. Tech., 1, 9, 1984. Richter, K. A cryoglue to mount vitreous biological specimens for cryoultramicrotomy at 110K, J. Microsc, 173, 143, 1994.

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Slam-Freezing, Metal-Mirror Freezing

21. 22. 23. 24. 25. 26. 27. 28.

47

Roth, J., Taatjes, D.J., and Tokuyasu, K.T. Contrasting of Lowicryl K4M thin sections, Histochemistry, 95, 123, 1990. Ryan, K.P. and Knoll, G. Time-resolved cryofixation methods for the study of dynamic cellular events by electron microscopy: a review, Scanning Microsc., 8, 259, 1994. Sitte, H., Edelmann, L., and Neumann, K. Cryofixation without pretreatment at ambient pressure, in Cryotechniques in Biological Electron Microscopy, Steinbrecht, R.A. and Zierold, K., eds., Springer-Verlag, Berlin, Germany, 1987, 87. Sitte, H., Neumann, K., and Edelmann, L. Safety rules for cryopreparation, in Cryotechniques in Biological Electron Microscopy, Steinbrecht, R.A. and Zierold, K., eds., Springer-Verlag, Berlin, Germany, 1987, 285. Stadie, W.C. and Riggs, B.C. Microtome for the preparation of tissue slices for metabolic studies of surviving tissues in vitro, J. Biol. Chem., 154, 687, 1944. Studer, D. and Gnaegi, H. Minimal compression of ultrathin sections with use of an oscillating diamond knife, J. Microsc, 197, 94, 2000. van Harreveld, A. and Crowell, J. Electron microscopy after rapid freezing on a metal surface and substitution fixation, Anat. Rec., 149, 381, 1964. Walther, P. and Ziegler, A. Freeze substitution of high-pressure frozen samples: The visibility of biological membranes is improved when the subsitution medium contains water, J. Microsc., 208, 2002, 2002.

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Plunge-Freezing (Holey Carbon Method)

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CONTENTS

1.

PRINCIPLES OF PLUNGE-FREEZING ......................................................... 53

2.

SUMMARY OF THE DIFFERENT STEPS ..................................................... 53

3.

MATERIALS/SOLUTIONS............................................................................... 54 3.1. 3.2.

4.

PROTOCOLS ...................................................................................................... 55 4.1.

4.2.

5.

Preparing Holey Carbon Grids ................................................................... 55 4.1.1. Making the plastic solution ........................................................... 55 4.1.2. Making “holey” plastic film.......................................................... 55 4.1.3. Choosing the plastic film .............................................................. 56 4.1.4. Covering grids with the plastic film.............................................. 56 4.1.5. Busting the holes........................................................................... 57 4.1.6. Finishing up .................................................................................. 57 Plunge-Freezing Grids................................................................................ 59 4.2.1. Glow-discharge treatment ............................................................. 59 4.2.2. Preparing the cryogen/vitrification device .................................... 60 4.2.3. Pipetting the sample on the freshly prepared grid......................... 64 4.2.4. Removing excess liquid ................................................................ 65 4.2.5. Vitrification................................................................................... 66 4.2.6. Specimen mounting and transfer................................................... 66

ADVANTAGES/DISADVANTAGES................................................................ 67 5.1. 5.2.

6.

Materials ..................................................................................................... 54 Solutions ..................................................................................................... 54

Advantages ................................................................................................. 67 Disadvantages............................................................................................. 68

REFERENCES .................................................................................................... 68

 

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1. PRINCIPLES OF PLUNGE-FREEZING Cryo-electron microscopy of frozen-hydrated macromolecules embedded in a thin layer of vitreous water is nowadays a well-established method. It was developed more than 20 years ago at the European Molecular Biology Laboratory (EMBL) in Heidelberg, by the pioneering group led by Jacques Dubochet.1 It is currently used in hundreds of labs worldwide in order to study biological complexes in their near-native state3-5 (see Chapter 1). The main advantage of cryo-EM versus air-drying negative staining is that the biological object is fully embedded in its native environment, namely the vitrified buffer surrounding it, thus its three-dimensional structure is fully preserved down to atomic scale. Typically, the frozen-hydrated specimen is observed suspended across the holes of a carbon support. The first part of this chapter describes how to prepare the holey carbon support grids. The second part describes how to apply the sample to the grid and the vitrification process in order to obtain a thin layer of suspension, to be observed in lowdose mode in the cryo-electron microscope. 

2. SUMMARY OF THE DIFFERENT STEPS 1. Preparing holey carbon grids      



Making the plastic solution Making “holey” plastic film Choosing the plastic film Covering grids with the plastic film Busting the holes Finishing up

   Glow-discharge treatment.  Preparing the cryogen/vitrification  device.  Pipetting the sample on the freshly  prepared grid.  Removing excess liquid.  Vitrification.  Specimen mounting and transfer.

2. Plunge-freezing grids      

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3. MATERIALS/SOLUTIONS 3.1. Materials  With grate

   

Acetone chamber COLD aluminum block Copper grids EM-grade tweezers

      

Filter paper, wiping paper Forceps Glass microscope slides Glass Petri dish Glass pipette with rubber bulb Glow-discharge apparatus Grid-boxes

   

Humidity chamber Humidity reader Parafilm Plastic container/liquid Dewar Razor blades Screwdriver Small plastic grid boxes Stop watch Vitrification device

    

 200 or 400 mesh  Dumont biology N5; Ted Pella Inc., Redding, California  E.g., Whatman grade 15  Clean

 For storage (Ted Pella Inc., Redding, California)  With humidity between 45 and 50%

nitrogen

 Vitrobot  Water container

 Ted Pella Inc., Redding, California  Homemade or commercially available  Or other commercially available plunging devices  Bigger dimensions than filter paper



3.2. Solutions  90% acetone  Cellulose acetate solution (plastic)  Ethane-35 gas bottle    

90% ethanol 99% ethyl acetate 25 L or 50 L liquid nitrogen Dewar Liquid nitrogen

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 With a pressure gauge and two security valves   For grid storage  4 L bottle

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Plunge-Freezing (Holey Carbon Method)

4. PROTOCOLS 4.1. Preparing Holey Carbon Grids 4.1.1. Making the plastic solution To make the cellulose acetate solution,2 mix the dry cellulose acetate-butyrate with ethyl acetate to a final concentration of 0.15% (w/v).

 As an example, if you use 0.026 g of the dry cellulose acetate, you would need to add 17.3 mL of ethyl-acetate so that the solution would be 0.15% (w/v).

4.1.2. Making “holey” plastic film To be performed in the humidity chamber if lab’s humidity is not within the 45% to 50% range. 1. Remove aluminum block from refrigerator. 2. Put it on ice to keep it cold and wipe dry. 3. Wait for about 23 minutes before  2 to 3 minutes waiting time is to form placing glass slide onto block. fine droplets (water condensation). 4. While waiting, clean a few (3 or 4) Use pure or 90% ethanol to clean glass slides with ethanol. glass slides. 5. Mark on which side of each slide the  Mark slides with a permanent marker plastic film is to be deposited. (a fine dot is sufficient). 6. Place the glass slide onto the  aluminum block, designated side up, for 10 seconds. 7. Remove slide from block.



8. While tilting the slide at a 60angle,  pipette a layer of plastic onto it. 9. Set the slide aside beveled and allow  to dry.   10. REPEAT steps 59 with no more  than 3 slides before wiping the block  dry and starting over again with step  3. 

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4.1.3. Choosing the plastic film 1. Hold the slides up to the light to make  sure that the plastic has dried evenly  Discard slides that do not meet this throughout. criterion. 2. Observe the slides in a phase contrast microscope with an EM grid on top of it for reference. The bubbles should be of homogenous shape and size. About 20 bubbles should fit across each side of each square (or more reasonably, most of the bubbles should be of that size, regardless of orientation).

 Check to make sure that the entire length of plastic has bubbles of the appropriate size. If not, discard the section with poor bubble formation. 

4.1.4. Covering grids with the plastic film 1. Fill the water container to the top with distilled water. 2. Scrape the edges of the plastic film to facilitate floating off the film upon entry into water. 3. Breathe on the slide and gently introduce it into the water container, designated side up, at a 30angle with the water (the plastic should detach from the slide and float on the water surface). 4. Delicately arrange copper grids dull side up on the plastic film (avoid the edges of the plastic). 5. To retrieve the grids, overlay filter  The plastic film/grids should have paper onto them. adhered to the underside of the filter paper.  Before the filter paper has an opportunity to slowly begin to sink into the water, securely grab a portion without grids under it and meticulously pull the filter paper out of the water as if peeling the skin off a fruit. 6. Dry flat for 34 hours (overnight is even better) with the grids facing up (the shiny side should be up with the plastic over it).

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4.1.5. Busting the holes 1. Fill the acetone chamber with acetone, replace grate once this is done. 2. Take one grid off the filter paper and place it on a microscope slide; this is the pretreated grid. 3. Place the filter paper with the grids in the acetone chamber for one minute. 4. After removing the filter paper with the grids from the chamber, transfer a representative grid from the filter paper to the microscope slide containing the pretreated grid. 5. Compare the two grids: The representative grid should have thinner plastic between the bubbles/holes (i.e., bigger holes in the representative grid than the bubbles in the pretreated grid) compared to the pretreated grid.

 The plastic between the holes should be quite thin. If it is not thin enough replace the representative grid onto the filter paper and put the filter paper with grids into the acetone chamber for 30 seconds and then compare again. Repeat if necessary.

Figure 3.1. Good homemade holey carbon (the big holes have an approximate diameter of 5 to 6 m).  4.1.6. Finishing up 1. Coat the grids with carbon using the carbon discharge protocol (see Harris.4). When done, you should see a little bit of carbon deposited underneath the grids (showing that the holes have popped).

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The thickness of the carbon may vary depending on your own needs. Typically a 10 to 20 nm layer is a good thickness for holey carbon, but thicker layers may work too, depending on the application and the sample.

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Handbook of Cryo-Preparation Methods for Electron Microscopy

2. To dissolve the remaining plastic Wash the grids with pure ethyl-acetate from the grids, remove the grids from or chloroform to completely remove the the filter paper and put them onto the plastic. grate in the acetone chamber, dull side up, overnight. 3. Eventually, use a sputter-coater to deposit a very thin layer of gold/palladium (15 nm). 4. To make sure you removed the plastic completely, wash the grids with ethylacetate and rinse with water right before use. ALTERNATIVE METHOD 1. Use precleaned glass slides or clean Materials and chemicals for the them with ethanol or methanol (90%) alternative method: and Kimwipes (delicate task wipers) beforehand.  Beaker 250 mL  Filter paper, wiping paper (e.g., 2. In a small glass beaker, prepare a Whatman grade 1) 0.5% Formvar solution in chloroform,  Forceps e.g., 0.25 g in 50 mL chloroform (use  Glass microscope slides (clean) agitation + cover; it takes a while to  Glass pipette with rubber bulb dissolve).  Glow-discharge apparatus  Kimwipes wipers 3. Add about 0.5 mL of 50% glycerol  Optical microscope with a Pasteur pipette on the surface of the Formvar/chloroform solution.  Pasteur pipette You can adjust the volume of glycerol  Ultrasonicator with tip added to get more or fewer holes (higher ratio of glycerol/Formvar solution gives smaller holes).  90% acetone  99% chloroform 4. Use ultrasonic treatment to make an  90% ethanol and methanol emulsion of glycerol droplets in the  99% ethyl acetate Formvar.  Formvar solution (plastic); Ladd Research Williston, Vermont 5. Dip an ultrasonicator tip in mix,  50% glycerol approximately 1 inch deep, sonicate 1  Water (distilled, nanopure, etc) minute at maximum power (50 W; 20-30 kHz). Quickly dip the glass slides, one by one, vertically in this  emulsion, blot the sides and let them  dry vertically (you can use, for  example, a big beaker with filter Vary the glycerol/Formvar ratio in the paper at the bottom). You can prepare solution and sonication times to adjust hole ~ 20 slides each time. Make sure to number and size. dip the glass slides immediately after the ultrasonic treatment

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6. Check the size of the holes with an optical microscope. You can adjust the number of glycerol drops and the sonication time to obtain the hole number and hole size you want. 7. When the slides are dry, cut the edges of the holey membrane on one side and float it on the surface of distilled water. Cover with copper grids and pick them up with a piece of filter paper (or whatever works best). Let them dry on filter paper. Soak the entire sheet in methanol for 30 minutes to get rid of the glycerol to form the holes, and then let them dry. 8. Evaporate carbon onto the grids, which are covered with the holey plastic membrane (as described before). 9. Remove the Formvar by soaking the grids in chloroform right before use. Let them dry on filter paper. 10. Eventually use a sputter-coater in order to deposit a thin layer of gold/palladium (15 nm). 11. Wash the grids with full-strength ethyl-acetate (pure) and then with water right before use.

4.2. Plunge-Freezing Grids 4.2.1. Glow-discharge treatment  Glow-discharge treatment of the grids right before use can help to obtain a good spreading of the sample across the holes in the carbon support. Apply right before use because the charging effect of the carbon film is only temporary (lasting time may depend on the apparatus used). It also cleans the carbon support surface. Glowdischarging time may depend on the apparatus you are currently using in your lab. Please refer to the user’s manual of the equipment for further operating instructions.

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4.2.2. Preparing the cryogen/vitrification device 1. In the meantime, prepare vitrification system. Prepare material you are going to need on bench (see Figure 3.2), near vitrification device, as timing important in the next few steps.

the the the the is



Figure 3.2 Forceps, EM-grade tweezers, Petri dish, Whatman filter paper, EM grids, Parafilm and, of course, the sample are displayed on the bench.

2. Make sure the Styrofoam box is placed  on the vitrification device at the right  place (see Figure 3.3).    

Figure 3.3 Placement of the Styrofoam box.   

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Plunge-Freezing (Holey Carbon Method)

3. In order to make your life easier, it is  useful to have a direct light coming  from behind, typically from a desk   lamp (see Figure 3.4).              Figure 3.4 Direct lightening coming from behind is advisable.   4. If you are using a commercially  available vitrification device (e.g., the Vitrobot, (see Figure 3.5 and Chapter 4)), please refer to the user’s manual for further instructions. 5. The small metal cup (typically aluminum) that will contain the cryogen should also be placed in the box; keep the aluminum cup in the center where the tweezers will fall later (see Figure 3.6). Test before use. 

Figure 3.5 Vitrobot vitrification device.

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Handbook of Cryo-Preparation Methods for Electron Microscopy

6. Prepare the gridbox and place it in the area that will be filled with liquid nitrogen (see Figure 3.7). 7. The plastic grid storage box should also be placed underneath the liquid nitrogen level; make sure it stays on the bottom by using a metal holder. Vitrified samples will be placed in this storage box.

Figure 3.6 Small metal cup is placed in the box. 8. Make sure you wear lab-certified protection eyewear BEFORE you start using ethane (see Figure 3.8). 9. Use your tweezers to pickup a grid (see Figure 3.9).

Figure 3.7 Prepared grid box 10. Fill the whole box with liquid nitrogen, do it slowly, no nitrogen should splash into the aluminum cup that will contain ethane (see Figure 3.10).

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Plunge-Freezing (Holey Carbon Method)

Figure 3.8 Protection eyewear.

11. Start filling the ethane cup (see Figure 3.11) and wait for the liquid ethane to reach the right temperature (see Figure 3.12).

Figure 3.9 Tweezers for picking up a grid.

 The freezing point is a good way to make sure the vitrification temperature is reached (liquid nitrogen will be at about 192°C).

Figure 3.10 Box filled with liquid nitrogen

12. Typically, one would wait until the  ethane starts to become solid and then add some more ethane to the cup to fill it to the top (see Figure 3.13).

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Handbook of Cryo-Preparation Methods for Electron Microscopy

 By repeating this several times the ethane remains liquid, at the right temperature to vitrify biological samples. Figure 3.11 Filling cup with ethane.

Figure 3.12 liquid ethane reaching the right temperature

Figure 3.13 When ethane starts to become solid, more is added to fill the cup.

4.2.3. Pipetting the sample on the freshly prepared grid 1. Typically 35 L of the sample solution are applied to the freshly glow-discharged EM grid (see Figure 3.9).

     2. Mount the tweezers, holding the grid,  in the vitrification device (see Figure  3.14).  

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Plunge-Freezing (Holey Carbon Method)

 The grid should already be held with the tweezers that will be mounted on the vitrification apparatus.  There is no need to wait further, as the sample is not adsorbed to the carbon support, but suspended in a droplet above the support.

Figure 3.14 Tweezers vitrification device.

mounted

in

4.2.4. Removing excess liquid 1. This is the key step. Use filter paper in  E.g., Whatman type I. order to remove excess liquid of the suspension on the grid. Typical blotting time is 23 seconds at room temperature and in a relatively dry (< 60% relative humidity) environment (see Figure 3.15) 2. The blotting time is also dependent on the concentration of the protein solution and the presence of lipids or detergents in the buffer.

 The backlight should help one to see a “halo” forming on the filter paper; release the plunging mechanism when the halo starts to disappear.  If you are using a temperature and humidity controlled chamber, blotting times could be as long as 10 seconds.  You can use blotting paper that has been preexposed to water (or buffer) by dipping or spraying (whichever is more convenient). Figure 3.15 Using filter paper to remove excess liquid on the grid. 

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 You can also set up a homemade system to blow humid air at 37°C right onto the grid (typically from behind the grid) while you apply the blotting paper to the front side.  Please refer to the lab equipment’s user’s manual if you are using commercially available vitrification devices. 4.2.5. Vitrification The cryogen usually used for vitrification is liquid ethane (in some labs liquid propane is used). Immediately after blotting the grid (previous step), the sample is plunged into the cryogen (see Figure 3.16).  Make sure the sample-releasing mechanism works efficiently, as the cooling process must be fast enough in order to guarantee vitrification.  Cubic and hexagonal ice will be observed if the cooling process was too slow, or if the sample was warmed up to a temperature above 135°C. Figure 3.16 Sample plunged into cryogen. 4.2.6. Specimen mounting and transfer 1. After plunging, keep the grid in liquid  ethane and transfer it quickly to liquid nitrogen, move it underneath the nitrogen level to the storage area (see Figure 3.17).   The grid must always be kept at liquid nitrogen temperature after vitrification.

Figure 3.17 Transferring the grid to the storage area. 

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Plunge-Freezing (Holey Carbon Method)

2. Use the precooled screwdriver to close  the grid box firmly (see Figure 3.18).

 The grid can be stored in a Dewar under liquid nitrogen, or directly mounted on the cryo-specimen holder and, subsequently, observed at liquid nitrogen (or liquid helium) temperature in the cryo-electron microscope. Figure 3.18 Closing the grid box with a screwdriver.  

5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  Preservation Unstained, frozen-hydrated biological macromolecules are prepared in a fully hydrated environment, e.g., water or physiological buffer. Therefore, they are observed in a near-native state.

 Artifact-free Unstained, frozen-hydrated biological macromolecules, prepared and observed using the method described here are fully suspended in an aqueous environment. Provided that the specimen is fully embedded in the vitreous layer, flattening and stainrelated artifacts, often observed with conventional air-dried, negatively stained samples, are thus avoided. 

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 The electron microscope must be equipped with an anticontaminator in the column and a low-dose beam-deflection unit to avoid beam damage when the specimen is observed at liquid nitrogen temperature.   The low-dose exposure for unstained, frozen-hydrated biological specimens is usually below 15 electrons/Å2.    Because the protein density is very close to that of vitreous water, the amplitude contrast arising from scattering is very low. Thus, object visibility in cryoEM is mainly obtained by defocusing (increasing phase contrast).  Defocusing alters the contrast transfer function (CTF) of the electron microscope, usually leading to loss of resolution in the data.

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Handbook of Cryo-Preparation Methods for Electron Microscopy

5.2. Disadvantages  Beam damage Unstained, frozen-hydrated biological complexes are highly sensitive to the electron beam. Low-dose exposure techniques are required to avoid beaminduced damage.   Low signal-to-noise ratio (contrast) Because of the low-dose mode required to image unstained protein complexes, the final visibility of the object (more precisely signal-to-noise ratio) is very low. 

 A field-emission gun (FEG) electron source and computer-assisted CTF correction are usually required to obtain medium to high resolution results.

 FEG-equipped microscopes operating at 200 to 300 kV are nowadays the standard in many EM labs where single-particle cryoEM and cryo-electron tomography are routinely employed. 

6. REFERENCES 1. 2.

3. 4. 5.

Adrian, M. et al. Cryo-electron microscopy of viruses. Nature, 308, 32, 1984. Bradley, D.E. The preparation of specimen support films, in Techniques for Electron Microscopy, Kay D., ed., Blackwell Scientific Publications, Oxford and Edinburgh, UK, 57, 1965. Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys., 21, 129, 1988. Harris, J.R. Negative Staining and Cryoelectron Microscopy. BIOS Scientific Publishers Limited, Oxford, UK, 1997. Kasas, et al. Vitrification of cryoelectron microscopy specimens revealed by high speed photographic imaging. J. Microsc., 211, 48, 2003.

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Controlled Vitrification

71

CONTENTS

GENERAL INTRODUCTION ...................................................................................... 73 1.

PRINCIPLES OF CONTROLLED VITRIFICATION ................................... 74 1.1. 1.2.

Effect of Environmental Conditions ........................................................... 74 Process Control prior to Vitrification ......................................................... 76 1.2.1. Thin film formation upon blotting ................................................ 76 1.2.2. Vitrification in melting ethane ...................................................... 77 1.2.3. Set up the temperature conditions for vitrification........................ 78 1.2.4. Set up humidity conditions for vitrification .................................. 78 1.2.5. Condensing ethane ........................................................................ 78 1.2.6. Checklist ....................................................................................... 79

2.

SUMMARY OF THE DIFFERENT STEPS ..................................................... 79

3.

MATERIALS/PRODUCTS/SAMPLES ............................................................ 82 3.1. 3.2. 3.3.

Materials ..................................................................................................... 82 Products ...................................................................................................... 83 Samples ...................................................................................................... 84

4.

PROTOCOLS ...................................................................................................... 84

5.

ADVANTAGES/DISADVANTAGES................................................................ 93 5.1. 5.2.

6.

Advantages ................................................................................................. 93 Disadvantages............................................................................................. 94

WHY AND WHEN TO USE A SPECIFIC METHOD .................................... 94 6.1. 6.2.

6.3.

Preparation of a Suspension........................................................................ 94 Preparation of a Viscous Sample ................................................................ 95 6.2.1. Gels ............................................................................................... 95 6.2.2. Creams .......................................................................................... 95 Preparation of Cells .................................................................................... 96 6.3.1. Bacteria ......................................................................................... 96 6.3.2. Cells growing on an EM grid........................................................ 96

7.

OBSERVED RESULTS ...................................................................................... 97

8.

REFERENCES .................................................................................................... 99

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GENERAL INTRODUCTION Fundamental research within the scope of cell, structural biology and nanotechnology is increasingly focusing on unraveling interactive biological and biochemical processes and pathways at the macromolecular level. For this, high resolution transmission electron microscopy (TEM) is indispensable. Of paramount importance is the three-dimensional visualization of macromolecular structures and molecular machines in their native hydrated state. Their physical fixation within ultra-thin vitrified ice layers is the crucial starting point. This chapter describes the essentials of controlled vitrification, a crucial step for cryoobservation in particular and a starting point for various cryo-preparative methods in general. Cryo-observation of vitrified samples allows the ultrastructural study of macromolecules, molecular assemblies and cells in their natural (= hydrated) environment (see Chapters 3, 7 – 10, 17). 3-D reconstructions can be obtained from vitrified specimens by applying careful microscopy and data analysis. 3-D data are based on single particle analysis (SPA; resolution better than 1 nm) or tomography (resolution better than 3 nm). Controlled vitrification is the starting point for these 3-D studies and has the potential advantage of accurate timing of the vitrification process (1 msec precision), thus enabling time resolved studies. Prior to vitrification, samples are vulnerable and sensitive to environmental conditions. Control over humidity and temperature of the environment is the cornerstone of sample preparation in a critical vitrification procedure,1,2 as will be outlined in this chapter. Practical procedures will be illustrated using the Vitrobot™ as an instrument for computer- controlled vitrification.

Figure 4.1: Modern cryo-electron microscope: (FEI Titan) tailored for 3-D cryo-EM by tomography and single particle analysis.

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1. PRINCIPLES OF CONTROLLED VITRIFICATION 1.1. Effect of Environmental Conditions  For cryo-electron microscopy, samples (macromolecules, molecular assemblies, virus particles, bacteria etc.) are suspended in buffer and cells cultured on a flat support.

 Thin samples are needed for vitrification: less than 100 nm thick for optimum resolution, less than a few micrometers for good vitrification (see Chapters 3, 7).

 A three microliter droplet is applied to a hydrophilic grid (e.g., glow discharged).  By blotting away most of the liquid, a 100 nm thick film is formed which comprises about 1/5000 of the applied volume.  The thin specimen that forms is shot into melting ethane.

 A thin specimen is vulnerable to environmental conditions as heat and mass exchange occur quickly because the surface to volume ratio is rather extreme  A high-entry velocity in ethane is required to optimize heat exchange to vitrify the specimen. To obtain such velocities over a short traveling distance (about 7 cm) requires an acceleration of ca. 3 times the gravitational force.  The vulnerability to the environment  The fragility at a nanometer scale will be further explained below with contrasts with the robustness at the its consequences for molecular and millimeter scale where the grid is subjected to blot forces and acceleration forces. cellular interactions.  Depending on the thickness of the specimen, the temperature decreases to about the dew point in seconds or split seconds. The temperature difference thus established between specimen and environment depends on environmental humidity.

 A thin specimen (typically around 0.1 μm), as used for cryo-EM, will thus attain its dew point temperature within 0.1 sec. The temperature of a thin film in equilibrium will be lower than the temperature of the environment unless the environment is saturated with water vapor.

Figure 4.2 Dew point effect and thickness. Image shows the relation between time and temperature with a thin specimen at 40oC and 40% relative humidity (rH). (Reproduced with permission.3)

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75  Depending on the temperature and the environmental humidity, a temperature gradient will be established. This results in a heat influx toward the specimen and in further evaporation of water. The evaporation velocity is independent of the thickness of the specimen. Evaporation is calculated according to the kinetic theory of gass and assumes evaporation from a free spanning thin film (evaporation from two flat surfaces). Figure 4.3 Evaporation velocity versus temperature and humidity. (Reproduced with permission.3,4)  Preparation at 20oC and 40% rH (typical environmental conditions in our laboratory) will result in the evaporation of water at a rate of 40 nm/sec). A thin film of 100 nm thus losing water will have an increasing solute (and particle) concentration. Liposomes, permeable to water, are exposed to an increasing salt concentration and will lose internal volume to maintain the same osmotic pressure internally as externally. The internal volume may decrease by some 50% during “open air” preparation of a thin film (concomitant with a two-fold increase of the salt/solute concentration). The loss of internal volume is accompanied by a change in shape ― spherical vesicles turn into concentric vesicles connected through an “orifice” that keeps the bilayer continuous. Figure 4.4 Osmotic effects of lab environment. Effect of evaporation on the internal volume of vesicles. (Reproduced with permission.3,4)

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 DLVO theory (Derjaguin-LandauVerwey-Overbeek): Molecular distance related to attraction/repulsion. Relation depending on salt concentration. Balance between van der Waals attraction and electrostatic repulsion.

 An increase in salt concentration has not only osmotic effects, but also an effect on molecular interactions (DLVO theory). The balance between electrostatic repulsion and attraction forces is modulated by the salt concentration (and valence of the ions involved). For the study of macromolecular assemblies, molecular machines and cells, it is essential to maintain the “original” salt concentration. This can only be achieved in an environment saturated with a solvent (e.g., water).

1.2. Process Control prior to Vitrification  Although vitrification of a specimen is a rather simple and straightforward 3step process (apply, blot, and plunge), every step requires careful attention.

 The final result in this chain of events depends on the weakest link and in the next paragraphs a concise description of the principles behind the critical steps will be discussed.

1.2.1. Thin film formation upon blotting  Preparing a thin vitrified object from bulk material is an important step for cryo-EM. The sample can be a solution/suspension (macromolecules, macromolecular assemblies, or particles) (see Chapters 3, 7 - 10, 17) or small, thin cells (see Chapter 12). A convenient (small) volume of liquid is used to obtain a “representative” thin film in which the object is suspended. Producing the thin film in a controlled and, therefore, reproducible manner is an asset of the Vitrobot™ technology.  The blotting action is defined by time and pressure and has to be optimized for each specimen under investigation.

 An applied volume of 3 µL would form a cylinder of about 0.5 mm on a diameter of 3 mm carrier grid and upon blotting the thickness is reduced to a thin film some 100 nm thick.

 The reproducible production of a thin film is thus an important processing step for cryo-EM.

 An optimal thin film is formed when it is continuous over the whole grid and thick enough to suspend the specimen in a pseudomonolayer.  A gentle and controlled movement of  The movement of the blot pads is the blot pads toward the grid was symmetric and perpendicular to the grid found to be important as well as the surface. final pressure they exert onto the grid in the stationary phase (maximum contact/pressure).

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 The liquid is removed from one side by gently pressing the blotting paper. The blot pad on the other side is used as backing to prevent damage by lateral or bending forces.  Both the blot pressure and blotting time can be varied and selected by the user interface.  Blotting is done with filter paper.

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 For viscous solutions, the blotting time can be increased and the pressure should be higher than for more liquid samples.  The absorbing properties of the filter paper are important as they also influence the blotting time and pressure.  Other types and brands of paper than those provided with the Vitrobot™ can be used, but the optimum blotting parameters have to be established.  Note: The blotting paper may release components, e.g., Ca2+, as contaminants that can influence the structure of the specimen.

1.2.2. Vitrification in melting ethane  For vitrification of aqueous solutions, a high freezing velocity (104oC/sec) is the key and a coolant with high thermal conductivity is necessary. In addition, the specimen is plunged into the coolant at a high velocity.  Plunging blotted specimens into melting ethane (liquid between 88 and 172°C) became the standard for vitrification following the monumental first observations on vitrified, aqueous solutions/suspensions by Dubochet at al.5,6 (see Chapters 1, 3, 7).

 Other hydrocarbons seem to be less suited, e.g., liquid methane (161 and 183°C). However, methane is liquid over a limited temperature range compared to liquid nitrogen (196 and 210°C) or liquid propane (42 and 187°C), which leaves residues on the specimen.

 Ethane residues may protect the specimen during transfer, which evaporates in the airlock and vacuum of the microscope.

 Most of the excess of liquid ethane originates from the liquid inbetween the tweezers’ tips or from the fact that the grid is removed from the ethane too abruptly.

 The excess liquid ethane on the grid can be limited by performing two manipulations:

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1. Squeeze the tweezers’ tips together while removing the grid from ethane. 2. Slowly lift the grid from the solution through the ethane surface while observing how the liquid film detaches from the grid.

 The ethane will form a meniscus of the liquid from the solution to the grid that detaches perfectly when the movements are done slowly.

1.2.3. Set up the temperature conditions for vitrification  The Vitrobot is designed to vitrify samples from temperatures between 4oC and 60oC.  The temperature and humidity prior to vitrification should be set using the user interface of the Vitrobot.  The bulk of the sample is normally at the same temperature as the chamber of the Vitrobo and, therefore, the sample solutions should be thermally equilibrated as well.  Special care and attention should be given to the thermal equilibration of the tweezers that are holding the specimen grid.

 A Peltier element is chosen for heating or cooling the instrument with appropriate accuracy.  When the conditions chosen deviate from ambient conditions, time has to be allotted for equilibration; 5 minutes at 25°C, 45 min at 4°C or 55°C are typical figures.  The chamber of the Vitrobot could be used for equilibration or any incubator or water bath.

 At a high relative humidity, a slight difference in temperature of the tweezers and the environment may result in condensation of water on the tweezers. The condensed water may flow down on the grid and dilute the solution.  We strongly advise preheating the  Slightly overheating keeps you on the tweezers before they enter the safe side. chamber.

1.2.4. Set up humidity conditions for vitrification  The ultrasonic humidifier has to be  Note that switching on the humidifier filled with distilled water or has an effect on the temperature as demineralized water following the evaporation of water extracts heat. instructions of the manual. Although the software critically adjusts heat input and water evaporation, it can take some time to reach equilibrium temperature (> 40°C) and humidity (> 95% rH). 1.2.5. Condensing ethane  The ethane container should be filled up to the rim to prevent precooling of the sample in cold gas before it enters the liquid ethane.

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 Note: Liquid ethane can cause severe burns and gaseous ethane may explode. Always work in a fume hood. Follow the safety rules of the manufacturer.

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1.2.6. Checklist  Control the temperature of ethane,  Temperature can be checked with a thermocouple or by visual inspection. The which has to be about 172°C. coexistence of liquid and solid ethane indicates that ethane is at its melting point (see Chapter 3, Figures 3.12, 3.13).  Control (or load) all parameters in the  Are the set temperature and humidity values already attained and stable? user interface of the Vitrobot.  Check the tweezers of the Vitrobot.  Sample and pipette should be ready for application and eventually brought to the same temperature as the chamber.  Replace blotting paper to ensure reproducible working conditions. Upon every blotting action, the blotpads are turned to expose a fresh area of filter paper (16 blot actions to make a full turn).

 Are they clean and at the same temperature as the chamber?  Liquid nitrogen, cryo-storage box and all necessary tools and utensils should be at hand.  The instrument keeps a record of the number of blotting actions and gives a message (“replace blot papers”) when a full turn is made.  Blotting papers are mounted on the foam pads with a clamping ring around the central hole.

2. SUMMARY OF THE DIFFERENT STEPS



   

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       Figure 4.5 Glow discharge grids. 

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Figure 4.6 Condense ethane (aluminum spindle placed on top of the chamber).



Figure 4.7 Replace filter papers.



Figure 4.8 Mount the grids onto tweezers.

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Figure 4.9 Set vitrification parameters.

Figure 4.10 Apply the sample to grid.

Figure 4.11 Blot the sample.

Figure 4.12 Vitrify the sample.

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Figure 4.13 container.

Put

grids

into

storage

Figure 4.14 Remove the storage container and transfer the grid to a cryo-holder. Images are taken at low temperature (< 170oC) at low dose conditions (see Chapter 7).

3. MATERIALS/PRODUCTS/SAMPLES 3.1. Materials  Vitrobot

 Automated device for reproducible vitrification of thin films (FEI Company, Eindhoven, The Netherlands).

 Cryo-holder and transfer system

 The vitrified grid is mounted in a cryoholder and then inserted into a cryo-TEM.  Alternatively, grids may be loaded into the special cartridge of a Tecnai Polara EM.

 Electron microscope with low-dose  Cryo-samples are very sensitive to imaging capabilities and low-dose radiation damage caused by electrons. software  The Low Dose SW suite has been developed to minimize the electron dose during searching, focusing and image acquisition of the specimen.

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 Image acquisition

 Image reconstruction

83  For single particle analysis (see Chapter 7), the Leginon SW suite allows automated particle picking and acquisition in the most optimal areas of vitrification.  For cryo-tomography (see Chapter 12), FEI’s Xplore 3D and Inspect 3D may be used for acquisition and reconstruction of low-dose tomograms.  Various reconstruction packages exist for particle averaging and/or tomographic reconstruction analysis, e.g., Spider, eMan, Imagic (see Chapters, 7, 12, 24).

3.2. Products  Grids

 Grid box

 Liquid nitrogen  Ethane

 Filter paper

 Homemade punch

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 Quantifoil R2/2 (Quantifoil GmbH, Jena, Germany) or Lacey carbon film grids (various suppliers) are used for vitrification.  A typical mesh size is 300.  Grids need to be glow-discharged in order to make them hydrophilic.  Circular grid boxes that fit in the metal grid box holder of the ethane container are used. The grid boxes are provided with the Vitrobot (FEI Company), but can be easily self-made from a square grid box.  Liquid nitrogen is used for precooling the container and for condensation of the coolant (e.g., propane or ethane).  Ethane with a purity > 99.9%.  Liquid ethane enables a very fast cooling rate (dT> 105K) in order to generate an amorphous ice layer.  Note: Liquid ethane can cause severe burns and gaseous ethane may explode. Always work in a fume hood. Follow the safety rules of the manufacturer.  Provided with the instrument, Schleicher & Schuell 595 or Whatman/Schleicher & Schuell 597; both Ø 55 mm.  Any other filter paper may be used, but has to be tested for its blotting properties.  Used to prepare blotting paper by punching a hole in standard Ø 5 cm filter papers.

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3.3. Samples  Various aqueous suspensions: Concentration range may vary depending on original concentration/density.  Isolated cells.

 E.g., proteins, protein complexes, viruses, bacteria or soft matter chemical substances, such as synthetic polymers.  Cells in suspension or cultured as monolayers on carbon coated grids.

4. PROTOCOLS 1. Glow discharging the grids

 Glow discharge helps to spread the applied solute and prevents the liquid film from breaking into smaller (micro) droplets after blotting.  Upon glow discharge, a grid will remain hydrophilic for several hours.  In extreme cases, the material may adhere to the support film instead of spanning the holes in solution.  Aging of the grids may reduce this tendency. Figure 4. 15 Bombardment of the grid with ionized air.

The surface of the support film should be hydrophilic. A droplet applied should spread out and behave as one entity transforming into a continuous thin film. 2. Filling the humidifier.  Prior to activation of the Vitrobot, the humidifier must be filled with 60 mL distilled water through the plastic tube at the bottom of the apparatus using a syringe (see Figures 4.16, 4.17).  NOTE 1: Be aware to fill the humidifier beaker with sufficient distilled water prior to enabling the ultrasonic humidification.  NOTE 2: The humidifier has to operate on distilled water. Figure 4.16 Filling the syringe.

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 It is important that a “vacuum” is created inside the syringe (by “pulling” the piston) to remove the “air” from the humidifier.  The humidifier is bayonet-attached to the bottom of the climate chamber. By turning and pulling, the humidifier can be removed. Figure 4.17 Filling the humidifier.

3. Starting up the Vitrobot.

 Before starting, make sure all cables and wires are properly connected including the pressurized air link.  Activate the pressurized air switch and make sure pressurized air flows into the Vitrobot.  The Vitrobot is switched on with the hard lock switch on the backside of the machine. Figure 4.18. Switching on the Vitrobot.

4. Defining the Vitrobot user interface.  The Vitrobot User Interface consists of two pages: The Console and the Options screen (see Figures 4.19, 4.20). A variety of vitrification parameters can be set in both pages.

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 After activating the hard lock switch, the embedded computer, with MS Windows® operating system, automatically starts up. The start-up page appears shortly followed by the Vitrobot User Interface (see Figure 4.19).  On the console screen, the temperature can be set to any value between 4° and 60°C with the + and – buttons. The actual temperature read-out is displayed in red (21.8).  The humidity — displayed in blue (41.1) — in the climate chamber can be set with the + and – buttons.  Enable the humidity switchbox to start the evaporation (On/Off switch).  The light in the climate chamber can be switched on.  A chronometer records the experimental time. Once a specific time is set, the chronometer counts down displaying a counterclockwise movement.

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 In the Controls box on the right, the sequence of the complete vitrification process (Place New Grid, Start Process, etc.) or to Exit is displayed and operated.  After 16 sequential blottings, the “reset blot paper” button becomes red pointing out that the blot papers need to be replaced.  The Memo box on the left side of the interface functions as an event logger. All major actions and warnings are displayed. Figure 4.19. The Console page.  E.g., the time that the grid is dipped into the vial (plunge time), the relaxation time before blotting (wait time) and the level of  In the Options screen, additional the liquid in the vial (in case of dipping). vitrification parameters can be set.  Alternatively, manual application of a drop of suspension onto the grid through the right or left side entry port can be selected.  In the Miscellaneous box, the use of a foot pedal switch (instead of the mouse) and the possibility to switch off the humidifier during manual application and plunge-freezing can be selected. Figure 4.20 The Options page.

 The semiautomatic grid transfer (i.e., the automatic movement of the grid from the liquid ethane/propane toward the grid box in the liquid nitrogen atmosphere) is default activated, but can be deactivated by checking the skip box.

 In addition, all essential freezing parameters can be saved (Save) or loaded (Load) depending on the type of sample or experiment.  If the “auto raise ethane lift” option is checked, the coolant container will be lifted toward the bottom of the climate chamber simultaneously with the uplift of the tweezers (this can also be done in separate actions).

 Parameters that affect the blotting process are the number of blottings (blot total), the time of each individual blot (blot time) and the position of the grid between the blot pads (blot offset).

 Blot offset determines the force with which the excess fluid is removed from the grid.  Another factor of significance is the drain time, the time between blotting and plunge-freezing.

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 One of the features in the Mark III Vitrobot is the option to do repetitive sample application onto the grid and subsequent blotting prior to plungefreezing.

 To activate this function, press ADD and define the application parameters for the first substance that is to be applied on the grid.  In the Processes section, the Process ID number, including subsequent parameters, are displayed.  By pressing ADD again, the application parameters for the second substance can be defined and displayed in Processes as Process ID 2.  Up to 20 application cycles can be added in this way (1, 2, 3, 4 to 20) – Figure 4.23. Figure 4.21 Repetitive Sample Application Processes.

5. Preparation of the climate chamber.

 INS allows for inserting application parameters at a specific position in the sequence of events that has been defined (1, 2, 3, 4 to 20). By pressing DEL, selected application parameters can be removed from the sequence list.  Besides setting the proper parameter conditions (see Section 4.4.), the LED lights are switched on, the pneumatic pressure control must be active and the blotting papers attached to the blot pads by using the white clipping rings (see Figure 4.23). Figure 4.22 Mounting the filter papers.

6. Preparation of the climate chamber.  A glow-discharged grid is attached to the tweezers. Make sure that the black clamping ring is fixed in such a way that the grid does not fall off in vertical position.

Figure 4.23 Picking up the glow-discharged grid.

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Figure 4.24 Mounting onto the connection groove of the plunge axis.  Mount the tweezers onto the connection grove in the central axis. To do this, first select the ‘Place New Grid’ button in the Vitrobot User Interface to put the central axis into the loading position. 7. Preparation and lifting of the coolant container.  Prior to setting the coolant container in the proper position for vitrification, it needs to be precooled and filled with liquid ethane.

 The tweezers with grid are subsequently lifted into the climate chamber by selecting the Start Process button in the User Interface, or alternatively, use the foot pedal switch.

 The outer ring of the container must be filled with liquid nitrogen. Cooling is a two-step process to be carried out in a fume hood; first, the peripheral reservoir will attain liquid nitrogen temperature; then the central part having a higher heat capacity will cool down. Vigorous boiling (“Leidenfrost effect”) followed by a “calm” equilibrium indicates that the metal parts have attained liquid nitrogen temperature.  The central cup can be precooled with liquid nitrogen before filling with ethane or propane. Figure 4.25 Cooling down the container.  Note: Wait for complete evaporation of the remaining liquid nitrogen in the central part.  To improve heat exchange/cooling of the condensing ethane, an aluminum “spindle” is placed on top of the cup. The spindle is a temporary heat conductor between the liquid nitrogen and the liquid ethane and should be positioned during condensing ethane and further cooling of ethane down to 172oC. Figure 4.26 Filling the container with ethane.

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 When the central cup is at liquid nitrogen temperature, ethane or propane can be condensed. With appropriate pressure reduction, a gentle stream of gas is brought into contact with the cold metal surface where it condenses into a liquid. When enough ethane is condensed, the gas flow can be adjusted (slightly increased) to prevent clogging in the feed line (visual inspection will then tell that solid ethane is formed).

 The ethane container should be filled up to the brim to prevent precooling of the sample in cold gas before it enters the liquid ethane.

 Immediately after the appearance of the halo of solid ethane, the metal spindle must be removed.  The spindle may interfere by creating a cold gas atmosphere (about –160oC) that may precool the specimen before it enters the liquid coolant.  Thawing the frozen ethane between spindle and ethane cup, by placing a bolt on the spindle for 10 seconds, is a more careful way to remove the spindle.

Figure 4.27 Removal of the spindle.  When the ethane/propane container is ready for vitrification, it is placed on the platform ring under the Vitrobot. Then the foot pedal switch or the Continue button in the User Interface must be pressed in order to raise the container toward the bottom of the climate chamber. Figure 4.28 Placing the container onto the platform ring.

8. Sample application  For manual application of the sample, select Manual Application in the Process parameters of the Options page.

Figure 4.29 parameters.

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 The liquid sample can be manually applied to the grid through the leftand/or right-hand side entry port using a pipette.  When the foot pedal switch is pushed or the mouse bottom (Continue to proceed) is pressed, the tweezers slightly lower to allow the application of suspension through the side-entry port using a pipette.  As a consequence, the suspension is applied on one side of the grid only.  The advantage of this method is that only small volumes of the sample (typically 3 µL) are used. Figure 4.30 Manual sample application. 9. Blotting and vitrification.  Excess suspension must be removed  Select Continue in the user interface or from the grid prior to plunge-freezing. use the foot pedal. This activates a slight uptake of the grid toward the correct position between the blot pads and a subsequent blotting of the grid.  After each blot, the blot pads undergo a slight rotation to ensure a clean, new area of filter paper for the next blotting.

Figure 4.31 The blotting process.

 The blotting procedure is immediately followed by injection of the tweezers with the grid into the liquid ethane or propane. The only delay between blotting and plunge-freezing is determined by the drain time that can be set in the Options page and the time required for removing the shutter from the hole in the climate chamber.  The actual plunging mechanism is mediated by pneumatics at the central axis combined with the gravitational force.

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 After plunge-freezing, both the liquid coolant container and the tweezers with the grid are automatically and simultaneously lowered while keeping the grid in liquid ethane. This prevents contamination and rewarming of the freshly frozen sample. 10. Transfer of the vitrified sample.  After vitrification, the frozen grid must  When the “semiautomated grid be transferred into a storage box or transfer” is active, the grid is automatically mounted into the cryo-holder. transferred from the liquid ethane into the liquid nitrogen.  The tweezers must be carefully disconnected from the central axis prior to positioning the grid into the grid box.

Figure 4.32 Removal of the tweezers.  To make the grid transfer more convenient, the coolant container may be lifted from the support ring and positioned next to the Vitrobot.  The excess of liquid ethane on the grid can be limited by squeezing the tweezers’ tips together and lifting the grid slowly from the ethane surface while observing how the liquid film detaches from the grid.  The anticontamination ring, which floats on the liquid nitrogen, creates a cold gas atmosphere, which facilitates the transfer and minimizes possible ice contamination on and rewarming of the grid. Figure 4.33 Transfer of the grid from the liquid ethane into the liquid nitrogen.  The outer ring contains a circular storage grid box for four grids underneath a layer of liquid nitrogen.

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 After the grids have been transferred into the grid box, the box is sealed with a special screw and stored in a Dewar with liquid nitrogen.  The transfer of the grid box into the Dewar should be done swiftly to minimize the risk of rewarming.

Figure 4.34 Loading the grid into the grid box.

11. Shutting down/switching off the Vitrobot  After removal of the tweezers, the central axis moves into its parking position (inside the climate chamber), the LED is switched off and the user interface shuts down.  The computer may be shut down. Figure 4.35 Switching off message.  When pressing the Exit button in the interface, two questions appear:  Pair of tweezers removed? Confirm.  Pair of Confirm.

tweezers

not

removed?

 After shutting down the Vitrobo PC, the remaining water in the humidifier should be removed. Pull the metal ring connector downward to disconnect the electronic cable from the humidifier.

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 NOTE: To prevent bacterial growth in the preheated water of the humidifier, it is advisable to drain the water supply at the end of each working day.

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 Twist and pull the humidifier to unleash the bayonet connection.  Emptying the humidifier is performed in two steps:  Drain the humidifier to empty the central reservoir.  Reconnect the syringe to the plastic tube at the bottom and remove the water from the outside reservoir. Figure 4.36 Emptying the humidifier.

5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  Fully automated and reproducible  All essential vitrification parameters — vitrification of aqueous suspensions. temperature, relative humidity, number of blottings, blotting pressure and drain time — can be programmed for each individual application and stored.  High sample throughput.  High vitrification quality controlled environment.

through  The Vitrobot provides a controlled environment, preventing cooling of the specimen and concentration of solutes due to evaporation before freezing.  These artifacts are inevitable when using conventional freezing apparatus.

 The liquid coolant container with an  More efficient “after-freezing” handling. anticontamination device.  The transfer from the vitrification  More constant and high yield sample medium into the liquid nitrogen has output. been automated.  Easy to Use.

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 Cryo-fixation has become easier with the newly designed and software controlled Vitrobot User Interface.

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5.2. Disadvantages  The Vitrobot is designed for  Monolayer cell cultures can also be vitrification of aqueous suspensions vitrified. only.  In special cases, material suspended in organic solutes can be vitrified as well.  Ethane, however, cannot be used as coolant because it is a “lipid solvent” and it often dissolves organic solutes even at low temperatures.  Decalin and acetone are examples of organic solutes that can be vitrified upon cooling in (the inert) liquid nitrogen, but not all organic solutes will vitrify under these conditions7 (see Chapter 17).

6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Preparation of a Suspension  Use of the standard vitrification technique (see Section 4.2) that avoids adsorption, staining and dehydration artifacts.

 Best structural preservation.  Most suitable to recover excellent structural information upon image analysis.  Most suitable to detect functional conformational changes and to analyse specimen dynamics. Figure 4.37 Cowpea mosaic virus (CPMV) embedded in a thin layer of plunge-frozen water.  Recommended starting conditions for suspensions of macromolecules (2 mg/mL) in aqueous buffers; blotting time of two seconds, one single blotting operation at 100% humidity, a temperature of 21°C. Setting blot offset at 0 mm is also a good point to start.  Depending on the observed results, the parameters can be changed accordingly.  In case the ice layer is too thick, change the blotting time to three seconds.

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 If ice is too thin or no sample is left, change the blotting time to one second.  Also, the blot offset, i.e., blotting pressure on the grid, can be adjusted. Going up in offset (positive offset values) will leave more sample on the carbon film because the pressure on the grid will be lower. Going down (negative offset values) will remove more of the sample. Figure 4.38 Herpes virus capsids imaged in a patch of vitreous ice.

6.2. Preparation of a Viscous Sample 6.2.1. Gels  For a sample with higher viscosity water, longer blotting time (e.g., seconds) is advised.  A sample containing long fibers DNA will have a gel-like behavior most often needs to be blotted longer.

than five like and

Figure 4.39 Shampoo at 25,000×.

6.2.2. Creams  For even more viscous material, an extra number of blots may be helpful.  Creams are difficult to prepare in a film thin enough to be penetrated with electrons.  Good results have been obtained with one second blotting time and three blots.  Sometimes it is useful to use 300 mesh or 400 mesh bare grids to obtain a large area of thin material in the center of a grid square. Figure 4.40 Handcream at 25 000×.

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6.3. Preparation of Cells 6.3.1. Bacteria  A suspension of Escherichia coli bacteria can be vitrified under similar conditions as a diluted suspension of macromolecules.

Figure 4.41 Bacterium in a patch of a frozen sample.

6.3.2. Cells growing on an EM grid  Growing cells directly on the EM-grid covered with a thin carbon film is a fast way of observing cellular structures.  This is, however, only possible in thin parts of the cell, e.g., at its circumference or in filopodiae.

Figure 4.42 Rat liver endothelial cell, isolated and cultured on an EM grid8 and frozen in liquid ethane.

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7. OBSERVED RESULTS

 Single particle image reconstruction of a set of 700 Cowpea mosaic virus (CPMV) images.  A typical 3-D map of 700 views has a resolution of 2 nm. Clear structural features can be observed.

Figure 4.43 CPMV from 700 views.

 Vitrified microtubules imaged using low-dose mode.

Figure 4.44 Microtubules are reconstructed by single particle reconstruction.

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 T4-phage GP23 proteins form tubes of different diameters. Tomograms of these tubes provide more information on their organization.9

Figure 4.45 Tubes from GP23 of T4phage.

 Polymer-layered silicate (PLS) nanocomposites were subjected to plungefreezing and low-dose cryo-electron tomography.  The field of nanomaterials is a fast developing area where the unique properties of inorganic-layered silicates are investigated.10

Figure 4.46 Laponite clay-enriched nanoparticles by cryo-electron tomography and reconstruction (see Chapter 12).

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8. REFERENCES 1. 2.

3. 4.

5. 6. 7. 8. 9.

10.

Bellare, J.R. et al. Controlled environment vitrification system: An improved sample preparation technique, J. Electron Microsc. Tech., 10, 87, 1988. Talmon, Y. Electron beam radiation damage to organic and biological cryospecimens, in Cryotechniques in Biological Electron Microscopy, Steinbrecht, R.A. and Zierold, K., eds., Springer-Verlag, Berlin, Heidelberg, Germany, 1987, 64. Frederik, P.M. and Hubert, D.H.W. Cryoelectron microscopy of liposomes, Methods Enzymol., 391, 431, 2005. Frederik, P.M. and Storms, M.M.H. Automated, robotic preparation of vitrified samples for 2D and 3D cryo electron microscopy, Microsc. Today, November 2005, 32, 2005. Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens, Q. Rev. Biophys., 21, 129, 1988. Dubochet, J. and McDowall, A.W. Vitrification of pure water for electron microscopy, J. Microsc., 124, RP3, 1981. Butter, K. et al. Direct observation of dipolar chains in iron ferrofluids by cryogenic electron microscopy, Nature Mat., 2, 88, 2003. Braet, F. et al. The observation of intact hepatic endothelial cells by cryo-electron microscopy, J. Microsc., 212, 175, 2003. Kükrer Kaletas, B. et al. Structural analysis of self-assembled nanotubes of bacteriophage T4 capsid protein gp23 by cryo electron microscopy and mass spectrometry, Microsc. Microanal., 12, 658, 2006. Negrete-Herrera, N. et al. Polymer/Laponite composite latexes: Particle morphology, film microstructure, and properties, Macromol. Rapid Commun., 28, 1567–1573, 2007.



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CONTENTS GENERAL INTRODUCTION 1.

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PRINCIPLES OF HIGH-PRESSURE FREEZING ....................................... 106 1.1. 1.2.

Fundamentals............................................................................................ 106 Basic Demands ......................................................................................... 106

1.3.

BALTEC HPM 010................................................................................ 107 1.3.1. Introduction to the principle .......................................................... 107 1.3.2. Components and function.............................................................. 107 General Rules for Obtaining Good Results with High-Pressure Freezing 109

1.4. 2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 110

3.

MATERIALS/PRODUCTS .............................................................................. 111 3.1. 3.2.

4.

PROTOCOLS .................................................................................................... 113 4.1. 4.2.

4.3.

4.4. 4.5. 5.

Materials ................................................................................................... 111 Products .................................................................................................... 112

General Tips and Hints ............................................................................. 113 Plant and Animal Tissue........................................................................... 114 4.2.1. Excision and sizing of plant tissue ................................................ 114 4.2.2. Excision and sizing of animal tissue ............................................. 114 4.2.3. Assembly of the specimen sandwich for subsequent freezesubstitution (FS) ............................................................................ 115 4.2.4. Assembly of the specimen sandwich for subsequent freezefracturing (FF), freeze-drying (FD) and cryo-sectioning (CS) (I) . 115 4.2.5. Assembly of the specimen sandwich for subsequent FF, FD and CS (II)..................................................................................... 116 Suspensions .............................................................................................. 116 4.3.1. Preparation of cell suspensions or small organisms (bacteria, algae, nematodes, etc.) for subsequent FS ............................................... 116 4.3.2. Preparation of cell suspensions, small organisms, liposomes and emulsions for subsequent FF, FD and CS ..................................... 118 Monolayer Cell Cultures .......................................................................... 120 Loading, Freezing, Unloading and Storage of the Specimen Sandwich... 122

ADVANTAGES/DISADVANTAGES.............................................................. 124 5.1. 5.2.

Advantages ............................................................................................... 124 Disadvantages........................................................................................... 124

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6.

WHY AND WHEN TO USE A SPECIFIC METHOD.................................. 125

7.

OBSERVED RESULTS.................................................................................... 126

8.

REFERENCES .................................................................................................. 128

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GENERAL INTRODUCTION Optimum preservation of biological ultrastructure is achieved by cryo-immobilization, provided that the biological material is not distorted by formation and growth of ice crystals. At ambient pressure, this requires high cooling rates (> 10,000 K/s). Specimens of up to 10 µm in thickness can be frozen without visible ice crystal damage and without the use of intracellular antifreezing agents by using the propane-jet freezing technique. Thicker samples can be adequately frozen only when the physical properties of water are changed, e.g., using antifreezing agents or under high pressure (2100 bars). The thickness of biological specimens to be adequately frozen by high-pressure freezing is limited to about 200 µm due to the thermal properties of water.15 The high-pressure freezing technique was developed in the 1960s by Moor and Riehle at the ETH Zurich.7,10 Two different approaches to implement high-pressure freezing were described: 1.

Pressure is applied to the specimen in a small container by a transmission fluid. The pressurized sample is then cooled.

2.

The specimen in the container is located in a pressure chamber. Liquid nitrogen at high pressure is shot into the chamber where it completes pressurizing and cooling sequentially (see Section 1, Principles of High-Pressure Freezing).

The first prototype was built using the first approach and patented by Balzers AG in 1968 (GB1230120). This approach showed that ice crystal formation and crystal size are efficiently reduced by the effect of high pressure. It proved, however, to be unsuitable for bulky, aqueous specimens such as animal and plant tissue. The scope of application was limited by the physical properties and geometry of the container (e.g., required thickness of specimen container to withstand pressure). This system was discontinued by BAL-TEC (a new model of type I has been constructed by Studer, see Chapter 6) and a high-pressure freezer according to the second approach was implemented. The new machine provided superior results for all kinds of aqueous specimens. Today, it is manufactured by ABRA-Fluid AG as the HPM 010 high-pressure freezing machine. The vast majority of successful results so far published have been obtained using this system.

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1. PRINCIPLES OF HIGH-PRESSURE FREEZING 1.1. Fundamentals Applying 2100 bars to the specimen prior to the freezing process reduces ice crystal growth drastically and permits adequate freezing of aqueous specimens up to 200 µm thickness.



Figure 5.1 The diagram4 shows the stable state of water as a function of pressure and temperature. Melting temperature and homogeneous nucleation temperature reach a minimum at ~ 2100 bars. Water can be supercooled to 90°C at 2100 bars, turning it into a very viscous liquid. I, II and III are forms of hexagonal ice.

1.2. Basic Demands Pressure buildup must be as rapid as possible. Upon reaching 2100 bars, the cooling process should start and proceed as quickly as possible to a temperature below 140°C where amorphous (vitreous) ice is stable.

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 Pressure leads to changes in the physical/chemical equilibrium, e.g., dissociation constant, enzymatic reaction. A slow increase of the pressure and an extended maintenance of the high pressure would change the specimen unpredictably. This must be avoided because the specimen should be immobilized in a state as natural as possible.

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1.3. BALTEC HPM 010 1.3.1. Introduction to the principle The specimen is enclosed and protected in a small volume between two specimen carriers and frozen in the high-pressure chamber of the HPM 010. Liquid nitrogen is used as a pressurizing and cooling medium. In order to coordinate pressure increase and temperature decrease, a small volume of alcohol (room temperature) is injected into the pressure chamber so that the specimen remains at room temperature during the pressure buildup period. 

 Now available from Boeckeler Instruments, Inc., Tucson, Arizona, USA or ABRA-Fluid AG, Widnau, Switzerland. www.abra-fluid.ch  A new modified apparatus HPM 100 is available from Leica Microsystems, Vienna, Austria.

1.3.2. Components and function  1. Oil pressure pump 2. Bladder-type pressure accumulator 3. Pressure valve (electromagnetic) 4. Low pressure line 5. Low pressure cylinder 6. Pressure piston 7. High-pressure cylinder 8. LN2 Dewar 9. Nonreturn valve   Figure 5.2 Pressure system and Dewar.

10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

High-pressure line Nonreturn valves Specimen holder Specimen pressure chamber Quick fastening bolt Specimen LN2 entry lines Pressure sensor Temperature sensor N2 exhaust with silencer Outlet apertures

Figure 5.3 Object head and alcohol container.

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The bladder-type accumulator (2) is prestressed with nitrogen gas to about 150 bars. This pressure is increased to 300 bars using the oil pressure pump (1). After starting a freezing cycle, a small volume of alcohol is injected into the specimen pressure chamber (13). Subsequently, the 300 bars are converted to high pressure pushing liquid nitrogen from the highpressure cylinder (7) through the highpressure line (10) to the specimen pressure chamber (13). The stream of the pressurized liquid nitrogen compresses the alcohol volume and forces it through the outlet apertures (20) into the environment. The pressure in the chamber containing the specimen (15) increases to about 2100 bars at room temperature before the stream of pressurized liquid nitrogen cools the specimen (15). Figure 5.4 Complete description of the process.

system

and

Pressure maintenance indicates the time of the pressure exceeding 2100 bars (560 ms). Temperature drops below 0°C only after pressure has reached 2100 bars. The flow rate of liquid nitrogen can be controlled by the size of the outlet apertures — Higher flow rate of liquid nitrogen = higher cooling rate = shorter pressure maintenance.

Figure 5.5 The diagram indicates a typical course of temperature and pressure during the process.

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1.4. General Rules for Obtaining Good Results with High-Pressure Freezing  Always use a “fresh” specimen of  It is absolutely crucial that the raw which as much as possible is known material is of highest possible and known (e.g., how it was prepared or grown). (defined) quality.  Always use the smallest possible  The thinner a specimen, the better the aqueous specimen surrounded by the freezing quality. thinnest possible metal support. The thickness of the metal support (carrier membrane) has higher impact on the cooling rate than the material from which it is made.14 Figure 5.6 Effect of wall thickness and material of the specimen carrier on the cooling rate at the center of a 200 µm thick specimen.  Extracellular fluid surrounding the specimen must be used to optimize heat transfer and prevent ice crystal formation (transmission fluid or extracellular antifreezing agents). Excess water and air in the specimen carriers and air in the specimen itself (e.g., plant leaves, antennae of insects) need to be removed and substituted with one of the following extracellular fluids.  Paraffin oils, e.g., 1-hexadecene.

 Dextran/BSA solutions.

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 Water in its liquid and solid (frozen) state is a poor heat conductor. Excess water (and later ice) surrounding the specimen decreases the cooling rate and, therefore, the quality of freezing.  Air acts as an insulator, which is compressed during high-pressure freezing, damaging the specimen mechanically.

 1-Hexadecene is a universal transmission fluid, which can be used for most applications due to its favorable properties such as low surface tension, good heat transfer coefficient in liquid and solid state, no osmotic effect, no miscibility with water.  Dextran and bovine serum albumin (BSA) are used as antifreezing agents usually at concentrations of 20% in a buffer solution.  Frozen dextran and BSA solutions are said to have better cutting properties than 1hexadecene with regard to cryo-sectioning (see Chapter 11).

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 Polyvinylpyrrolidone (PVP) solution  Hydroxyethyl starch solution

NOTE: These solutions dry quickly at ambient conditions leading to an increase in the dextran / BSA concentration. Preparation is preferably performed in a humid environment (e.g., on a wet filter paper or on agar). NOTE: BSA dissolves cholesterol from membranes (defatting).  Alternative antifreezing agents, e.g., for specimens that are affected by 1-hexadecene (e.g., cheese).

 During freezing, the specimen  The specimen may be squeezed out of sandwich must be kept firmly in place the carrier sandwich if it is not closed by the sample holder. properly, e.g., suspensions. In addition, alcohol may penetrate the specimen causing damage.

2. SUMMARY OF THE DIFFERENT STEPS 1. Sample excision and sizing  The sample is excised and sized to fit into the cavity of the specimen carrier (max. 2 mm diameter, 200 µm thickness) using a biopsy gun,2 hand microtome, vibratome, scalpel, razor blade, etc. 2. Assembly of the specimen sandwich  Cavity size is selected according to the dimension of the specimen.  The residual space between carrier cavity and specimen must be filled with an extracellular fluid (e.g., 1-hexadecene). Specimen assembly is completed with a second carrier (e.g., flat carrier). 3. Loading of the specimen holder  The complete specimen sandwich is transferred to the specimen holder of the high-pressure freezer.

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4. Freezing of the specimen  The holder is inserted into the specimen pressure chamber of the high-pressure freezer and locked. Subsequently, the specimen is frozen by a one-button operation.

5. Unloading of the specimen  Immediately after the freezing process the holder is pulled out and transferred to liquid nitrogen in the unloading device. The specimen sandwich is removed from the holder. 6. Storage of the specimens  The specimen in the carrier is stored in liquid nitrogen until further processing. Figure 5.7 (1-6) Summary of the particular steps.

3. MATERIALS/PRODUCTS 3.1. Materials  Aluminum Type A specimen carrier Note: The BAL-TEC specimen carriers and consumables are now supplied by Leica. (100 µm/200 µm cavity)

Figure 5.8 BAL-TEC LZ 02135 VN.  Aluminum Type B specimen carrier (flat, 300 µm cavity)

Figure 5.9 BAL-TEC LZ 02136 V.

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 Aluminum Type C specimen carrier (100 µm/200 µm cavity)

Figure 5.10 BAL-TEC LZ 02137 VN  Gold specimen carrier, cylinder  BAL-TEC LZ 02125 VN shaped indentation (bottom carrier) (3 mm diameter, 0.8 mm thickness)  Gold specimen carrier, dome-shaped indentation (top carrier) (3 mm diameter, 0.8 mm thickness)  Gold tubes (diameter: outside 300 µm, inside 200 µm)  Cellulose capillary tubes  Promag biopsy gun (Set)  Tissue puncher 2 mm  Spacer rings 0.5 mm     

 BAL-TEC LZ 02124 VN

 BAL-TEC LH 01846 VN

 BAL-TEC LH 01843 VN  BAL-TEC BU 012 139 –T  BAL-TEC LH01847 KN  BAL-TEC LH 01842 VN  Athene G209G, BAL-TEC LZ 04884 Gold grids, 8…10 µm thickness KN Single hole EM grids, 50 µm thickness  Agar G2620C, BAL-TEC LZ 04885 KN EM grids, 400 mesh 12…15 µm  Pelco 1GC400, BAL-TEC LZ 04886 thickness KN Sapphire discs (50 µm thickness,  BAL-TEC LH 01845 VN 3 mm) Universal holder for freeze-fracturing  BAL-TEC LZ 04746 VN or cryo-scanning electron microscope (SEM).

3.2. Products  1Hexadecene  Bovine serum albumin (BSA)  Dextran  Ethanol  Isopropanol

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 Fluka 52278, 02079 KN  Sigma A4503  Sigma D4133  Merck 100971   Merck 109634  

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LZ

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4. PROTOCOLS 4.1. General Tips and Hints 1. Stereomicroscope

 A stereomicroscope with low magnification and a flat base plate is useful for the preparation and handling of specimens.

2. Loading station  A plastic Petri dish is helpful as a loading station for the specimen holder. The rim has the proper height to stabilize the tip of the holder. The Petri dish must be pinned down with a weight or tape to prevent tilting during operation. Figure 5.11 Simple loading station using a plastic Petri dish.  3. Freezing cycle

4. Specimen carriers

 Extracellular fluids 

 Time between sample excision and freezing should be as short as possible in order to keep the specimen in its native state. Working in pairs facilitates processing. Usually, one person prepares and loads the specimen while a second person operates the high-pressure freezer (freezing, unloading, and drying of holder).   Always use clean specimen carriers. Aluminum carriers are cleaned in detergent solution and/or ethanol in an ultrasonic bath. NOTE: Do not use chloroform, acetone or acids. These chemicals disintegrate the aluminum carriers.  NOTE: Remember that dextran or BSA solutions dry very quickly increasing the BSA/dextran concentration. A Petri dish with agar or a wet filter paper is helpful to prevent drying of the solution and specimens.  

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6. Preparation of the HPM 010 machine

 The machine must be started up and checked for faultless operation.  Some test shots must be performed using the temperature measuring holder. Pressure maintenance and cooling time/maintenance should meet specifications.  Check the level of alcohol in the container. A lack of alcohol will lead to severe ice crystal damage. NOTE: For subsequent freezefracturing, isopropanol should be used (melting point 88°C) instead of ethanol (melting point 114°C). 

4.2. Plant and Animal Tissue 4.2.1. Excision and sizing of plant tissue Plant leaves not exceeding a thickness of  1-Hexadecene ideally is evacuated 200 µm can be excised using a 2 mm before use because air can dissolve it.  Plant tissue containing air needs to be biopsy punch. submitted to a slight vacuum in 1Plant leaves thicker than 200 µm or other hexadecene to remove the air (see Chapter plant material must be sized and trimmed 6). using a scalpel, razor blade or hand  The choice of extracellular fluid may microtome to fit the cavity of the specimen vary depending on the scientist’s carrier. preferences. A variety of different protocols  are found in the literature.   4.2.2. Excision and sizing of animal tissue Animal tissue is dissected and sized using a NOTE: The Promag biopsy gun system scalpel, razor blade or biopsy gun. has proven to be very suitable.2 The indentation of the smallest available biopsy The time between excision of the tissue and needle (8 mm × 0.4 mm × 0.25 mm) is the freezing process must be reduced to a larger than the space offered by the minimum to prevent artifacts caused by the specimen carriers for high-pressure freezing separation of the tissue from its natural (2 mm diameter, 0.2 mm thickness). environment. However, the excised tissue is often smaller (shorter) than the indentation of the needle, The use of a biopsy gun allows fast excision e.g., depending on the size of the tissue and and sizing of the tissue at the same time.2 its consistency. In many cases, the excised Dissection of tissues using a scalpel or tissue “sausage” may be transferred directly razor blade can be performed in a buffer to the specimen carrier containing 1solution dedicated for use with tissues or in hexadecene. The “sausage” must be cut into 1-hexadecene. shorter pieces if it is too long. Tissue can be squeezed slightly without causing damage due to its flexibility.

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4.2.3. Assembly of the specimen sandwich for subsequent freeze-substitution (FS)

For subsequent freeze-substitution (see Chapter 13), the sandwich is usually composed of a Type A carrier and a flat cover Type B carrier. Whenever possible, the 100 µm cavity is selected to carry the specimen for superior freezing quality. Punched leaf discs of 2 mm diameter fit directly into the cavity of the Type A aluminum carrier (one side 100 µm cavity, other side 200 µm). Tissues excised with a biopsy gun can be transferred directly from the needle into the cavity of the carrier. After freezing the flat cover can be shifted away and the sample stored in liquid nitrogen until further processing.

Figure 5.12 Assembly of the specimen sandwich for subsequent FS. Left, bottom to top: type A carrier with 1hexadecene (e.g., 200 µm cavity), tissue, flat type B carrier wetted with 1-hexadecene. Right, assembled specimen sandwich.  NOTE: The cover carrier is dipped into 1-hexadecene before completing the sandwich, regardless of the kind of extracellular fluid used. This eases the removal of the cover carrier after freezing. NOTE: When using 1-hexadecene, crack the frozen 1-hexadecene by tapping the surface with pointy tweezers. This ensures that the freeze-substitution mixture penetrates the specimen. This is especially important when the specimen is completely embedded in 1-hexadecene after freezing.1 NOTE: Excess 1-hexadecene does not affect the freezing.

4.2.4. Assembly of the specimen sandwich for subsequent freeze-fracturing (FF), freeze-drying (FD) and cryo-sectioning (CS) (I)

For freeze-fracturing (cryo-SEM or replica, see Chapter 22), freeze-drying (see Chapter 15) and cryo-sectioning (see Chapter 11), the sandwich is composed differently than for FS. The specimen is enclosed between two Type A carriers with the 100 µm cavities facing each other.

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Figure 5.13 Assembly of the specimen sandwich for subsequent FF, FD and CS. Left (bottom to top): Type A carrier with 1hexadecene in 100 µm cavity facing up, tissue, type A carrier with 1-hexadecene in 100 µm cavity facing down. Right, assembled specimen sandwich.  NOTE: Pure mechanical attachment is essential if the specimen is to be “heated” for sublimation after fracturing or for freeze-drying. Hence, the carrier containing the specimen or the complete sandwich must be mechanically attached onto a special holder for freeze-fracturing or onto a holder adapted for the cryoultramicrotome .

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After freezing, the sandwich is either kept together for freeze-fracturing (sandwich fracturing) or opened to get access to the specimen with the knife in the cryoultramicrotome or freeze-fracturing device. For subsequent cutting of frozen hydrated sections by cryo-sectioning, the specimen can be also removed from the carrier and glued (using a cryoglue5) to a stub for the cryo-ultramicrotome.

NOTE: High-pressure frozen aqueous material is very brittle. Often, the specimen is cracked or even lost during the fracturing process. This problem is reduced by using the alternative approach described below.

4.2.5. Assembly of the specimen sandwich for subsequent FF, FD and CS (II) Figure 5.14 Assembly of the specimen sandwich for subsequent FF, FD and CS. Left, bottom to top: type C carrier with 1hexadecene in 100 µm cavity facing up, tissue, type C carrier with 1-hexadecene in 100 µm cavity facing down. Right, assembled specimen sandwich.   For FF, FD and CS, the sandwich can be NOTE: This approach is useful for formed alternatively by using type C slit subsequent freeze-fracturing because the carriers. The specimen is enclosed in the slit specimen is mechanically stabilized. between two carriers with the 100 µm cavities facing each other. Holes in the type C carrier surface enable proper alignment.

4.3. Suspensions 4.3.1. Preparation of cell suspensions or small organisms (bacteria, algae, nematodes, etc.) for subsequent FS 

 Concentration by centrifugation

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 Cell suspensions or organisms normally must be concentrated before freezing in order to achieve a reasonable density for EM investigation.   The suspension is centrifuged into a viscous paste at a centrifugal force relevant to the sensitivity of the specimen. Subsequently, the paste is either placed onto the specimen carrier directly or first drawn into a cellulose capillary tube.

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 Assembly of the specimen sandwich: direct loading of specimen carriers. Figure 5.15 Assembly of the specimen sandwich for subsequent FS. Left (bottom to top): Type A carrier with suspension in 100 µm/200 µm cavity facing up, flat type B carrier with 1-hexadecene facing down. Right, assembled specimen sandwich. The specimen paste is transferred into the 100 µm (or 200 µm) cavity of a type A specimen carrier with a spatula or toothpick. The sandwich is completed with a flat type B carrier dipped previously into 1-hexadecene. Centrifugation of a suspension can also be performed in a closed plastic pipette tip (closed by heat). After centrifugation, the pipette tip is opened with a razor blade and the paste is squeezed directly into the specimen carrier using the appropriate pipette.

NOTE: It is important that no air is included during transfer of the paste into the carrier. NOTE: The cell suspension aggregate usually stays together during freezesubstitution and embedding. The aggregate may break into a few pieces, but will not dislocate as single cells in the mixture.

 Assembly of the specimen sandwich: loading of specimen carriers using cellulose capillary tubes.1 Figure 5.16 Assembly of the specimen sandwich for subsequent FS. Left (bottom to top): Type A carrier with 1hexadecene with the 200 µm cavity facing up, filled cellulose capillary tube, flat type B carrier with 1hexadecene facing down. Right, assembled specimen sandwich.

The viscous specimen paste is normally drawn into cellulose capillary tubes by capillary force. The tube may be attached to a pipette tip using wax or glue to increase suction with an attached pipette. This approach is helpful for very thick and viscous suspensions.

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NOTE: Depending on the suspension, it is often difficult to close both ends. However, freezing is not affected and only a minor part of the suspension is lost during freeze-substitution or embedding. Moreover, an open end supports the freezesubstitution process. For this purpose, tubes may be closed on one side only.

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The filled tube is immediately immersed in 1-hexadecene or another medium to prevent drying of the suspension. The tube is cut into small pieces (<2 mm) that fit in the specimen carrier. Cutting is best performed with a blunt scalpel blade. The tube is then cut and squeezed (closed) at the same time. Tube pieces (1 to 4) are transferred into the 200 µm cavity of a type A carrier dipped in 1-hexadecene. The sandwich is completed with a flat type B carrier wetted with 1hexadecene.

NOTE: Dry, fresh cellulose tubes may be squeezed at one end. In that case, the ends of the tube are cut off with sharp scissors before use. NOTE: Always prepare a fresh specimen tube for freezing. Do not leave the tubes in 1-hexadecene for an extended time period prior to freezing. NOTE: Do not use cavities smaller than 150 µm. The tubes would be completely squeezed in a 100 µm cavity.

2. Alternative concentration method.  Assembly of the specimen sandwich. Figure 5.17 Assembly of the specimen sandwich for subsequent FS (alternative). Left ‘bottom to top): Type A carrier with specimen, flat type B carrier with 1hexadecene facing down. Excess medium is Suspensions consisting of cells or blotted off with filter paper. Right, organisms may be prepared in the following assembled specimen sandwich. way: Cells or organisms (e.g., nematodes, cell aggregates) are transferred individually or in groups onto the specimen carrier containing the original medium. After transfer, the excess medium is blotted off with filter paper and the sandwich is completed with a flat type B carrier dipped into 1-hexadecene. 4.3.2. Preparation of cell suspensions, small organisms, liposomes and emulsions for subsequent FF, FD and CS Preparation of cell suspensions, small  Concentration of the specimen (except organisms, liposomes and emulsions for liposomes, emulsions) is performed as subsequent FF, FD and CS. described above.  Assembly of the specimen sandwich for “thin” suspensions. Figure 5.18 Assembly of the specimen sandwich for subsequent FF, FD. Left (bottom to top): Scored flat type B carrier with flat side up, spacer grid with suspension, scored flat type B carrier facing down. Right, assembled specimen sandwich.

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119 Figure 5.19 The flat side of the carriers should be scored with a scalpel in order to enhance the adhesion of the water/ice. By using this method, the majority of the sandwiches will adhere after high-pressure freezing for subsequent freeze-fracturing.

Suspensions consisting of particles or NOTE: No 1-hexadecene is needed. structures smaller than 10 µm (bacteria, cells, liposomes) are frozen in a sandwich NOTE: This approach is not suited for of two flat type B specimen carriers with a subsequent CS. transmission electron microscope (TEM) grid as a spacer (12 to 15 µm) in between. The grid is simply plunged into the suspension and clamped between the two flat sides of the type B carriers.9  Assembly of the specimen sandwich for “thick” and ”thin” suspensions. Figure 5.20 Assembly of the specimen sandwich for subsequent FF, FD and CS. Left (bottom to top): Type A carrier with specimen in the 100 µm cavity facing up, type A carrier with specimen in the 100 µm cavity facing down. Right, assembled specimen sandwich. Suspensions consisting of particles or structures larger than 10 µm (cosmetic emulsions, cement solutions) are frozen in a sandwich consisting of two type A specimen carriers with the 100 µm cavities facing each other. This approach may be applied to CS of any type of suspension. In this case, the sandwich is opened and the carrier(s) containing the specimen is mounted on a corresponding holder for CS.

NOTE: Ensure that no air is trapped in the carrier cavity. Depending on the sensitivity of the specimen, toothpicks or a pipette may be used for filling the carriers. Both carriers are filled with the specimen before assembling the sandwich. NOTE: No 1-hexadecene is needed. NOTE: Specimens may also be frozen with the gold specimen carriers for subsequent FS.

 Assembly of the specimen sandwich for “thin” suspensions for CS (alternFigure 5.21 Assembly of the specimen ative). sandwich for subsequent CS. Left (bottom to top): Stainless steel spacer ring 0.5 mm, filled gold tube, stainless steel spacer ring 0.5 mm. Right, assembled specimen sandwich.14

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Alternatively, suspensions can be frozen in gold tubes for subsequent CS. The gold tubes have an inner diameter of 200 µm and an outer diameter of 300 µm. The suspension is simply drawn into the tube by capillary forces. Thereafter, the tube is clamped between two 0.5 mm spacer rings for freezing.

NOTE: The tube is cut into pieces of about 5 mm length using a 200 µm thick wire and a scalpel. The wire with a diameter corresponding to the inner diameter of the tube (200 µm) must be inserted into the tube prior to cutting in order to prevent squeezing of the gold tube.

NOTE: Sensitive structures of some Turbulence in the pressure chamber of the suspensions (e.g., certain emulsions) may high-pressure freezer can sweep the tube be damaged by the capillary forces of the away. Therefore, the tip of the specimen tube. holder is turned 45° clockwise: Primary stream of liquid nitrogen will hit the small face of the tip (rather than the tube) and, therefore, reduce the shearing forces.

4.4. Monolayer Cell Cultures Varying substrates have been developed for growing and processing monolayer cell cultures by high-pressure freezing: Sapphire discs,13 Matrigel,12 PET discs,8 and Aclar discs.3 The assembly of the specimen sandwich is basically the same for all substrates (discs of the corresponding material) except for the Matrigel approach where cells are grown on the specimen carrier itself. The alternatives I to IV described here are sorted by increasing freezing quality.  Assembly of the specimen sandwich (I).

 Preparation of the Sapphire discs (50 µm thickness, 3 mm diameter): Sapphire discs are thoroughly cleaned with sulfuric acid, rinsed with water and ethanol, and further scoured in a plasma cleaner. Subsequently, the disc is coated with a carbon layer of about 10 nm and a “2” is scratched into the surface. Carbon is a good substrate for various cell types and it creates a visual contrast on the disc for easier manipulation. The asymmetric “2” helps to identify the side with the cells. 

Figure 5.22 Assembly of the specimen sandwich, alternative I. Left (bottom to top): Flat type B carrier with layer of 1hexadecene face up, Sapphire disc with cells facing upwards, type A carrier with 100 µm cavity dipped in 1-hexadecene facing cells. Right, assembled specimen sandwich. This assembly is the easiest one with NOTE: Excess medium is blotted off respect to handling because it consists of with filter paper in order to reduce the only three pieces. aqueous layer to be frozen. NOTE: The layer of 1-hexadecene on the flat carrier ensures good thermal contact between disc and carrier surface.

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 Assembly of the specimen sandwich (II). Figure 5.23 Assembly of the specimen sandwich, alternative II. Left (bottom to top): Flat type B carrier with layer of 1hexadecene, Sapphire disc with cells facing up, spacer (one hole grid with appropriate thickness), flat type B carrier optionally with a film of 1-hexadecene facing cells. Right, assembled specimen sandwich. NOTE: 1-Hexadecene is optional between cells and carrier cover for alternatives II and III because the volume is reduced to a few µm by using the spacer.  Assembly of the specimen sandwich (III). Figure 5.24 Assembly of the specimen sandwich, alternative III. Left (bottom to top): Spacer ring 0.5 mm, Sapphire disc with cells facing up, spacer (one hole grid with appropriate thickness), flat type B carrier optionally with a film of 1hexadecene facing cells. Right, assembled specimen sandwich. Liquid nitrogen hits the support, e.g., Sapphire disc directly, which improves the cooling rate and freezing quality.  Assembly of the specimen sandwich (IV). Figure 5.25 Assembly of the specimen sandwich, alternative IV.6 Left (bottom to top): Spacer ring 0.5 mm, Sapphire disc with cells facing up, spacer (one hole grid with appropriate thickness), and Sapphire disc with cells facing down, spacer ring 0.4 mm. Right, assembled specimen sandwich. Liquid nitrogen hits the support, e.g., Sapphire discs directly from both sides, remarkably improving the cooling rate and freezing quality. Additionally, two discs are frozen at the same time.

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4.5. Loading, Freezing, Unloading and Storage of the Specimen Sandwich  Transfer of the sandwich to the NOTE: The holder tip has to be dried specimen holder for high-pressure after each freezing cycle with a hair dryer. freezing. 1. The specimen sandwich is transferred to the specimen holder for high-pressure freezing.

2. The specimen sandwich is inserted into the cavity of the specimen holder and then closed.

3. Tighten the specimen holder. The specimen holder is held in the left hand or with tweezers and the holder bolt is turned clockwise.

4. The specimen sandwich must be firmly attached in the holder. The carriers should not move after tightening. This can be checked with tweezers.

Figure 5.26 (1-4) Loading of the specimen holder.

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 Freezing of the specimen. 5. Specimen holder is inserted in the specimen pressure chamber of the highpressure freezer. 6. Specimen holder is secured with the locking bolt and the freezing process is started by pushing the corresponding button. Figure 5.27 (5-6) Freezing of the specimen.  Unloading of the specimen after freezing. 7. Immediately after freezing, the specimen holder is removed from the specimen pressure chamber and transferred to the designated slit in the unloading box filled with liquid nitrogen. The tip containing the specimen is unscrewed by turning the holder counter clockwise. NOTE: The holder can be removed as soon as the locking bolt can be retracted. Transfer of the specimen holder to liquid nitrogen should be performed within five seconds after the freezing shot. 8. The specimen holder is opened in liquid nitrogen using tweezers and the specimen sandwich is pushed out from behind. 9. Example of a frozen specimen: cellulose capillary tubes embedded in 1-hexadecene.

10. The specimen is stored in liquid nitrogen until further processing, e.g., an aluminum box.

Figure 5.28 (7-10) Unloading of the specimen. IMPORTANT: Always precool tweezers or other tools prior to handling frozen specimens.

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5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  Very good preservation of ultrastructure without the use of intracellular antifreezing agents or any other chemical treatment.

 Preservation of high-pressure frozen (cryo-immobilized) specimens is superior to conventional, chemical fixation techniques.  Processing of cryo-immobilized specimens is no more time consuming than the conventional room temperature processing.  Quality of freezing depends on the size of the specimen and its composition, e.g., content of natural antifreezing agents.   All kinds of specimens can be frozen  Handling is easy. using the same set of carriers for all  No special tools are necessary. subsequent cryo-preparation methods.  High-pressure freezing technique using the HPM 010 allows the use of very thin-walled specimen carriers. Any type of specimen sandwich can be used, e.g., assembling a sandwich with spacer rings and 10 µm thick stainless steel discs.11

 Pressure in the pressure chamber of the HPM 010 is the same throughout the whole chamber. This makes the use of thin-walled carriers possible without collapsing the sandwich.  Freezing quality is remarkably enhanced by using thin-walled carriers or discs.

 Large specimen size (2 mm diameter).

 Compared to other high-pressure freezing systems, the HPM 010 (system II, see General Introduction) allows the largest specimens to be frozen.

5.2. Disadvantages  Maximum specimen size feasible for high-pressure freezing (2 mm in diameter and 200 µm in thickness) is small compared to conventional chemical fixation techniques.

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 Sizing of the specimen to the required dimensions is a very important step and may often be challenging and should be performed as fast as possible.  Although freezing of thicker specimens is possible, freezing without visible ice crystal damage for most biological material is restricted to about 200 µm.

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6. WHY AND WHEN TO USE A SPECIFIC METHOD Desired subsequent cryotechnique

High-pressure freezing methods 

 Freeze-substitution (FS)

 Freeze-fracturing (FF)  Freeze-drying (FD)

 Cryo-sectioning (CS)

Figure 5.29 Specimen carrier assembly in relation to further processing.

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7. OBSERVED RESULTS  Figure 5.30

 All specimens were high-pressure frozen in their native state (no chemical pretreatment).



 Figure 5.30.A Thin section of mouse kidney biopsy.

 Figure 5.30.B Cryo-SEM micrograph of fractured mouse kidney biopsy.

freeze-

 Figure 5.30.C Thin section of human lung fibroblast, grown as monolayer on Sapphire disc.

 Figure 5.30.D Thin section of Scenedesmus vacuolatus alga.

 Figure 5.30.E Thin section of Caenorhabditis elegans frozen in a cellulose capillary tube.

 Figure 5.30.F Freeze-fracture replica of a liposome suspension.

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 Figures A, C, D, E:  Freeze-substituted in acetone with 2% OsO4  Embedded in Epon/Araldite  Thin sectioned  Stained with uranyl acetate 2% and Reynolds lead citrate  All images:  Electron Microscopy Center Zurich (EMEZ), ETH Zurich, Switzerland.

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8. REFERENCES

1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11.

12. 13.

14. 15.

Hohenberg, H., Mannweiler, K., and Müller, M. High-pressure freezing of cell suspensions in cellulose capillary tubes, J. Microsc., 175, 34, 1994. Hohenberg, H., Tobler, M., and Müller, M. High-pressure freezing of tissue obtained by fine-needle biopsy, J. Microsc., 183, 133, 1996. Jiménez, N. et al. Aclar discs: A versatile substrate for routine high-pressure freezing of mammalian cell monolayers, J. Microsc., 221, 216, 2006. Kanno, H., Speedy, R.J., and Angell, C.A. Supercooling of water to -92 degrees C under pressure, Science, 189, 880, 1975. Michel, M., Hillmann, T., and Müller, M. Cryosectioning of plant material frozen at high pressure, J. Microsc., 163, 3, 1991. Monaghan, P. et al. High-pressure freezing in the study of animal pathogens, J. Microsc., 212, 62, 2003. Moor, H. and Riehle, U. Snap-freezing under high pressure: A new fixation technique for freeze-etching, Proc. Fourth Europ. Reg. Conf. Elect. Microsc., 2, 33, 1968. Morphew, M.K. and Mcintosh, J.R. The use of filter membranes for high-pressure freezing of cell monolayers, J. Microsc., 212, 21, 2003. Müller, M., Meister, N., and Moor, H. Freezing in a propane jet and its application in freeze-fracturing, Mikroskopie, 36, 129, 1980. Riehle, U. Über die Vitrifizierung verdünnter wässriger Lösungen. Federal Institute of Technology, Zurich, Switzerland, 1968. Sawaguchi, A., Ide, S., and Suganuma, T. Application of 10-microm thin stainless foil to a new assembly of the specimen carrier in high-pressure freezing, J. Electron Microsc. (Tokyo), 54, 143, 2005. Sawaguchi, A. et al. Direct attachment of cell suspensions to high-pressure freezing specimen planchettes, J. Microsc., 212, 13, 2003. Schwarb, P. Morphologische Grundlagen zur Zell-Zell Interaktion bei adulten Herzmuskelzellen in Kultur. Federal Institute of Technology, Zurich, Switzerland, 1990. Shimoni, E. and Müller, M. On optimizing high-pressure freezing: From heat transfer theory to a new microbiopsy device, J. Microsc., 192, 236, 1998. Studer, D. et al. Vitrification of articular cartilage by high-pressure freezing, J. Microsc., 179, 321, 1995.



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CONTENTS

CONTENTS................................................................................................................... 131 GENERAL INTRODUCTION .................................................................................... 133 1.

PRINCIPLES OF HIGH-PRESSURE FREEZING ....................................... 135 Simultaneous Pressurizing and Cooling of Biological Samples .......................... 135

2.

SUMMARY OF THE DIFFERENT HPF TOOLS......................................... 137

3.

MATERIALS/PRODUCTS .............................................................................. 138 3.1. 3.2.

4.

PROTOCOLS .................................................................................................... 143 4.1. 4.2. 4.3. 4.4. 4.5. 4.6.

5.

Materials ................................................................................................... 138 Products .................................................................................................... 143

The Tube Holder System .......................................................................... 143 The Flat Carrier System............................................................................ 144 The Biopsy Carrier ................................................................................... 146 The Ring Carrier....................................................................................... 147 The Membrane Carrier ............................................................................. 148 The Live Cell Carrier................................................................................ 148

ADVANTAGES/DISADVANTAGES.............................................................. 149 5.1. 5.2.

Advantages ............................................................................................... 149 Disadvantages........................................................................................... 150

6.

WHY AND WHEN TO USE HIGH-PRESSURE FREEZING ..................... 150

7.

OBSERVED RESULTS .................................................................................... 152

8.

REFERENCES .................................................................................................. 156



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GENERAL INTRODUCTION High-pressure freezing is a means to produce vitreous or well-frozen bulk biological samples.6 Successful highpressure freezing leads to improved ultrastructural preservation.

 Vitrification: Samples that are cryofixed without ice crystal formation during cooling (usually very high cooling rates are necessary). Vitrification can only be proven by diffraction of cryo-sections (electron or x-ray; see Chapters 1, 11).  Well-frozen: Samples show no detectable ice crystal-induced segregations after embedding in polymers.  Bulk samples have to be small. The typical size is a disc of 1 to 3 mm in diameter with a thickness of 200 m.

Figure 6.1 The Leica EMPACT highpressure freezer. Bar = 10 cm

High pressure (2000 bars = 200 MPa) is a physical cryoprotectant that suppresses ice nucleation and ice crystal growth to a certain degree during cooling. The cryoprotective influence can be quantified.8 In practice, biological sample discs 200 m thick (or less) and a few millimeters in diameter are well-frozen or vitrified.12

 High pressure lowers the freezing point of water. At 204.8 MPa, the freezing point is at a minimum of 251 K (22°C). Applying more pressure increases the freezing point again.

The thinner the sample, the higher the cooling rates within the sample. Slamfreezing (see Chapter 2) on a copper block may supply an almost infinitely high superficial cooling rate;3 however, within the sample (depending on its thickness) this cooling rate decreases rapidly.

 A 200 m-thick aqueous sample has in its center a cooling rate of about 5000 K/sec. This cooling rate is dictated by sample size. To achieve optimal cooling rates in the center of a 200 mthick sample, the cooling rate applied at the surface has to be 10,000 K/sec or more.

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 Sartori et al.8 showed that 10 times thicker samples can be vitrified when highpressure freezing is applied compared to ambient pressure freezing, e.g., slamfreezing and plunge-freezing.

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High-pressure freezing machines can generate superficial cooling rates in the range of 10,000 to 30,000 K/sec. Pressure and cold are simultaneously applied to the sample.

NOTE: Cooling rates higher than 10,000 K/sec do not increase the cooling rate in the center.10,12  The measured cooling rate (10,000 to 30,000 K/sec) depends more upon the thermocouple than on the machine’s performance. Cooling rate measurements depend on each thermocouple. To have a series of identical sensors is almost impossible.

Figure 6.2 Calculations-based on the properties of water show that in 200 mthick samples (shown only from surface to centre, 100 m, the other half is exactly mirrored) the highest cooling rates are generated when the sample has reached a temperature, of 253 K (20°C). At this temperature water in the sample is still liquid at 200 MPa. A high-pressure frozen sample shows improved structural preservation when the sample does not contain gaseous compartments and is well prepared prior to freezing.

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 Gaseous compartments are compressed to almost zero volume. The collapse of gas-filled compartments can induce heavy distortions in the sample. The aqueous phase, however, is almost incompressible.

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1. PRINCIPLES OF HIGH-PRESSURE FREEZING Simultaneous Pressurizing and Cooling of Biological Samples We consider all samples that are highpressure frozen as aqueous samples because water is the main constituent (around 80%) of living biological structures.

 Samples frozen under ambient conditions are in most cases heavily segregated by ice crystal formation. Cryoprotectants have to be added.

Pressure is an efficient “physical” cryoprotectant. However, it can be harmful when applied for too long a period of time (>100 ms7,9). Therefore, one has simultaneously to pressurize and cool the sample to vitrify or freeze it well. Best results are achieved at a pressure of 2048 bars and cooling rates higher than 10,000 K/sec applied at the surface of a 200 µm-thick sample.

 All statements made are based on measured and calculated physical facts.12 Therefore it is always important to correctly declare what one is doing. To achieve maximum cooling rates in the centre of a 200 µm-thick sample, a surface cooling rate of 10,000 K/sec is sufficient; however, for optimal cooling rates in a 100 µm-thick sample, about 50,000 K/sec are needed at the surface.

A correct high-pressure freezing cycle is provided by the EMPACT. The sample is surrounded by a metal carrier (usually goldplated copper able to withstand 2048 bars). This carrier is connected via a small tube to a pressure system. Once the pressure is applied, a double jet of liquid nitrogen cools the sample. The high-pressure and cooling systems are separate from one another.

 The total force generated by pressure onto an object depends on the surface area of the object in contact with the pressure source. The EMPACT is, on one hand, applying about 2000 bars onto a sample and, on the other hand, it cools the sample from outside. For efficient cooling of the sample, the enveloping container should have minimal mass. However, the container has to withstand pressure and, therefore, the mass cannot be reduced ad infinitum.

Figure 6.3 An optimal high-pressure cooling cycle: The pressure slope reaches 2045 bars in 20 msec. The pressure increase is nicely synchronized with the temperature decrease. The cooling rate is indicated as dT/dt: 23,650 K/sec.

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F S

Figure 6.4 The diagram illustrates how the EMPACT is working. The inset shows a cross section through a copper tube sealed on both sides by cones.  PF = Pressure fluid  LN2 = Liquid nitrogen

The pressure is transmitted by a liquid, methyl cyclohexane. This solvent is liquid in the temperature range of 150 to 370 K (123°C to +97°C), thus transmitting pressure correctly over a wide range of temperatures. The high boiling point helps in that methyl cyclohexane does not evaporate quickly at room temperature. Furthermore, due to its hydrophobic properties, methyl cyclohexane does not mix with water. Because the interface between sample and hydraulic fluid is small (spatial as well as temporal), the impact of methyl cyclohexane is considered to have no detrimental effects on the sample. In the case of membrane carriers, no contact is made between sample and methyl cyclohexane.

 The sample container is represented by the copper tube system. The copper tube surrounded by the envelope and filled with a cell suspension is introduced into the EMPACT with the help of a manipulator. The tube is connected to the pressure system by pushing the copper tube end onto the cone-shaped end of a pressure tube (with pneumatic piston C). The opposite end of the copper tube is sealed with a stainless steel cone integrated into the manipulator. When the sample is located in position and consequently sealed, piston D, generating high pressure, is loaded with compressed air. However, bar F stops piston D so that no pressure is applied to the sample as of yet.

Technically, one can exchange methyl  Piston B jets the cryogen toward the cyclohexane with any other solvent. sample; however, in the very first moment, However, there is no experience available. the jet is deviated by shutter S, not hitting the sample and, consequently, the sample is not cooled at this step. After a cycle time of 600 msec, the sample  Piston A coordinates the high-pressure is released automatically into a reservoir freezing process. The connecting bar (F) filled with liquid nitrogen. releases the pressure piston (D) applying 2048 bars pressure to the sample via the pressure fluid (PF). The jets are synchronized with the same rod to redirect the liquid nitrogen onto the surface of the sample carrier immediately after pressurization.

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2. SUMMARY OF THE DIFFERENT HPF TOOLS The handling of the EMPACT and EMPACT2/RTS is well described in the manuals supplied by Leica. Their operation is supported step-by-step on the integrated touch screen.Because a wide variety of sample types exists and different follow-up procedures — freeze-substitution (see Chapter 13), freeze-fracturing (see Chapter 22), freeze-drying (see Chapter 15) and cryo-sectioning (see Chapter 11) — can be chosen, a variety of sample carriers is available.   The tube holder system.

 The flat carrier.

 The biopsy carrier.  The ring carrier.

 The membrane carrier.

 The live cell carrier.

 The freeze-fracture carrier.

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When nothing else is indicated, the tools and small parts are supplied by Leica Microsystems (Vienna, Austria)

 One system is based on hollow cylindrical holders (the tube holder system), the other six on disk-like carriers (the flat specimen system).

 The tube holder system allows very efficient cooling of the sample, and is the best suited carrier for cryo-sectioning, CEMOVIS (Cryo-electron microscopy of vitreous sections), if the sample allows it.  The carrier is used to high-pressure freeze samples that can easily be trimmed to a disc of 200 m in thickness and a diameter of 1.2 to 1.5 mm (depending on the carrier) or for suspensions. It is dedicated to freeze-substitution.  Part of the microbiopsy system and designed for biopsies of tissues. Dedicated to freeze-substitution.  It is originally a consumable of the freeze-fracture system. It is suitable for dense suspensions. Dedicated to freezesubstitution.  Has the same function as the flat carrier; however, it rules out that the biological sample comes into contact with the hydraulic fluid (methyl cyclohexane). Dedicated to freeze-substitution.  Especially suitable for cell cultures grown on sapphire discs. They can be investigated in the light microscope and then high-pressure frozen within five seconds with the help of the rapid transfer system (RTS) mounted on the EMPACT2. Dedicated to freeze-substitution.  A platelet system suitable to perform cryo-fixation prior to freeze-fracturing in the machines available on the market.

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3. MATERIALS/PRODUCTS 3.1. Materials  The high-pressure freezing system Figure 6.5 Illustration of the Leica EMPACT (left) and the Leica EMPACT2 with the rapid transfer system (right). Apart from several small updates (Dewar with drain, new software with USB connection, bath led illumination), the most prominent difference between the two machines is the rapid transfer system (RTS). The RTS allows specimen carrier loading in less than five seconds, a prerequisite for correlative light and electron microscopy (CLEM). The EMPACT is described in Studer et al.11 and functioning of EMPACT2 and RTS in Manninen et al.5 Bar = 20 cm  The sample loading system  Envelop of tube holder system

 Dedicated to CEMOVIS samples and small samples in suspension (single cells, invertebrates, embryos, etc.). Figure 6.6 A copper tube carrier is inserted into the tube holder (top), which in turn is connected to the loading device (bottom). Bar = 1 cm

 Dedicated to all other types of samples.  The flat specimen system Five kinds of carriers exist, depending on the type of sample. All five types fit in the carrier holder (pod). Figure 6.7 The carriers are locked into the pod by means of a torque wrench. Bar = 1 cm

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 The carriers  The tube holder system

 Copper tube  Length = 15.9 mm  Outer diameter = 0.650 mm  Inner diameter = 0.350 mm Figure 6.8 The copper tubes are fixed in the tube holder with the help of a recycling kit. The tube is introduced into the holder and cones are pushed into both ends to form funnels. Bar = 2 mm  The system is dedicated to vitrifying suspensions for cryo-sectioning (CEMOVIS, see Chapter 11).

 The carriers of the flat specimen  All carriers have a gold-coated copper system body.  Flat carrier  Outer diameter = 2.8 mm  Inner diameter = 1.25 to 1.5 mm  Central opening diameter = 0.2 mm  Inner wall height = 0.2 mm  Carrier thickness = 0.5 mm  For suspensions and easy to prepare tissue samples (e.g., cartilage, leaves).  Biopsy carrier  Outer diameter = 2.8 mm  Cavity length = 1.2 mm  Cavity width = 0.3 mm  Carrier thickness = 0.6 mm  For high-pressure freezing of biopsies (tissue).  Ring carrier  Outer diameter = 2.8 mm  Inner diameter = 1.2 mm  Carrier thickness = 0.2 mm  Used as carriers above; not so easy to prepare sample, but much easier to handle sample in follow-up procedures. Figure 6.9 Available carriers as mentioned above. Bar = 1 mm

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 Membrane carriers  Outer diameter = 2.8 mm  Inner diameter = 1.5 mm  Wall height = 0.2 mm or 0.1mm  Carrier thickness = 0.26 mm or 0.16 mm  Same use as flat carrier. However, the pressure solvent is not in contact with the sample.  Live cell carriers, sapphire discs and finder grid

Outer diameter Large inner diameter Small inner diameter Wall height Thickness

Live cell carriers 2.8 mm

Sapphire disks 1.4 mm

Finder grid 1.5 mm

1.5 mm

–

–

1.0 mm

–

–

0.2 mm 0.4 mm

– 0.05 mm

– 0.01 mm

Figure 6.10 Membrane carrier, live cell carrier and sapphire discs (from top to bottom). Bar = 1 mm  Dedicated for correlative light and electron microscopy.  Freeze-fracture carriers Large outer diameter Small outer diameter Inner diameter Wall height Thickness

Carriers 2.8 mm 2.0 mm 1.15 mm 0.2 mm 1.0 mm

Ring 2.8 mm – 1.2 mm – 0.4 mm

Figure 6.11 Carrier and ring are mounted on top of one another and loaded into a pod. These carriers provide a link between highpressure freezing with the EMPACT and the freeze-fracture technique (BAL-TEC freeze-fracture machine). Bar = 1 mm

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 The sampling systems Sampling systems are tools that are necessary to excise the biological material from its environment (in vivo or in vitro) and transfer it to an EMPACTcompatible carrier.  Piston (tube holder system). A piston is a thin wire (diameter 0.2 to 0.3 mm, less than the inner diameter of a copper tube), long enough to handle easily (4 to 5 cm).

 Stainless steel wires of the correct diameter (0.25 mm) are available. The likelihood of vitrifying the medium surrounding single cells can be improved by adding 20% dextran.

 Cellulose tubes (tube holder system). The cellulose tubes were introduced by Hohenberg et al.4 The tubes fit in the copper tubes and are porous, ensuring that the substitution medium reaches the sample inside.

 The cellulose tubes can be filled with samples (e.g., single cells, small invertebrates, embryos, etc.) using a suction pressure pipette. Sealing a tube is easily done by pressing with the back (i.e., the opposite side of the blade) of a surgical blade.2 Figure 6.12 A sealed cellulose tube containing nematodes is sticking out of a copper tube. Bar = 500 µm

 Commercial pipette system (ring  A volume of 2 to 5 L per carrier is carrier, flat carrier, membrane carrier). sufficient. Use micropipettes (maximum For the transfer of solutions, fluids and volume) 20 L with extra thin (white) tips. suspensions.  Punch (flat carrier, membrane carrier). The punches are available in two sizes: 1.2 mm or 1.5 mm diameter.  Sapphire discs (live cell carriers). 0.05 mm-thick sapphire discs (diameter 1.4 mm) allow monolayer culture. These discs withstand the physical conditions of high-pressure freezing and are good heat transducers.  Microbiopsy system (biopsy carrier).

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Figure 6.13 The microbiopsy needle has a notch in its central rod. By inserting the needle into the tissue, the notch is filled with tissue and excised upon releasing the sharp hollow tube. Bar = 1 mm

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 The transfer station Figure 6.14 The transfer station is the tool for transferring the biological sample into the biopsy platelet.  Top: Transfer station with RTS adapter installed for EMPACT2.  Middle: The transfer station with biopsy needle for EMPACT.

Bar = 2 cm

 Inset below: a higher magnification of the transfer area. The needle is made transparent for a better view. Bar = 5 mm The transfer station provides a situation  When an EMPACT2 (with RTS) is used, that allows a swift transfer of the sample the loading of the platelet in the pod is done into the biopsy carrier, the carrier into the automatically. pod and the pod into the EMPACT.  The rapid transfer system (RTS)

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Figure 6.15 The rapid transfer system (RTS) is an addition to the EMPACT2. It speeds up the loading of the flat carrier system by automation of a number of steps. It was developed specifically for correlative light and electron microscopy studies and rapid biopsy work. The RTS system uses a new type of pod, but it does not differ in functionality from the pod described herein. The adaptor provides the link between the transfer station (see Figure 6.14) and the high-pressure freezer. Bars = 10 cm and 1 cm  The adapter has a clamp diameter of 2.4 mm, stretchable to 3.0 mm. Hence, the adapter can hold all types of flat carriers (diameter 2.8 mm).

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3.2. Products  EMPACT, EMPACT2, EM RTS and  Leica Microsystems, Vienna, Austria tools.  Liquid Nitrogen (LN2). 15 L are required to fill the EMPACT Dewar.  Methyl cyclohexane as hydraulic fluid.  Merck (VWR International), Germany Ord. no.: 806147  Merck (VWR International), Germany  1-Hexadecene. Ord. no.: 822064  High molecular glycans, such as  Fluka, Switzerland, Ord. no.: 31390 dextran (4075 kDa).  Biological samples depending on investigators interest.

4. PROTOCOLS 4.1. The Tube Holder System The tube holder system allows highpressure freezing of small organisms (cell suspensions, Caenorhabditis elegans, etc.) as well as fluids (blood, milk). Addition of 20% dextran (40 to 75 kDa) enables vitrification of the medium around the cells. 1. A copper tube is loaded into the tube holder.

 The tube holder system is the most elegant way to prepare samples for frozen hydrated sectioning and subsequent CEMOVIS1 see Chapter 11).   A copper tube is inserted into a holder and fixed into place with the loading jig included with the EMPACT tool set. The outer tips of the copper tube are funneled by tightening the screw.

2. Prepare your sample. Place a 20 L  The hydrophobic Parafilm permits the drop on a Parafilm sheet. formation of stable drops. 3. A thin wire used as a piston is inserted in the copper tube and directed through the tube into the suspension. The bottom end of the copper tube has to be inserted into the suspension drop.

 Make new drops for every new run as the small drops are very sensitive to drying out. To ensure vitrification around the cells, use 20% dextran (40 to 75 kDa).

 Because the copper tube has a funnel4. The piston is pulled out at a constant shaped end, it is not difficult to insert the speed of about 1 cm/sec. wire in the tube. 5. The tube is turned 180° (upside down), i.e., the bottom end becomes the top and the top becomes bottom. Repeat Step 3 to 4.

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Figure 6.16 (Left) Diagram of the aspiration of a fluid into the copper tube (encased by a tube holder) using a wire (piston). (Right) Diagram of the tube holder attached to the loading device prior to highpressure freezing.

Bars = 1 cm 6. The copper tube is now filled with suspension and can be loaded into the loading device and high-pressure frozen. 7. After freezing, the tube is removed by cutting out the central 5 mm. This is done with the tube cutter included in the EMPACT tools. Be aware that only the sample in the central 5 mm of the tube is vitreous and will be further processed.

 To prevent the loss of biological samples (e.g., due to their fluidity) during freezesubstitution, they can be enclosed in cellulose tubes. The cellulose tubes were introduced by Hohenberg et al.4 They are thin walled, water permeable and have a diameter less than the inner diameter of the copper tubes. For additional sampling information on the cellulose tubes, consult Claeys et al.2

4.2. The Flat Carrier System 1. A punch produces discs of a leaf or of  The flat carrier is intended as an alltissue sliced to 200 m thick sections. purpose carrier. Its use is explained using leaves as an example.  Figure 6.17 Drawing of the excision of leaf discs with a punch. Bar (left) = 1 cm Bar (right) = 1 mm

2. Gaseous reservoirs in the sample (e.g.,  1-Hexadecene was introduced by Studer leaves or lung) must be replaced by an et al.13 in 1989. inert, noncompressible fluid. 1-Hexadecene is a suitable and often used inert fluid for replacing gaseous volumes.4

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Figure 6.18 Air evacuation  The leaf samples are collected in a 5 mL syringe, which is half filled with 1-hexadecene.  Air is removed from the syringe.  The syringe is airtight closed at the needle exit (Parafilm and finger) and the piston is pulled. Gas bubbles will appear around the biological sample. Continue until no more gas bubbles appear. Bar = 2 cm 3. Prepare the transfer stage. Figure 6.19 Install the pod (P) onto the transfer stage (tool supplied with the EMPACT). Insert a flat carrier into the fork of the handle (H), making sure the cavity points upward. Bar = 2 cm

4. Introduce sample in carrier. Figure 6.20 Diagram of the leaf disc in a carrier. The leaf discs can now be introduced into the flat carrier (one leaf disc per carrier). A drop of 1-hexadecene can be added to ensure all gas is removed from the carrier cavity. Bar = 1 mm 5. Once the sample is introduced into the carrier, push the fork into the pod and lock it. Attach the pod to the loading device and freeze.

 Move the handler to the end stop.  Close the pod with the torque wrench. A torque of 30 N/m is applied.  With the RTS, Step 5 is automatic. Figure 6.21 Drawing of the pod attached to the loading device, prior to high-pressure freezing. Bar = 1 cm

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4.3. The Biopsy Carrier  Only take biopsies of exposed organs. Do not stick the needle through the skin to take a biopsy of an organ.  If notch and carrier cavity are not aligned, adjust the needle position with the screw.  The cavity in the biopsy carrier has the 2. Retract the outer tube of the biopsy same size as the biopsies obtained with the gun. The gun is now “loaded.” Leica microbiopsy system. This allows fast tissue sampling and little gaseous volume to fill prior to high-pressure freezing (HPF).  Figure 6.22 The orientation of the cavity of the biopsy carrier must be in line with the notch of the biopsy needle. 1. Load the biopsy carrier into the loading fork and push the fork through the pod to the end of the sampling bench. Now the biopsy needle notch and the carrier cavity should be aligned on top of each other.

Bar = 1 mm 3. Introduce the tip of needle in the organ and take the biopsy.

Figure 6.23 The microbiopsy needle  In essence, a solid rod surrounded by a hollow tube. Both are sharp.  The rod has a notch at its tip (0.3 mm high × 0.6 mm wide × 1.2 mm long), which will contain the biopsy.  Upon inserting the rod in the organ of interest, the notch is filled with tissue due to its natural movement.  Upon taking a biopsy, the outer tube moves along the rod and cuts away the content of the notch from the rest of the organ. Bar = 1 mm

4. Quickly dip the tip of the biopsy gun  Dipping in 1-hexadecene will prevent (with the tissue) in 1-hexadecene. the sample from drying out.

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5. Install the gun on the microbiopsy transfer station and transfer the tissue from the needle into the biopsy carrier with the transfer tool. Figure 6.24 Close-up (left) of the transfer station with the pod, the biopsy carrier and the transfer tool in place and ready to receive a biopsy. The needle containing the biopsy is not shown. (right) Once the biopsy gun is installed on the transfer station, the notch in the needle will be positioned exactly above the cavity of the biopsy carrier. Bar = 1 mm 6. Proceed as explained in Step 4 to 5 of  The biopsy station ensures the alignment of the tools to one another (needle notch, the flat carrier. biopsy carrier, pod, transfer tool). (See Vanhecke et al. 14 )  This procedure takes less than 30 seconds from obtaining the biopsy until it is frozen. With the EM RTS on the EMPACT2, the procedure takes less time.

4.4. The Ring Carrier  The main advantage over the copper tube is the possibility to observe the sample under the microscope or stereoscope during every step of the follow-up procedure. The mammalian (rat) cell line Walker carcino sarcoma was grown in suspension and is used as an example. 1. Install a ring carrier in the tip of the  Sample loading is still easy, although a fork on the transfer stage. little more difficult than for flat carriers.

The surface tension of water allows fluids to be introduced into a bottomless carrier. The ring carrier can be used for cell suspensions or fluids. The ring carriers are the same rings supplied for freezefracturing.

2. Prepare tools as in Step 3 in the flat  The advantage is that handling after carrier system. freeze-substitution is easier. 3. Pipette a small volume of the  The theoretical cavity volume is concentrated cell suspension or fluid in 0.226 L, but overfill (2 to 5 L) is needed the central cavity of the carrier. to overcome the surface tension of water. The excess liquid will be pushed into the pressure tube of the pod during locking.

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Figure 6.25 The filling of a ring carrier. Bar = 1 mm

4. Proceed as explained in Step 4 of the flat carrier system.

4.5. The Membrane Carrier The membrane carrier is used in the same  The base of the carrier is thin and way as explained for the flat carrier. flexible and, therefore, allows transmission of the pressure. The compression factor of water is: 5 × 10-6 cm2/N At 2000 bars, this equals a volume change of 0.01 (or 1%), which in 3-D is very small. Assume a cube with an edge of 1000 m; this edge is reduced to 996.6 m at 2000 bars. 

4.6. The Live Cell Carrier  Sapphire discs are, after carbon coating, well suited for cultivation of cells.  Furthermore, sapphire discs are transparent and very good thermal 2. Load a live cell carrier. conductors and perfect for high-pressure freezing. 3. Introduce (in medium) a sapphire disc  Cultivated Madin-Darby canine kidney with cells into the live cell carrier. (MDCK) cells are used as an example of the live cell carrier functionality. 4. Clamp a finder grid. 1. Cultivate cells on sapphire discs.

5. Observe the cells in the light microscope (e.g., confocal scanning laser microscopy). 6. Proceed by using the RTS. All necessary manipulations are performed automatically.

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Figure 6.26 Drawing of the loading of a sapphire disc in a live cell carrier, allowing observation in a light microscope. Bar = 1 mm

The follow-up procedures discussed in this chapter.

are

not  Freeze-substitution, and cryo-sectioning. 

freeze-fracturing

5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  High-pressure freezing best preserves  Chemical fixation is comparatively slow and during cross-linking destroys gradients bulk biological samples. essential for life processes.  Handling is easy and very safe.  All steps necessary to perform highpressure freezing are listed step-by-step on the control panel of the EMPACT machine.  Specimen preparation is easy with  Biopsies obtained with the microbiopsy system are frozen within 30 sec. Cell dedicated tools. cultures grown on sapphire discs and sampled with the RTS are frozen within 5 sec.  For all follow-up methods, suitable  All follow-up procedures are possible sample carriers and holders are with the EMPACT frozen samples (cryoavailable. substitution, freeze-fracture, cryo-SEM). The tube system is the most convenient for cryo-sectioning.  The EMPACT is small and easy to  In the case of a centrally organized transport on a cart. electron microscopy unit, it is often easier to fix the samples where they originated. It takes little effort to move the EMPACT around a campus.  The EMPACT consumes only a small  15 liters of liquid nitrogen are sufficient amount of liquid nitrogen. to run the EMPACT for at least 20 samples. The remaining nitrogen can be used for, for example, freeze-substitution.  Information on temperature and pressure  Data management changes is saved digitally on the storage device of the machine. The curves can be retrieved via a USB key and printed or added to a protocol log.

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5.2. Disadvantages  All high-pressure freezing machines are unable to vitrify or adequately freeze native biological samples thicker than 200 m.

 This statement is valid for most biological samples. The limit of 200 m thickness is given by physics and not by any technical insufficiency.10,12 However, eye lenses, for example, are enriched with proteoglycan and this natural cryoprotectant allows vitrification of these samples thicker than 200 m.   The success of the approach has to be  Water content and its state (bound vs. experienced with every new sample. free water) is the most limiting factor for Also this statement holds for all high- successful high-pressure freezing. There are pressure freezing machines. “easy,” “difficult” and “impossible” samples. What the outcome will be is nearly impossible to predict. However, most well prepared biological samples with a thickness of 200 m or less are, in general, segregation free (well-frozen).  

6. WHY AND WHEN TO USE HIGH-PRESSURE FREEZING The dream of all microscopists is to achieve optimal ultrastructural preservation of biological samples in electron microscopy. Up to now there is no better method available than cryo-fixation or cryoimmobilization. Bulk samples in their native state have to be high-pressure frozen to obtain satisfactory (segregation free) cryo-fixation. Therefore, high-pressure freezing is a prerequisite in achieving this. Chemical fixation has to be avoided whenever possible. The size of the sample is a major limiting factor of high-pressure freezing. With few exceptions, there is no physical fixation available to date for samples thicker than 200 m.

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 Optimal cryo-fixation is vitrification (see Chapter 1). A vitreous sample is, in theory, preserved at the atomic level. A prerequisite is that the sample itself is not changed during specimen preparation. This means no chemical prefixation, no addition of cryoprotectants. Specimen preparation prior to freezing must be gentle and as fast as possible.

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7. OBSERVED RESULTS  Figure on the Chapter’s title page

 Connective tissue of the endomysium of a rat soleus muscle is shown.

 Figure 6.27 Brain tissue

 E = endothelium Brain tissue obtained from an  M = mitochondrion anaesthetized rat (Rattus norvegicus)  PoN = postsynaptic neuron using the microbiopsy system (including  PrN = presynaptic neuron the biopsy carrier). The tissue was freeze RBC = red blood cell substituted and embedded in Epon.  S = synapse (Samples prepared and images acquired by Werner Graber, Institute of Anatomy, Bar = 1 µm Bern.)

 Figure 6.28 Ivy leaf 1.2 mm-diameter ivy leaf discs were excised from fresh ivy leaves (Hedera sp.) using a punch, evacuated in 1-hexa decene prior to cryo-fixation in a flat carrier. The tissue was freeze-substituted and embedded in Epon. (Samples prepared and images acquired by Werner Graber, Institute of Anatomy, Bern.)

        

CP = chloroplast DL = double lipid layer M = mitochondrion G = granum N = nucleus NE = nuclear envelope NP = nuclear pore PNS = perinuclear space S = stroma

Bar = 250 nm Bar (inset) = 100 nm

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 Figure 6.29 Yeast Cryo-section of a vitreous yeast sample. The yeast cells (Saccharomyces cerevisiae) were vitrified using the copper tube system.

 B = bubbling induced by the electron beam  M = mitochondrion  RER = rough endoplasmatic reticulum

Bar = 1 m (left) Bar = 500 nm (right) Arrow = cutting direction

 Figure 6.30 Walker carcinosarcoma cells.

 R = ribosome

The cells grew in vitro in suspension and were cryofixed in a ring carrier, followed by freeze-substitution and Epon embedding.

 RER = rough endoplasmatic reticulum  V = vesicle

Bar = 1 m

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8. REFERENCES

1. 2.

3. 4. 5. 6.

7.

8. 9.

10. 11. 12. 13. 14.

Al-Amoudi, A. et al. Cryo-electron microscopy of vitreous sections, Embo J., 23, 3583, 2004. Claeys, M. et al. High-pressure freezing and freeze substitution of gravid Caenorhabditis elegans (Nematoda: Rhabditida) for transmission electron microscopy, Nematology, 6, 319, 2004. Escaig, J. New instruments which facilitate rapid freezing at 83K and 6K, J. Microsc., 126, 221, 1982. Hohenberg, H., Mannweiler, K., and Müller, M. High-pressure freezing of cell suspensions in cellulose capillary tubes, J. Microsc., 175, 34, 1994. Manninen, A. et al. Caveolin-1 is not essential for biosynthetic apical membrane transport, Mol. Cell Biol., 25, 10087, 2005. Moor, H. Theory and practice of high-pressure freezing, in Cryotechniques in Biological Electron Microscopy, Steinbrecht, R.A. and Zierold, K., eds., Springer, Berlin, Germany, 1987, 175. Moor, H. and Hoechli, M. The influence of high-pressure freezing on living cells, in Electron Microscopy: Proceedings of the 7th International Congress on Electron Microscopy, Favard, P., ed, Grenoble, France, 1970, 445. Sartori, N., Richter, K., and Dubochet, J. Vitrification depth can be increased more than 10-fold by high-pressure freezing, J. Microsc., 172, 55, 1993. Sato, N. et al. Ultrastructural effects of pressure stress to Saccharomyces cerevisiae cells revealed by immunoelectron microscopy using frozen thin sectioning, in HighPressure Bioscience and Biotechnology, Hayashi, R. and Balny, C., eds., Elsevier B.V., Amsterdam, The Netherlands, 1996, 109. Shimoni, E. and Müller, M. On optimizing high-pressure freezing: from heat transfer theory to a new microbiopsy device, J. Microsc., 192, 236, 1998. Studer, D. et al. A new approach for cryo-fixation by high-pressure freezing, J. Microsc., 203, 285, 2001. Studer, D. et al. Vitrification of articular cartilage by high-pressure freezing, J. Microsc., 179, 321, 1995. Studer D., Michel, M., and Müller, M. High pressure freezing comes of age. Scanning Microsc. Suppl. 3, 253, 1989. Vanhecke, D. et al. A rapid microbiopsy system to improve the preservation of biological samples prior to high-pressure freezing, J. Microsc., 212, 3, 2003.



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Part II Cryo-Electron Microscopy

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Frozen Hydrated Macromolecules for Structural Analysis

161

CONTENTS

GENERAL INTRODUCTION .................................................................................... 163 1.

PRINCIPLES OF THE METHOD .................................................................. 164 1.1. 1.2.

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 166 2.1. 2.2.

3.

Standard Method ...................................................................................... 166 Reduce Surface Effects............................................................................. 168

MATERIALS/PRODUCTS/SOLUTIONS ...................................................... 169 3.1. 3.2. 3.3.

4.

Vitrification .............................................................................................. 164 Thin, Aqueous Water Layers.................................................................... 164

Materials ................................................................................................... 169 Products .................................................................................................... 172 Solutions ................................................................................................... 172

PROTOCOLS .................................................................................................... 173 Preparation of Holey Carbon Films8 ......................................................... 173 Vitrification of a Thin Layer..................................................................... 174 Preparation of a Holey Carbon Film Covered with a Thin, Continuous Carbon Foil ............................................................................................... 176 4.4. Adsorption of a Specimen on a Carbon Film............................................ 176 4.5. Adsorption of Poorly Concentrated Protein Samples9 .............................. 177 4.6. Chemical Cross-Linking........................................................................... 177 4.7. Preparation of a Sandwich9 ....................................................................... 178 4.8. Incubation with a Lipid Monolayer1,12,15,16 ............................................... 178 4.9. Specimen Transfer.................................................................................... 179 4.10. Low-Dose Imaging ................................................................................... 180 4.11. Image Analysis ......................................................................................... 181 4.12. Troubleshooting........................................................................................ 182 4.1. 4.2. 4.3.

5.

ADVANTAGES/DISADVANTAGES.............................................................. 183 5.1.

5.2.

5.3.

Standard Method ...................................................................................... 183 5.1.1. Advantages.................................................................................. 183 5.1.2. Disadvantages ............................................................................. 183 Adsorption on Carbon .............................................................................. 183 5.2.1. Advantages.................................................................................. 183 5.2.2. Disadvantages ............................................................................. 184 Glutaraldehyde Cross-Linking.................................................................. 184

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5.4.

6.

5.3.1. Advantages ................................................................................. 184 5.3.2. Disadvantages............................................................................. 184 Interaction with Lipid Layers ................................................................... 184 5.4.1. Advantages ................................................................................. 184 5.4.2. Disadvantages............................................................................. 184

WHY AND WHEN TO USE A SPECIFIC METHOD.................................. 185 6.1. 6.2.

6.3.

Stable Specimen Available at High Protein Concentration...................... 185 Stable Specimen Available at a Concentration Lower Than 0.3 mg/mL . 185 6.2.1. Adsorption on a carbon film (see Section 4.4) ........................... 185 6.2.2. Interaction with a lipid layer (see Section 4.8) ........................... 185 Unstable Specimen................................................................................... 185 6.3.1. Chemical stabilisation ................................................................ 186 6.3.2. Adsorption of the specimen ........................................................ 186 6.3.3. Use of lipid layers....................................................................... 186

7.

OBSERVED RESULTS.................................................................................... 187

8.

REFERENCES .................................................................................................. 189



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Frozen Hydrated Macromolecules for Structural Analysis

GENERAL INTRODUCTION  Imaging of biological macromolecules by electron microscopy has developed into a major method for structural biology during the last decade. This approach aims at understanding the mode of action of molecular assemblies by correlating their structure with their function. A prerequisite for such structure-function studies is to observe the molecules in a state as close as possible to their physiological state.  A major obstacle in reaching this goal is keeping the specimen hydrated in an electron microscope that operates under vacuum. Methods were developed in the 1980s to preserve the hydration of the specimen by vitrifying a macromolecular suspension through rapid freezing.10,13 The specimen is observed by its own contrast in the absence of stain so that the collected structural information is comparable to other structural methods, such as x-ray crystallography. The direct observation of a thin layer of suspension avoids specimen adsorption onto a carbon support and, thus, allows the analysis of its dynamic behaviour.  It was observed, however, that the structure of some molecules can be affected during the formation of the thin layer of suspension, notably through surface effects.5,6 This chapter will describe the standard method used by the authors to prepare thin films of macromolecules and a special emphasis will be given to variants of the method that may help to reduce surface effects.

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 Structure-function analysis.

 Keep the specimen hydrated in the electron microscope.  Keep the specimen at a very low temperature.

 Needs a cold stage.  Needs an anticontaminator.

 A variety of methods adapted to fragile specimen.

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1. PRINCIPLES OF THE METHOD 1.1. Vitrification  Vitrification is the process by which a special form of solid water — vitreous ice — is formed by rapid freezing, thus preventing ice crystal formation.5 This form of water is required for optimal preservation and clear visibility of the specimen. The high cooling rate of about 10,000°C/sec is obtained by plunging the specimen into ethane slush (liquid ethane at the solidification temperature).

 Vitrification: A cryogenic immobilisation of the specimen in its hydrated state.

 Ideally, the molecules are randomly oriented and thus are observed through all directions. Figure 7.1 Schematic representation of randomly oriented particles within the frozen hydrated film.

 The thickness of the water suspension that can be cooled into a vitreous state depends on the freezing velocity,11 on the exact composition of the aqueous solution and on the ambient pressure. At atmospheric pressure and by using the standard freezing devices a thickness of about 200 nm of water can be converted into a vitreous state. This value is larger than the thickness generally acceptable for highresolution imaging of particles that should not exceed 100 nm.

1.2. Thin, Aqueous Water Layers  During specimen preparation for cryoEM, the suspension has to be reduced to a less than 100 nm thick layer, which is generally performed by blotting off the excess liquid with filter paper.  During the short interval between the removal of the filter paper and the actual vitrification, the suspension is in

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a metastable state. Effects related to dehydration are reported in Chapters 3 and 4 and will not be treated here.  Just before vitrification, the specimen has also the opportunity to interact with the air/water interface, which may generate various effects related to the high surface tension of the interface. 1. Concentration  Interaction with the surface may result in concentrating the specimen. This is likely to happen very often because at a specimen concentration of 1 mg/mL, the particles occupy only 0.1% of the volume and few particles would be visible.  In some instances the surface can also repel the particles and this gives rise to areas where particles are absent. This phenomenon is generally observed in the thinnest parts of the vitrified layer.

 Because the particles are immobilised at the surface, this effect can be used to remove undesirable agents, such as glycerol or high salt concentrations, by rinsing the grid with a drop of solution.

Figure 7.2 Locally concentrated particles at the surface of the frozen hydrated film.

2. Orientation  In several instances, it could be observed that the particles are preferentially oriented at the air–water interface, a phenomenon not only due to thinning of the film, but to the preferred interaction of one face of the particle with the interface.5

Figure 7.3 Partially oriented particles at the surface of the frozen hydrated film.

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3. Denaturation  In extreme cases, the interaction of the specimen with the air–water interface can severely affect the structure and the function of the molecule.17 Proteins can be unfolded to expose their hydrophobic residues or domains at the interface. Particles smaller than expected are then observed.

Figure 7.4 Partial denaturation of fragile particles at the air-water interface.

2. SUMMARY OF THE DIFFERENT STEPS 2.1. Standard Method 

    

1. Place the liquid ethane container and the grid box into the liquid nitrogen container.        2. Lock the EM grid on tweezers and fix the tweezers on the plunger. 3. Fill the liquid nitrogen container and wait 10 to 15 min for the system to equilibrate.

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  



4. Liquefy the ethane by inserting polyethylene tubing connected to an ethane gas cylinder into the ethane reservoir.

   5. Wait for the ethane to solidify at the rim of the container.

    6. Place 4 µL of the sample on the grid. 7. Remove the excess liquid by blotting with a filter paper.

    8. Plunge the grid into the ethane slush. 9. Store the grid in a grid storage device in LN2.    

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Figure 7.5 (1-9) Different steps of the vitrification procedure.

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2.2. Reduce Surface Effects  The structure of some macromolecules can be affected before vitrification and during the short exposure to surfaces. In this case, a number of methods may be used to reduce the effects the airwater interface exerts on the molecules. 1. Adsorption methods  Absorption to a solid surface such as a plain carbon film, rather than to a “mobile” air–water interface may help to reduce particle reorganisation. A deformation may still result from the interaction with carbon, but it is likely to be localised at the interaction interface.

 Adsorption onto a thin carbon film that has been previously glow-discharged.  In case the sample concentration is very low, the drop of solution can be left to interact with the carbon support for several hours.  In the sandwich method (see Section 2.4.), the specimen is placed between two carbon films, which may further limit specimen rearrangements.

2. Cross-linking methods  To prevent particle reorganisation, the  Use of glutaraldehyde stored at 20°C at specimen may be stabilised by a final concentration of 0.05 to 0.1%. chemical cross-linking with low concentrations of glutaraldehyde. 3. Methods using amphiphilic molecules  To prevent the particles from surface interaction, the air–water interface can be covered with amphiphilic lipid molecules that will instantly spread as a monolayer at the surface.1,12,15,16 The properties of the hydrophilic part of the lipid, such as its charge or the presence of a specific recognition function, will trigger the interaction of the protein of interest. Thus, the protein is concentrated at the interface, but cannot be denatured because the surface is covered by the lipid. Eventually this interaction can lead to a specific orientation of the protein and to its 2-D crystallisation.

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 A detergent or a solution of liposomes may be mixed with the particles of interest.  The particles may be incubated prior to vitrification with a monolayer of lipids spread at the air–water interface.

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3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Carbon evaporator  Produce holey carbon films and plain carbon films evaporated on mica. From Boc Edwards model Auto 306. A = Electrodes B = Carbon rods C = Shutter D = Work plate  The carbon evaporation device can be replaced by a metal sputtering device in case metal nanoparticles need to be deposited onto the thick holey carbon film as a defocalisation aid.

Figure 7.6 Carbon evaporator.

 Glow discharge apparatus   

    

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  Used to charge electrostatically the surface of the carbon films to favour specimen adsorption.14 A = Vacuum gauge B = Discharge current reading C = Air inlet D = Amylamine inlet E = Vacuum chamber air F = Vacuum chamber amylamine G = Rotary pump H = Vacuum selector I = Discharge selector J = Voltage setting K = Current setting Figure 7.7 Glow discharge apparatus 

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 Holey carbon grids 

 Used as a support for the thin layer of suspension to be vitrified. A = EM grid bar B = Hole C = Thick carbon mesh

 Cryo-plunger



Figure 7.8 Holey carbon grid.   Used to vitrify a thin layer of suspension. A = Sliding shaft B = Tuneable abutment to set the height C = Electromagnet for automatic release D = Fastening device to hold the tweezers E = LN2 container

Figure 7.9 Cryo-plunger  Nitrogen container

  Used to liquefy the ethane and to vitrify the sample. A = Nitrogen container B = Polyurethane foam isolating device C = Ethane container D = Grid storage device Figure 7.10 Nitrogen container.

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 Ethane container

 To keep the ethane for a longer time in a liquid state before solidification, the Lausanne group5 designed a double-walled container to prevent the inner ethane container from being in direct contact with LN2.

A = Outer container in contact with LN2 B = Inner ethane container C = Isolating air layer

Figure 7.11 Ethane container.

 Cryo-box for grid storage

 Storage device for four grids.  Rotationally indexed for unambiguous grid identification.  Screw cover numbered for device identification.

 Long term storage in liquid nitrogen.

Figure 7.12 Cryo-box for grid storage.

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3.2. Products  Filter paper Round ashless filter paper, 9 cm in diameter, cut into small sectors  Bare Cu/Rh grids, 300 or 400 mesh  Tweezers

 LN2 tank for storage of frozen grids

 Used for blotting. From MachereyNagel, Düren, Germany, grade. MN 640 W  From EMS, Hatfield, PA, USA.  Large size, cold-isolated tweezers to manipulate the cold grid storage device.  Small Dumont N5 tweezers for grid manipulation.

 Small plastic containers to pour LN2      

 Several are required to avoid contamination with moisture. Ruby red mica sheets for carbon  From EMS, Hatfield, PA, USA, Ref 71851. evaporation  Used to prepare plain carbon films and Carbon floating device to sandwich the specimen between two carbon films. Teflon® wells for incubation with  Wells 3 mm in diameter and 2.5 mm deep bored into a Teflon block. lipids  Used to deposit lipids at the air–water Hamilton syringe interface.  Used to keep stock solution of lipids. Lipid storage 2 mL glass vials  Used as a protection in case of ethane Protection glasses spilling.

3.3. Solutions  Ethane gas cylinder  LN2 supply  Glutaraldehyde  Incubation with lipids  Lipids: Preferably use oleyl C18:1 side chains, which combine good film stability and fluidity. Positive charges can be introduced by ammonium and trimethyl ammonium groups, and negative charges can be introduced by phosphatidyl serine. To reduce the amount of charges present in the monolayer, oleyl alcohol can be used as a dilution lipid.  Ethane: Chloroform 1:1 solution

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 Keep frozen aliquots to avoid polymerisation. From Sigma, Ref G-5882.  Lipids are available as powders or as solutions and can be ordered from most chemical companies. Lipids with derivatised polar groups may be found at Avanti Polar Lipids (Alabaster, Alabama, USA).

 To prepare dilutions of the lipids.

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 To produce grids covered with holey carbon films  From ICN Biomedicals, Aurora, Ohio,  7X®PF cleaning solution  Pasteur pipette USA, Ref. 76-671-21  Cellulose acetate  Benzalkonium chloride  Ethyl acetate  Dioctyl sulfo succinate

 From Aldrich, Ref 18,095-5  From Sigma, Ref B-1383  From Aldrich, Ref 27,052-0  From Sigma, Ref D-4422

4. PROTOCOLS 4.1. Preparation of Holey Carbon Films8 1. Place 10 glass slides in a 500 mL  The perfect cleaning of the glass slides is beaker, cover with 7 × detergent and essential to obtaining a homogeneous boil for 10 min. distribution of holes. 2. Wash extensively the slides with tap water for 30 min and then with 5 beaker volumes of distilled water. 3. Render the glass slides hydrophobic by immersion into Benzalkonium chloride 0.5% (w/v) in water for at least 30 min. From now on, each slide is treated individually.  The slide has to be perfectly dry when withdrawn. 4. Dip each individual slide into a beaker containing distilled water. Slowly draw the slide out of the water by holding it by one end with tweezers.  Pre cool a 10 mm-thick aluminium plate for a few hours at 20°C. 5. Place the glass slide onto the precooled aluminium plate. Small water  The size of the holes will increase with droplets will form on the cold glass the relative humidity and the length of time plate. the slide is on the cold plate. 6. Cover the glass slide with a layer of  The slide is held inclined and the cellulose acetate dissolved to 0.4% cellulose acetate solution is poured over the (w/v) in ethyl acetate. slide with a Pasteur pipette.

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7. Let the solvent evaporate.

 At this stage, the slide can be observed under a light microscope to inspect the holes and to select the region of interest.

8. To transfer the holey cellulose film  To peel off the cellulose film more onto bare grids, it has to be loosened easily, the dioctyl sulfosuccinate solution from the glass slide through an can be heated to 80°C. immersion into a dioctyl sulfosuccinate solution 1% (w/v) in water. 9. The loosened film can now be floated at the surface of a glass container in which bare grids were immersed and  In case the film sticks at the border of placed onto a filter paper positioned on the glass slide, the rim can be scratched a metal support. with a scalpel. 10. The cellulose film is deposited onto  The best area covered with suitable holes the grids by lowering the water level. can be marked and the grids can be deposited only under this area. 11. After drying, the filter paper is placed  At this stage, gold nanoparticles used as into the carbon evaporator and a 20 to a defocalisation aid can also be deposited 40 nm-thick carbon film is evaporated by evaporation. on top of the cellulose film. 12. The cellulose film can be dissolved  To dissolve the cellulose film, the grids, with ethyl acetate and the grids are still placed on the filter paper, are placed ready for use. overnight onto several layers of filter paper soaked with ethyl acetate in a glass Petri dish.

4.2. Vitrification of a Thin Layer 1. Select 3 to 4 EM grids coated with a  The grid may be glow-discharged in air holey carbon film. for 30 sec to render them hydrophilic. The electrodes are separated by 4.5 cm; a 2. Place the ethane container and the grid voltage of 110 V is applied at a partial air box into the LN2 container of the pressure of 0.2 mbar yielding a discharge plunging device. current of 2.5 mA. 3. Fill the container with LN2.

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4. Wait 10 min equilibration.

for

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temperature  In case of incubation with lipids, the lipid-protein film has to be transferred onto a grid.

5. Refill with LN2.

6. Liquefy the ethane by flowing a stream  LN2 has to be dry. Take care that few ice of gas through plastic tubing crystals accumulate in the LN2 containers. connected to an ethane flask into the ethane reservoir cooled to LN2 temperature. 7. Pick up the holey carbon-coated grid  Liquid ethane may spill and, therefore, with the tweezers and fasten the the operator should wear protective glasses. tweezers onto the plunger. 8. Wait for the ethane to solidify.

 When the ethane starts to solidify, a white solid rim appears around the container.

9. Apply 4 µL of the sample onto the grid or transfer the lipid-protein layer onto a holey carbon film. 10. Remove the excess solution by  The filter paper can be applied only on applying a filter paper onto the drop half of the grid. In this case, a gradient of of solution. solution thickness will be obtained and at least one area of the grid will have a suitable thickness. 11. Remove the filter paper and immediately plunge the grid into the ethane slush. 12. Rapidly transfer the frozen hydrated  Any delay at this stage will result in sample into the LN2 container and evaporation. place it into the storage device.

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4.3. Preparation of a Holey Carbon Film Covered with a Thin, Continuous Carbon Foil 1. Evaporate a 50 to 100 Ǻ-thick carbon  A continuous carbon film is required for film onto a freshly cleaved mica sheet. specimen adsorption. To reduce the signal arising from the carbon film, it is 2. Place a filter paper onto a metal recommended to use a film as thin as support immersed in a water- possible. Such a film is not stable enough containing glass dish. over the grid bars and needs to be supported by the thick holey film. 3. Place about 100 EM grids covered with a holey carbon film onto the immersed filter paper. 4. Dip the mica sheet into the liquid with an angle of about 45° in order to float the carbon film on the air–water interface. 5. Lower the water level in order to deposit the carbon film onto the holey grids.

4.4. Adsorption of a Specimen on a Carbon Film 1. If required, the carbon-coated holey film (see Section 4.3) can be glow discharged to render it hydrophilic. The grid is placed on an electrode into a vacuum chamber pumped down to a partial air pressure of 2 10-1 mbar. An alternative discharge current of 2.5 mA is applied for 30 sec to charge the grid. 2. Five µL of the sample at a protein concentration of about 30 µg/mL is applied onto the charged grid and left for 45 sec to allow adsorption. 3. Proceed as described in Section 4.2 for vitrification.

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 If required, exchange the buffer by placing the grid onto a drop of the new buffer deposited on a flexible laboratory film (Parafilm).

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4.5. Adsorption of Poorly Concentrated Protein Samples9 1. Cut the carbon coated mica into 4 mm  This method can be used for protein strips. concentrations lower than 5 µg/mL. 2. Deposit 75 µL of the protein sample into a Teflon well 5 mm wide, 5 mm long and 3 mm deep. 3. Float the carbon film from the mica strip at the surface of the protein sample placed in the Teflon well. 4. Leave the carbon in place for 12 hours to allow specimen adsorption. 5. Plunge an EM grid coated with a holey carbon film into the Teflon well and pick up the floating carbon onto which the specimen is adsorbed. 6. Proceed as described in Section 4.2 for vitrification.

4.6. Chemical Cross-Linking 1. The sample is incubated for 2 min at  This method is used to stabilise the room temperature with glutaraldehyde specimen independently of the subsequent at a final concentration of 0.1 to preparation method. 0.05%. 2. The incubation time and glutaraldehyde concentration depends on sample composition. It is strongly recommended to screen the crosslinking conditions by negative staining EM to find the optimal conditions in which the sample is stable and does not form aggregates.

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4.7. Preparation of a Sandwich9 1. Adsorb the specimen as described in  This method can be used to further Section 4.4. immobilise the specimen by sandwiching it between two carbon films. 2. Cut a carbon coated mica into 4 mm strips. 3. Deposit 75 µL of buffer into a Teflon  This method can be combined with well 5 mm wide, 5 mm long and 3 mm negative staining when the solution in the deep. Teflon well is replaced by a 2% uranyl acetate solution. The stained sample is dried 4. Float the carbon film from the mica for 2 min and plunged into LN2 before strip at the surface of the buffer placed observation to keep a hydration shell in the Teflon well. around the specimen. 5. Plunge the EM grid coated with a holey carbon film on which the specimen is adsorbed into the Teflon well and pick up the floating carbon. 6. Proceed as described in Section 4.2 for vitrification.

4.8. Incubation with a Lipid Monolayer1,3,12,15,16 1. Place the Teflon support into a Petri dish cover placed upside down; add 5 mL of buffer into the Petri dish to seal the incubation chamber once the Petri dish base is placed upside down on top of the chamber.

2. Place 10 µL of buffer into a Teflon well 3 mm in diameter and 2.5 mm deep.

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3. Place 1 µL of lipids dissolved in a 1:1 chloroform/hexane solution at a concentration of 0.5 mg/mL. 4. Add 5 µL of the sample. Different concentrations need to be tested between 30 and 500 µg/mL. 5. Incubate for various time periods ranging between 1 and 24 hours. 6. Place an EM grid coated with a plain or a holey carbon film on top of the drop for 5 min. 7. Lift the EM grid from the drop. A good transfer is witnessed by the presence of a thin layer of solution on the EM grid. 8. The transferred lipid-protein layer can be negatively stained or processed for cryo-EM as described in Section 4.2. Figure 7.13 Illustration of the protein-lipid layer incubation and transfer to the EM grid.

4.9. Specimen Transfer 1. Cool down the anticontaminator and the cryofork of the microscope. 2. Cool down the cryo-holder by pouring LN2 into the Dewar and into the grid mounting device.

A = Dewar of the cryo-holder B = Mounting device

Figure 7.14 Grid mounting station. 3. Retrieve a cryo-box containing the samples from its long-term storage device and place it into the grid mounting device.

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A = Position of the grid B = Place for the grid-box C = Cryo-shield D = Reservoir

Figure 7.15 Holder cryostation and grid mounting device

4. Take out a grid from the cryo-box and mount it onto the cryo-holder. Close the cryo-shield over the sample to prevent ice contamination during the transfer.

 Solid ethane may remain attached to the grid at this stage and may cause problems during transfer or EM observation. The solid ethane may be scratched off with the tweezers before specimen mounting onto 5. Transfer the cryo-holder into the the cryo-holder. electron microscope as recommended by the manufacturer.

4.10. Low-Dose Imaging 1. Recording images at the lowest possible electron dose is required to limit the irradiation damage of the specimen.

 Search mode: Select an area of interest (circle A) at low magnification (typically 5,000×) with the lowest possible irradiation.  Focus mode: Focus the objective lens at high resolution (typically 40,000×), on a neighbouring area (B), close enough to have little changes in height and far enough to avoid irradiation of the area of interest. This is performed using an image shift whose angle and amplitude can be selected.  Record mode: Record the image at the required magnification of the area of interest. Figure 7.16 Illustration of the principle of low dose imaging.

2. Images can be recorded on film plates  To improve sensitivity, data may be or on charged coupled device (CCD) recorded on SO163 films (Kodak) and cameras. developed for 12 min with undiluted D19 developer.

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4.11. Image Analysis 1. Image analysis of single molecules aims principally at improving the resolution of the noisy images and at determining a 3-D model of the particles. This section is not meant to describe the image analysis protocols, but to get some starting directions.2-

 Information on two commonly used image analysis software suites can be found on: http://www.wadsworth.org/spider_doc/spid er/docs/spider.htmL and http://www.imagescience.de/

2. Digitise the images. Unless recorded on CCD cameras, the silver halide emulsions have to be digitised using a high-resolution microdensitometer.

 Most widely used scanners are flat bed (Nikon Coolscan 8000) or drum scanners (Heidelberger Druck Maschinen, Primescan D7100). They should provide a linear response with at least 5000 dpi resolution.  Several free displaying systems are available on line. Among them, EMAN can be used for particle picking: http://blake.bcm.edu/EMAN/

4,7,18

3. Particle picking. An image representing a large field containing many particles (see Figure 7.1) has to be displayed on a monitor and the coordinates of the particles have to be determined.

4. Alignment. Particle images have to be  Cross correlation functions are generally iteratively aligned in rotation and in used to align images.19 translation against references to be placed in register. 5. Clustering. Similar images have to be  Multivariate statistical methods are used grouped into classes according to their to classify images. maximal resemblance. 6. Averaging. Aligned particle images  An image class corresponds generally to within each class are averaged to a particular view of the particle. improve the signal-to-noise-ratio. 7. 3-D reconstruction. The different views of the particle have to be combined to form a 3-D model of the particle.  An interactive molecular graphic 8. Visualisation. The 3-D model can be program can be found under: displayed and compared to other http://www.cgl.ucsf.edu/chimera/ existing models.  A protein structure data base is hosted at: http://www.wwpdb.org/  A macromolecular structure data base is found under: http://www.ebi.ac.uk/msd/

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4.12. Troubleshooting 1. No particles are observed  Is particle concentration in the correct  The concentration should be between range? 0.3 and 1 mg/mL.  Particles smaller than 50 kDa are difficult to spot at low dose.  Is the contrast sufficient?  The presence of glycerol or sucrose will increase the density of the media and will reduce the contrast of the particles.  Are the particles hidden in the thicker  Some particles do not like to be close to part of the frozen hydrated layer? the surface and escape into the thick parts of the vitreous water.  In this case, smaller particles are  Are the particles denatured? observed in the background indicating that  Are the particles aggregated? the large assembly is dissociated. 2. Ice contamination  The grid surface may be covered with long filamentous aggregates that arise from impurities present in the ethane. These aggregates tend to be more numerous when the grid is covered with a thick layer of solid ethane that evaporates during transfer into the microscope. Careful removal of the excess ethane before mounting the grid helps to avoid this problem.  The grid may be contaminated with hexagonal ice crystals that were present in LN2 or may have condensed during grid transfer. Working in a dry atmosphere, heating up the cryo-holder between successive transfers into the microscope and possibly emptying and drying the LN2 container generally limits this problem.  The grid surface may be covered by a thin, almost continuous cubic ice layer arising from the condensation of water present in the microscope. This form of contamination may appear like faint discontinuous spots. It may reflect high water pressure around the specimen either because the anticontaminator is not working properly or because poorly adsorbed ice crystals are melting or because there is a small leak in the air lock. Figure 7.17 Illustration of ice contamination.

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5. ADVANTAGES/DISADVANTAGES 5.1. Standard Method 5.1.1. Advantages  No adsorption effects.  No staining effects.  Physiological ionic strength.

 Use of holey carbon films.   Beware of the effects of evaporation after blotting.

 Fully hydrated structure.  Particles observed through all orient-  Important for image analysis of single tations. particles and 3-D model reconstruction.

5.1.2. Disadvantages  Low intrinsic contrast.

 High protein concentration.  Possible surface effects.

 Use of phase contrast and, thus, need to correct for the contrast transfer function (CTF) of the microscope to have a faithful representation of the particle.  0.3 to 1 mg/mL.  The structure of the specimen may be affected.  Particles pushed together may aggregate or be too close to be separated.

5.2. Adsorption on Carbon 5.2.1. Advantages  Adsorption concentrates the specimen.  Use plain carbon films.  10 to 30 µg/mL; concentration range.  Adsorption may reduce surface  The immobilisation of the particles induced aggregation. reduces particle redistribution during thin film formation.  Carbon film gives a strong signal to  determine the CTF of the microscope.  Uniform particle distribution.   All particles have the same CTF.  Particles in the same plane.

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5.2.2. Disadvantages  Reduces the angular range for 3-D reconstruction.  The surface of the particle in contact  Adsorption induced deformation. with the carbon may be altered.  Carbon film introduces a granularity  Use of thin carbon films. that may affect particle alignment.

 Preferred orientations.

5.3. Glutaraldehyde Cross-Linking 5.3.1. Advantages  Stabilises specimen structure.

 Prevents surface denaturation.

5.3.2. Disadvantages  May promote aggregation.

 Adjust glutaraldehyde concentration.

 Stabilise lowest energy conformation.

 Dynamic information is questionable.

5.4. Interaction with Lipid Layers 5.4.1. Advantages  Covers the air–water interface.

 Prevents surface denaturation.

 Mobile surface.  Two-dimensional crystallisation.

 Promotes particle organisation.

5.4.2. Disadvantages  Lipid–particle interaction.

 The structure of the molecules may be affected by this interaction.

 Possible orientation of the particles.

 Tilting the specimen may help to record all particle views.

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6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Stable Specimen Available at High Protein Concentration  Use of the standard vitrification technique (see Section 4.2) that avoids adsorption, staining and dehydration artifacts.

 Best structural preservation  Most suitable to recover excellent structural information upon image analysis.  Most suitable to detect functional conformational changes and to analyse specimen dynamics.



6.2. Stable Specimen Available at a Concentration Lower Than 0.3 mg/mL  In this case, an adsorption method is  For any method used, the interaction of required to concentrate the specimen. the specimen with the support may alter the specimen structure. However, high resolution has been obtained with both methods.

6.2.1. Adsorption on a carbon film (see Section 4.4)  The specimen will interact with the  The surface properties of the carbon film surface of the carbon film and will are generally hydrophobic and may be modified by glow-discharge. concentrate at the interface. 6.2.2. Interaction with a lipid layer (see Section 4.8)  The interaction of the specimen with  Lipids carrying an electrostatic charge or the lipids can be tuned by using a specific recognition function, such as a ligand of the protein to be studied, can be different lipid mixtures. used.5

6.3. Unstable Specimen  An unstable specimen will appear aggregated or dissociated when processed using the standard vitrification method. Different strategies may be used to prevent specimen dissociation or aggregation.

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 Additional tests may be performed by using buffers containing different ionic strengths in order to find conditions where the specimen might be stable.  The criteria for having little surface denaturation is the structural homogeneity of the sample.

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6.3.1. Chemical stabilisation  Glutaraldehyde or other cross-linking  Be aware that the stabilised species may agents can be used to stabilise the not correspond to the functional state of the complex. protein complex.

6.3.2. Adsorption of the specimen  Different adsorption methods can be used that may help to prevent surface denaturation. The sandwich method may further reduce molecular movements and aggregation prior to vitrification.

6.3.3. Use of lipid layers  Incubation with a spread lipid layer prevents interaction of the specimen with the air–water interface and subsequent denaturation.

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7. OBSERVED RESULTS  Figure on the Chapter’s title page (see Figure 7.19)

 Figure 7.18 Cryo-electron microscopy observation of a frozen hydrated suspension of yeast RNA polymerase at a concentration of 0.6 mg/mL in a buffer containing 10 mM Tris/HCl pH 7.4 and 100 mM NaCl.

  Figure 7.19 Image analysis of the molecular images shown on the title page. The top panel shows several characteristic class averages obtained upon several alignment/classification iterations. Each class average corresponds to a different view of the RNA polymerase I molecule. The lower panel shows the three-dimensional model obtained by combining the different class averages.

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 Figure 7.20 Panel of cryo-electron micrographs showing different preservation conditions and illustrating the effects of surface denaturation. The upper left panel shows well preserved RNA polymerase molecules with an optimal distribution. The molecules are represented as circles in the right panel. The middle panel shows partially denatured particles (represented as squares in the right panel) that appear heterogeneous in size and shape. The lower panel shows an inhomogeneous distribution of the particles that tend to stick to each other.

 Figure 7.21 Illustrations of yeast RNA polymerase I molecules incubated with positively charged lipids spread as a monolayer at the air–water interface. The upper panel represents a short incubation time (5 min) and shows that the molecules (initial concentration of 50 µg/mL) are concentrated at the lipid/air interface. This lipid/protein interaction favours or stabilises RNA polymerase dimers that are observed in large amounts. The middle panel was obtained after one hour incubation and shows the organisation of the dimeric molecules into rows. Finally, the lower panel shows an imperfectly ordered, two-dimensional crystal formed at the lipid interface.

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8. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18. 19.

Bischler, N. et al. Specific interaction and two-dimensional crystallization of histidine tagged yeast RNA polymerase I on nickel-chelating lipids, Biophys. J., 74, 1522, 1998. Bischler, N. et al. Localization of the yeast RNA polymerase I-specific subunits, Embo J., 21, 4136, 2002. Crucifix, C., Uhring, M., and Schultz, P. Immobilization of biotinylated DNA on 2-D streptavidin crystals, J. Struct. Biol., 146, 441, 2004. De Carlo, S. et al. Cryo-negative staining reveals conformational flexibility within yeast RNA polymerase I, J. Mol. Biol., 329, 891, 2003. Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens, Q. Rev. Biophys., 21, 129, 1988. Dubochet, J. et al. Emerging techniques: Cryo-electron microscopy of vitrified biological specimens, Trends in Biochem. Sci., 10, 143, 1985. Frank, J. Three-Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in Their Native State, Oxford University Press, Oxford, U.K.,2006. Fukami, A. and Adachi, K. A new method of preparation of a self-perforated micro plastic grid and its application, J. Electron Microsc. (Tokyo), 14, 112, 1965. Golas, M.M. et al. Major conformational change in the complex SF3b upon integration into the spliceosomal U11/U12 di-snRNP as revealed by electron cryomicroscopy, Mol. Cell., 17, 869, 2005. Jaffe, J.S. and Glaeser, R.M. Preparation of frozen-hydrated specimens for high resolution electron microscopy, Ultramicroscopy, 13, 373, 1984. Kasas, S. et al. Vitrification of cryoelectron microscopy specimens revealed by high-speed photographic imaging, J. Microsc., 211, 48, 2003. Lebeau, L. et al. Specifically designed lipid assemblies as tools for twodimensional crystallization of soluble biological macromolecules. Handbook of Nonmedical Applications of Liposomes, Vol. 2, Barenholz, Y. and Lasic, D.D. eds., CRC Press, Boca Raton, FL, USA, 1996, 155. Lepault, J., Booy, F.P., and Dubochet, J. Electron microscopy of frozen biological suspensions, J. Microsc., 129, 89, 1983. Oudet, P. et al. Electron microscopy of simian virus 40 minichromosomes, Methods Enzymol., 170, 166, 1989. Schultz, P., Bischler, N., and Lebeau, L. Two-dimensional crystallization of soluble protein complexes, Methods Mol. Biol, 148, 557, 2001. Schultz, P. et al. Three-dimensional model of yeast RNA polymerase I determined by electron microscopy of two-dimensional crystals, Embo J., 12, 2601, 1993. Tanford, C. Protein denaturation, Adv. Protein Chem., 23, 121, 1968. van Heel, M. et al. Single-particle electron cryo-microscopy: Towards atomic resolution, Q. Rev. Biophys., 33, 307, 2000. van Heel, M., Schatz, M., and Orlova, E.V. Correlation functions revisited, Ultramicroscopy, 46, 307, 1992.

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CONTENTS GENERAL INTRODUCTION .................................................................................... 195 1.

PRINCIPLES OF THE METHOD .................................................................. 196 1.1.

1.2.

1.3. 2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 200 2.1. 2.2.

2.3. 2.4. 3.

Materials ................................................................................................... 204 Products .................................................................................................... 205 Solutions ................................................................................................... 205

PROTOCOLS .................................................................................................... 206 4.1. 4.2. 4.3. 4.4. 4.5. 4.6.

5.

2-D Crystallization by Membrane Fusion ................................................ 200 2-D Crystallization by Reconstitution ...................................................... 200 2.2.1. Crystallization by dialysis........................................................... 200 2.2.2. Crystallization using BioBeads................................................... 201 Crystallization under a Lipid Monolayer.................................................. 202 Surface Crystallization ............................................................................. 203

MATERIALS/PRODUCTS/SOLUTIONS ...................................................... 204 3.1. 3.2. 3.3.

4.

2-D Crystallization of Membrane Proteins ............................................... 196 1.1.1. Naturally occurring crystals ........................................................ 196 1.1.2. 2-D crystallization by reconstitution........................................... 196 2-D Crystallization of Soluble Proteins .................................................... 198 1.2.1. Lipid monolayer crystallization .................................................. 198 1.2.2. Surface crystallization................................................................. 199 Cryo-Preparation of 2-D Crystal Samples ................................................ 199

Membrane Fusion of Purple Membranes.................................................. 206 2-D Crystallization by Dialysis ................................................................ 206 2-D Crystallization using BioBeads ......................................................... 207 Preparation of Cryo-Samples by the Back-Injection Method ................... 208 Lipid Monolayer Crystallization............................................................... 209 Surface Crystallization ............................................................................. 211

ADVANTAGES/DISADVANTAGES.............................................................. 213 5.1.

5.2.

Using Natural Crystals.............................................................................. 213 5.1.1. Advantages.................................................................................. 213 5.1.2. Disadvantages ............................................................................. 213 2-D Crystallization by Dialysis ................................................................ 213 5.2.1. Advantages.................................................................................. 213 5.2.2. Disadvantages ............................................................................. 213

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5.3.

5.4.

5.5.

6.

2-D Crystallization Using BioBeads ........................................................ 214 5.3.1. Advantages ................................................................................. 214 5.3.2. Disadvantages............................................................................. 214 Lipid Monolayer Crystallization .............................................................. 214 5.4.1. Advantages ................................................................................. 214 5.4.2. Disadvantages............................................................................. 214 Surface Crystallization ............................................................................. 214 5.5.1. Advantages ................................................................................. 214 5.5.2. Disadvantages............................................................................. 214

WHY AND WHEN TO USE A SPECIFIC METHOD.................................. 215 6.1.

6.2.

6.3.

2-D Crystallization of Membrane Proteins............................................... 215 6.1.1. Natural 2-D crystals.................................................................... 215 6.1.2. Detergent removal by dialysis .................................................... 215 6.1.3. Detergent removal using BioBeads ............................................ 215 2-D Crystallization of Soluble Proteins.................................................... 215 6.2.1. Lipid monolayer crystallization.................................................. 215 6.2.2. Surface crystallization ................................................................ 215 Specimen Preparation............................................................................... 215

7.

OBSERVED RESULTS.................................................................................... 216

8.

REFERENCES .................................................................................................. 218

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GENERAL INTRODUCTION Two-dimensional (2-D) crystallization of proteins is a powerful alternative to x-ray crystallography when 3-D crystals of sufficient quality cannot be grown or when proteins are not suitable for this technique; for instance, when a protein aggregates above a specific concentration. The aim of this technique is to force lateral interactions between proteins in a plane and thereby to induce the formation of 2-D crystals, as shown in Figure 8.1. Cryo-electron micrographs of 2-D crystals have very low contrast. However, it is possible to investigate the structure of proteins by image analysis based on Fourier methods because the molecules exist in thousands of identical copies. Developments in sample preparation, imaging and image analysis now make it possible to resolve protein structures from intermediate (6 to 10 Å) (see Figure 8.25) to high (2 to 4 Å) resolution from 2-D crystals.6 Initially, electron crystallography was mainly dedicated to the study of membrane proteins.7 Over the last decades, new methods have been developed to induce 2-D crystallization of both membrane and soluble proteins. The goal of this chapter is to present commonly used methods to induce 2-D crystallization of proteins and related cryopreparation techniques. Figure 8.1  A: HC-Pro, a soluble protein expressed with a 6His-tag, can interact with a lipid monolayer.  B: When the pH is raised to 8.4, lateral interactions between proteins induce formation of a regular array. Bar = 50 nm

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1. PRINCIPLES OF THE METHOD 2-D crystals can occur naturally or can be produced by different approaches, depending on the chemical properties of the  proteins. In this context, we will distinguish between membrane and soluble proteins. 

1.1. 2-D Crystallization of Membrane Proteins 1.1.1. Naturally occurring crystals A few membrane proteins occur naturally in 2-D arrays. These protein arrays can be purified and prepared for electron microscopy (EM) directly. When the patches are too small, they can be merged into larger arrays of several µm in diameter.

 Bacteriorhodopsin is a classic example.

 Bacteriorhodopsin membrane patches can be fused using detergents (see Protocol, Section 4.1).  

1.1.2. 2-D crystallization by reconstitution The most general method for 2-D crystallization of membrane proteins involves mixing detergent-solubilized proteins with suitable lipids, also in detergent solution, and the removal of detergent that induces the reconstitution of the protein in a lipid bilayer (see Figure 8.2). 

 The protein purification step is outside the scope of this book. We will suppose that the proteins are purified, solubilized in the presence of detergent and have a concentration of at least 1 mg/mL. 

 mp = membrane protein  d = detergent

Figure 8.2 Proteins are solubilized in detergent.

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Lipids used for reconstitution are prepared in detergent (see Figure 8.3).  l = lipid  d = detergent Figure 8.3 Lipids are solubilized in detergent.  Lipids commonly used are phosphatidyl choline (PC), dioleyl phosphatidyl choline (DOPC), dioleyl phosphatidyl glycerol (DOPG), dimyristoyl phosphatidyl choline (DMPC). For a review see Walz and Grigorieff.12 Proteins and lipids are mixed together (see Figure 8.4).  mp = membrane protein  d = detergent  l = lipid Figure 8.4 Membrane proteins and lipids are mixed together and incubated overnight.  The concentration of free detergent has to be above the critical micelle concentration (CMC). Detergent is removed inducing formation of a lipid bilayer (see Figure 8.5).  mp = membrane protein  d = detergent  l = lipid Figure 8.5 A lipid bilayer is reconstituted by detergent removal.  Proteins are usually inserted in an up and down orientation in the membrane. The detergent can be either removed by dialysis using a membrane that is permeable to detergent monomers but not to micelles  A 10 kDa molecular weight cut-off is (see Protocol, Section 4.2) or by absorption generally used. onto polystyrene beads (see Protocol, Section 4.3).

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The dialysis can take days to weeks, depending mainly on the critical micelle concentration (CMC) of the detergent. Under appropriate conditions, regular arrays will be formed. The most important parameters include the nature of the lipid (or lipid mixture), the lipid-to-protein ratio, the temperature, pH, ionic strength and presence of additives.

 Detergent removal by BioBeads® is much faster than by dialysis,10 which can be an advantage when the CMC of the detergent is very low or when the protein is very unstable.  Determination of crystallization conditions is empirical. This means that all parameters should be tested individually and in combination with others.

1.2. 2-D Crystallization of Soluble Proteins 1.2.1. Lipid monolayer crystallization 2-D crystallization can be induced under a monolayer of lipids (for a review, see Chiu et al.3). The interactions between proteins and lipids will force the proteins into a fixed orientation and increase the concentration of proteins under the monolayer (see Figure 8.6). 

 Proteins should have a concentration above 1 mg/mL in a suitable buffer.    Interactions of proteins with the lipid monolayer can be electrostatic or lipidspecific, as for instance with a nickel functionalized lipid.  Figure 8.6 The lipid monolayer is formed at the air-buffer interface. Proteins bind functionalized lipids.  The lipid monolayer consists of functionalized lipids and diluting lipids, which play a role in the lateral diffusion of the proteins.    

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Dl = Diluting lipid Fl = Functionalized lipid P = Protein Protein with its interaction region with the functionalized lipid.

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1.2.2. Surface crystallization During the 3-D crystallization of proteins,  Crystallization of the solubilized physical phenomena involving specific H+ATPase from Neurospora crassa has interactions between proteins occur at the been performed using this method.5 air-buffer interface. Thus, in a drop in which 3-D crystals are formed, it is interesting to observe what happens at the surface of the drop. Under favorable situations, 2-D crystallization can also occur at the airbuffer interface. 

1.3. Cryo-Preparation of 2-D Crystal Samples 2-D crystals are very large, but thin. For cryo-preparation it is important to have the crystals stretched out over the support film.  Depending on the size of the unit cell of the crystal and the expected resolution, a deviation of as low as 1o over several µm can be tolerated.11  For cryo-microscopy, most 2-D crystals can  The carbon film usually wrinkles after be deposited on carbon support films and freezing because the expansion coefficients plunged into liquid ethane using a classical of copper1 and amorphous carbon do not plunge-freezing device (see Chapters 3, 7). match. In practice, molybdenum is the grid material of choice because it maintains the film flatness best at liquid nitrogen (LN2) temperature.

Flatness of the crystals on the support film is essential for successful high-resolution data collection. 

A plunge-freezing device in a chamber with  At a very high humidity level, a thin controlled atmosphere (see Chapter 4) is layer of water is still preserved at the very useful in some cases. surface of 2-D crystals.

2-D crystals can alternatively be  Glucose, trehalose or tannin are cryoprotected by a sugar. This method, commonly used cryoprotectants. which is most suitable for 2-D crystals of membrane proteins, will be presented in this chapter (see Protocol, Section 4.4).

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2. SUMMARY OF THE DIFFERENT STEPS 2.1. 2-D Crystallization by Membrane Fusion Membrane patches are mixed together in the presence of detergents and incubated for several weeks.

2.2. 2-D Crystallization by Reconstitution 2.2.1. Crystallization by dialysis 1.

Figure 8.7

Figure 8.8

Mix detergent solubilized proteins, lipids and detergent. 2. Incubate with gentle magnetic stirring for 1 to 24 hours. 3. Transfer the mixture into a dialysis device without forming air bubbles on the dialysis membrane (see Figures 8.7 and 8.8). 4. Put the dialysis device into a detergent-free buffer (see Figure 8.9). 5. Incubate at fixed temperature with gentle magnetic stirring of the buffer. 6. Change buffer two or three times during the first 72 hours. 7. Incubate several days. 8. Take a sample. 9. Deposit a few µL of solution onto a carbon-coated grid (usually 300 or 400 mesh grids) and wait for two minutes. The grid can be glowdischarged before using it in order to improve the interaction and flatness of 2-D crystals with the carbon film of the EM grid. 10. Blot the grid and freeze it in liquid ethane using a plunge-freezing device. 9&10 Alternatively, a cryo-sample can be prepared with the back-injection method (see Protocol, Section 4.4). Figure 8.7 Dialysis device (Slide-ALyzer®) from Pierce. Figure 8.8 Bent glass capillary tube.

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 P = Parafilm  B = Buffer without detergent  M = Magnetic bar

Figure 8.9 Overview of the dialysis device.

2.2.2. Crystallization using BioBeads

Figure 8.10A

1. Mix proteins and lipids in detergent in a microcentrifuge tube. 2. Incubate with gentle magnetic stirring for 2 to 24 hours at 16°C. 3. Add wet BioBeads (see Figure 8.10A). 4. Incubate at 16°C with gentle magnetic stirring. 5. To stop adsorption of hydrophobic molecules to the BioBeads, remove the sample and transfer it to a new tube (see Figure 8.10B). 6. Freeze-thawing cycles can be used to induce 2-D crystallization (see 2-D crystallization of cytochrome b6f2). The sample is plunged into liquid nitrogen and thawed on ice. The cycle is repeated at least three times. Figure 8.10 BioBeads in detergent removal.  A: BioBeads are added to the solution.  B: Detergent removal is stopped by transferring solution into another tube.

Figure 8.10B

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7. Deposit a few µL of solution onto a carbon-coated grid and wait for two minutes. Depending on sample properties, the interaction and flatness of 2-D crystals with the carbon covering the grid can be improved by using glow-discharged grids. 8. Blot the grid and freeze it in liquid ethane using a plunge-freezing device. 7&8. Alternatively, a cryo-sample can be prepared with the back-injection method (see Protocol, Section 4.4).

2.3. Crystallization under a Lipid Monolayer 1. Wash a Teflon® crystallization plate (see Figure 8.11) and place it in a Petri dish that contains two layers of wet filter paper. 2. Inject the buffer into the incubation well. The surface of the well must be perfectly plane. The volume of the well is about 50 µL. 3. Deposit 0.5 µL of lipids at the surface of the incubation well. 4. Incubate at least 4 to 12 hours at 16°C. This temperature decreases the dehydration phenomenon. 5. Inject 3 to 8 µL protein solution using the injection well. 6. Protein incubation is performed under very gentle magnetic stirring for 24 to 48 hours at 16°C. Figure 8.11 The Teflon crystallization plate. The technical features of this crystallization plate are described in Levy et al.8

Figure 8.11

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7. Deposit a carbon-coated grid at the surface of the well and wait for two minutes. The grid can be glowdischarged before using it.

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203 8. Pick up the grid and transfer it to a plunge-freezing device. 9. Blot the grid and freeze it in liquid ethane.

2.4. Surface Crystallization 1. Take a cell culture plate and fill the wells with 1 mL of buffer. 2. Deposit 7 to 10 µL of the protein solution onto a siliconized cover slide. 3. If 2-D crystallization occurs in the first hours then a gold grid covered with a carbon film is deposited onto the drop. 4. Apply grease around the crystallization well for sealing. 5. Return the cover slide and seal it onto the well. 6. Incubate the plate at 16°C. For the H+ATPase, 2-D crystals appear within the first two hours of incubation. 7. Remove the cover slide, pick up the grid with standard tweezers and place it into a freezing device. 7’. When 2-D crystallization occurs slowly (>24 hours), the EM grid is only deposited onto the drop after having removed the cover slide. Wait for at least two hours at 16°C and then pick up the grid with tweezers and place it into a plunge-freezing device. 8. Blot the grid and freeze it in liquid ethane.

Figure 8.12 The crystallization plate.

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3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  A freezing device for cryo-samples.

 See Chapters 3, 4, 7

 BioBeads.

 SM2 (BioRad) www.bio-rad.com

 Dialysis devices:

 Any device that can dialyze volumes between 10 to 100 µL.  Bent-glass capillary is homemade. A piece of dialysis membrane is fixed at the short end with a rubber ring or a piece of plastic tubing. These vessels are easy to make, cheap and can be reused. The sticks should be prepared and placed in buffer several hours before filling to check for leaks in the membrane. www.hamptonresearch.com/ www.piercenet.com/

 Bent glass capillary  Dialysis button (Hampton Research)  Slide-a-Lyzer (Pierce)

 Dialysis membrane.

 10 kDa cut-off

 Fixed temperature incubating room.

 16°C is the commonly used temperature.

 Refridgerator.

 Storage of BioBeads, buffers, etc.

 Magnetic stirrers.

 Small magnetic stirrers are used for mixing proteins and lipids. Volumes usually used are in the 50 to 100 µL range.  2 mm × 2 mm magnetic stirrers are used in Teflon wells.

 Teflon crystallization plate.

 The depth of the well allows magnetic stirring. The injection well is used for protein injection.  Drawing and technical features are presented in Levy et al.8

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3.2. Products  Carbon-coated grids.  Gold-coated grids.  Molybdenum grids.

 Diluting lipids.  DOGS-NTA lipid.

 DTAC. (N, N-dodecyl trimethyl ammonium chloride).  Ethane gas.  Liquid nitrogen.

 Vacuum grease.

 Commercially available. For example:  Oxford Instruments www.oxford-instruments.com  E.M.S. www.emsdiasum.com/microscopy/ For molybdenum grids, 300 mesh is the smallest mesh size available  Pacific Grid Tech www.grid-tech.com.  Dioleyl phosphatidyl choline or dioleyl phosphatidyl glycerol for instance  Commercially available from Avanti Polar Lipids (www.avantilipids.com/)  In chloroform/methanol (9:1) (v/v) at 0.5 mg/mL. Synthetic lipids with chelated nickel that can interact with histidinetagged proteins.  DTAC can be supplied by: www.sigmaaldrich.com  Extremely inflammable. Always use ethane in well-ventilated areas.  May cause severe burns; wear protective clothes and gloves for handling LN2; always use LN2 in well-ventilated areas.  The grease is used to seal the cover slide onto the well of the cell culture plate. Can be supplied by: www.hamptonresearch.com



3.3. Solutions  Buffers  100 mM potassium phosphate buffer (pH 5.2) with octyl glucoside (6 mM) and DTAC (200 µM).  For cryoprotection  Glucose  Trehalose  Tannin

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 Used for fusion of purple membranes.

 Usually 2% (w/v) aqueous solution  A 4 to 8% (w/v) aqueous solution  0.5% (w/v) adjusted to pH 6.0 with KOH and clarified by centrifugation.

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4. PROTOCOLS 4.1. Membrane Fusion of Purple Membranes 1. Mix purple membranes in a suspension containing 3 mg/mL in 100 mM potassium phosphate buffer (pH 5.2) with octyl glucoside (6 mM) and DTAC (200 µM).

 Use 100 µL aliquots with slight variations in detergent concentration.  Adding 3 mM sodium azide will prevent bacterial growth.

2. Incubate for several weeks at room temperature. 3. Check for large sheets (10 µm or  2-D crystals of membrane proteins are more) by EM. often cryoprotected by sugars before freezing using the back injection method 4. Prepare crystals for cryo-electron (see Protocols, Section 4.4). microscopy. 

4.2. 2-D Crystallization by Dialysis 1. Mix together in a 10 to 100 µL  volume:    Membrane protein in detergent, about  Lipid/protein ratio: 0.1 to 10 (w/w). The 1 mg/mL. suitable ratio should be determined  Lipid in detergent. empirically.  The final free detergent concentration  Additional detergent, if necessary. has to be above its CMC.  10 to 15% glycerol, if desired.  Addition of glycerol often produces  Add buffer to obtain the final volume. better crystals.   2. Let the mixture equilibrate at room temperature for one hour. 3. Transfer the mixture to one of the  Use slow magnetic stirring. following dialysis devices:  Slide-A-Lyzers.  They have a large surface area, are suitable for 100 µL volumes or more, are very easy to use and a sample can be taken during dialysis.  Bent glass capillary.  The surface area is small, so dialysis is slow.  Dialysis button.  For very small sample volumes (down to 10 µL). They are not easy to handle. The ratio of surface-to-volume is low.

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4. Dialyze against detergent-free buffer  All dialysis installations are placed in 30 to 250 mL dialysis buffer and dialyzed at for several days or weeks. defined temperature, most commonly at 20 5. Check for 2-D crystals by negative to 37°C. staining. 6. Adjust crystallization parameters or  Dialyzing time can vary from two days prepare crystals for cryo-electron to several weeks or months for detergents with a low CMC. microscopy.  Online Fourier transform of charged coupled device (CCD) images can be used to check if 2-D crystals are formed.  See Figure 8.23.  2-D crystals of membrane proteins are often cryoprotected by sugars before freezing using the back injection method (see Protocol, Section 4.4).  Crystal quality can sometimes be improved by incubation at a higher temperature (37oC) for a few hours.

4.3. 2-D Crystallization using BioBeads 1. Mix proteins with lipids as described  (see Section 4.2, Step 1) in a 100 µL volume. 2. Wash BioBeads thoroughly with  BioBeads can be prepared and then methanol and then with water or stored at 4°C. buffer.  To avoid bacterial contamination, BioBeads need to be washed with filtered 3. Discard dry BioBeads. water before use.  4. Deposit a small amount of BioBeads  BioBeads must always remain wet. onto a filter paper. 5. Weigh the desired amount of beads  Use a precision balance. The amount of and add to protein/lipid mixture. BioBeads needed is in the mg range.  The amount of BioBeads needed to remove detergents is described in Rigaud et al.10  If detergent removal is too rapid, the beads can be added in small aliquots every hour. 6. When all the detergent is adsorbed to  Remove the sample from the bottom of the beads, the sample has to be the tube with a pipette. The BioBeads transferred to a new tube. remain in the tube.

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7. Incubate the sample at 16°C. 8. Check for 2-D crystals by EM after negative staining. 9. Adjust crystallization parameters or  2-D crystals of membrane proteins are prepare crystals for cryo-electron often cryo-protected by sugars before microscopy. freezing using the back-injection method (see Protocol below).

4.4. Preparation of Cryo-Samples by the Back-Injection Method 1. 200 µL of sugar solution is put on a  piece of Parafilm (see Figure 8.13).  Figure 8.13 Sugar solution onto Parafilm.  Sugars are used to cryoprotect the specimen.  Glucose, tannin or trehalose can be used.  2. A 3 mm × 3 mm piece of carbon film  on mica is floated off and picked up on a molybdenum grid (see Figure 8.14).

Figure 8.14 Carbon film is picked up on a EM grid.  3. The grid is turned over so that the carbon faces downwards (see Figure 8.15). Figure 8.15 Turning over of the grid. Some liquid remains on the grid (about 3 µL). Excess liquid can be removed from the grid side with a pipette. 1 to 2 µL of the crystal suspension is added to the grid side and mixed with the sugar by pipetting up and down (see Figure 8.16).

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209 Figure 8.16 Deposit of crystal solution on the grid.  The sugar forms a thin layer protecting the extra-membrane part of the proteins.  As the image contrast is very low, defocused diffraction mode can be used to identify 2-D crystals.

4. The grid is put on two sheets of filter paper to blot for 5 to 10 seconds and then frozen by plunging into liquid nitrogen. Figure 8.17 Deposit of the grid on filter paper.  2-D crystals of c-ring proteins are prepared according to this method (see Figures 8.23 to 8.25).

4.5. Lipid Monolayer Crystallization A crystallization plate is placed in a Petri  dish that contains two layers of wet filter paper (see Figure 8.18). .  Wfp = Wet filter papers  Tcp = Teflon crystallization plate  Pd = Petri dish

1.   

 Figure 8.18 Crystallization device.  Volume of the wells is about 50 to 60µL.  Wet filter paper is used to saturate the atmosphere with water vapor and prevent dehydration.  Prepare the Teflon plate.  Wash the plate with detergent, rinse  with distilled water.  Sonicate the plate in ethanol two times  for 15 minutes. Rinse with hot water at least  Hot water (>60°C) is used for removing 30 minutes. traces of ethanol.

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Fill wells with buffer. Place spherical stirrers into wells. Inject the buffer. Remove air bubbles by stirring. Adjust volume to obtain a plane surface.

3. Deposit the lipid monolayer.  The lipid mixture contained in chloroform/methanol solution should be at room temperature.  With a very thin syringe, deposit 0.5 µL lipid mixture that contains the ligand and the diluting lipid in the crystallization plate.  Put the plate in the Petri dish, cover it and incubate at 16°C.

     The surface of the well is observed with a horizontal light.   Take lipids from freezer at least 10 minutes before depositing the monolayer.  Lipid mixture is at 0.5 mg/mL.  In case of His-tagged proteins, the ligand lipid is DOGS-NTA-Ni.  Diluting lipids usually used are DOPC, DOPG and DOPS.  DOGS-NTA-Ni/DOPC ratio between 1:1 and 1:5.  Lipids must be sealed with dry nitrogen to avoid lipid oxidation. 

4. Protein injection.  Adjust volume with distilled water to obtain a perfectly plane surface.  Remove the necessary volume through the injection well.  Inject the protein solution through the injection well.  Check the planarity of the surface of the well. 5. Incubate at 16°C with very slow stirring.  If the crystals are extremely fragile, use gold grids covered with a carbon film and deposit the grid on the monolayer for at least two hours (see Figures 8.26 and 8.27).  If the grid slightly turns when it is deposited on the surface of the monolayer, Glow-discharge the grids. Check the planarity of the lipid this means that protein interactions with the lipid monolayer are weak. surface. Deposit the grid on the monolayer,  If there is no liquid at the surface of the carbon film in contact with the lipids. grid that was in contact with the monolayer, then it means that very few proteins have Wait for two minutes. interacted with lipids.

6. Grids.  Use copper grids (300 to 400 mesh) covered with a thin carbon film or a carbon-collodion film.    

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7. Freeze.  Take the grid with tweezers dedicated for freezing.  Mount the tweezers on the freezing device.  Remove excess liquid with a filter paper and quickly plunge the grid into liquid ethane.  Transfer the grid into a cryo-holder  see Chapter 7. and observe the grid in a cryo-electron microscope.

4.6. Surface Crystallization       Figure 8.19 The cell culture plate.  Use the basic 24-well cell culture plates.  1. Apply vacuum grease around the well.  2. Fill with buffer solution (~ 1 mL) (see Figure 8.20).  G = Grease  B = Buffer Figure 8.20 Grease is applied around the well.  The grease should not be too liquid.  3. Rinse the cover slide with 70%  Handle the cover slip with standard tweezers. ethanol and dry. 4. Add the protein solution containing  Be careful of a charging effect that precipitant agents (~ 7 to 8 µL). repulses the drop. 5. Deposit a carbon-coated gold grid (see Figure 8.21).

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Figure 8.21. The grid is deposited on the drop.  The carbon face is in contact with the drop.  Gold grids are used to avoid oxidation.

6. Carefully turn the cover slip over (see Figure 8.22).

Figure 8.22 The cover slide is turned over.  The drop must stay in the center of the cover slip. 7. Put the cover slide on the well and  For H+-ATPase, 2-D crystallization occurs within two hours. If crystallization press to seal it with grease. needs more time, do not put the EM grid on 8. Incubate at 4°C. the drop immediately. The grid should be placed on the drop two hours before 9. Remove the coverslip with the grid freezing. facing up. 10. Freeze the grid.  Handle the grid with tweezers dedicated for freezing.  Mount the tweezers on the freezing device.  Remove the excess liquid with a filter paper and quickly plunge the grid into liquid ethane.  Transfer the grid into a cryo-holder and observe the grid in a cryo-electron microscope.

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5. ADVANTAGES/DISADVANTAGES 5.1. Using Natural Crystals 5.1.1. Advantages  Membrane proteins are in their natural  Proteins have never been extracted from environment. the membrane.   Membrane proteins often need to interact with specific lipids to be functional.   Possibility of merging crystal patches  Very large crystals. (see Protocol, Section 4.1, 2-D crys-  Suitable for electron diffraction. tallization by membrane fusion of purple membranes). 5.1.2. Disadvantages



 Natural 2-D crystals usually form very  The quality of the crystal is poor. small arrays  Contaminant proteins can be included  This perturbs the periodic arrangement in arrays. in the array. 

5.2. 2-D Crystallization by Dialysis 5.2.1. Advantages



 General method for membrane pro-  Can be tried with any protein. teins   Crystallization parameters accurately controlled.  Detergent removal is slow.

can

be  Very highly ordered crystals can be formed.  Influence of detergent in 2-D crystal formation can be investigated accurately.

5.2.2. Disadvantages  Detergent removal is slow.

 Not suitable for sensitive proteins

 Detergent removal is not efficient for  Detergent concentration in dialysis buffer quickly reaches the CMC. detergent with low CMC.  

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5.3. 2-D Crystallization Using BioBeads 5.3.1. Advantages  Very fast detergent removal.

 Suitable for sensitive membrane proteins.  Possibility to quantify the amount of  Very reproducible. detergent to remove.  Small amounts of detergent can be  Allows accurate control of the added if detergent removal is too fast. detergent/protein/lipid ratio.

5.3.2. Disadvantages  Lipids and proteins can be adsorbed  The hydrophobic surface of BioBeads onto BioBeads. can bind lipids and proteins after a while.  Accurate weighing of BioBeads is  Beads dry very quickly. difficult.

5.4. Lipid Monolayer Crystallization 5.4.1. Advantages  Screening of crystallization conditions  Formation of the first 2-D arrays occurs is very fast. within 6 to 12 hours.  Requires very small amounts of  Usually between 2 to 5 µg protein/well. protein. 5.4.2. Disadvantages  Only one crystallization trial per well.  The lipid monolayer is picked up with an EM grid. 

5.5. Surface Crystallization 5.5.1. Advantages



 A very good alternative to obtain  Proton-ATPase from Neurospora crassa structural data when 3-D crystals are is a good example.5 not well ordered.

5.5.2. Disadvantages  No structural data better than 8 Å has been obtained.  The method is not very general.

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6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. 2-D Crystallization of Membrane Proteins 6.1.1. Natural 2-D crystals  Should be used when possible.  Membrane fusion can be tested with  To increase size of 2-D crystals. different detergents.  6.1.2. Detergent removal by dialysis  The process is time consuming, but  The most general method. usually gives large crystals suitable for electron crystallographic studies.  6.1.3. Detergent removal using BioBeads  Detergent removal by BioBeads  Useful with proteins solubilized in should be used with unstable proteins Triton X100 or dodecyl maltoside. or proteins solubilized with a detergent of low CMC. 

6.2. 2-D Crystallization of Soluble Proteins 6.2.1. Lipid monolayer crystallization  The lipid monolayer approach is ded-  Proteins can be used whatever their size.  Often used with soluble proteins that icated to proteins with a His-tag. aggregate at low concentrations.  The ligand lipid for His-tagged proteins is DOGS-NTA-Ni.  Charged lipids can be used to bind  His-tag is not necessary. proteins to the lipid monolayer by  The charge distribution does not have to electrostatic interactions. be homogeneous.  Positively charged lipid: DOTAP.  Negatively charged lipid: DOPG, DOPE. 6.2.2. Surface crystallization  Surface crystallization should be tried  This method can be used for both when small 3-D crystals appear in the soluble and detergent solubilized membrane drop. proteins.

6.3. Specimen Preparation  Sugar embedding often works well for  To avoid basic problems occurring membrane proteins without large during vitrification. extra-membranous domains.  The back-injection method yields very flat crystals for high-resolution data collection.

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7. OBSERVED RESULTS  Figure on the Chapter’s title page

 Image of negatively stained 2-D crystals of HC-Pro.9

 Figure 8.23 2-D crystal of ATPsynthase c-ring formed by detergent dialysis (see Protocol, Section 4.2), negatively stained with 1% uranyl acetate diluted in water. The crystal is in a closed tubular vesicle. The inset shows the crystalline structure (superposition of the two sides of the vesicle). Size of the inset = 300 nm   

 Figure 8.24 Fourier transform of an image of a similar crystal, cryoprotected in 4.5 % trehalose and prepared by the backinjection method (see Protocol, Section 4.4).

Figure 8.25 Projection map of this crystal calculated at 5 Å resolution. Image processing was performed using the MRC package.4 Bar = 2 nm



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217      Figure 8.26 2-D crystal of HC-Pro obtained by the lipid monolayer crystallization method (see Protocol, Section 4.5). A carbon-coated copper grid was applied for two minutes on the lipid monolayer and negatively stained with 1% uranyl acetate diluted in water. The 2-D crystals are extremely fragile and have fragmented due to surface tension.      Bar = 100 nm     

 Figure 8.27 A gold grid was used to prepare HC-Pro 2-D crystals. Two hours incubation of the grid on the drop prevents breakage of the 2-D crystals, which are now suitable for structural investigation. Gold is used to prevent grid oxidation during incubation.9

Bar = 100 nm

 

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8. REFERENCES

1. 2. 3.

4. 5. 6. 7.

8. 9. 10. 11. 12.

Booy, F.P. and Pawley, J.B. Cryo-crinkling: What happens to carbon films on copper grids at low temperature, Ultramicroscopy, 48, 273, 1993. Bron, P. et al. The 9 Å projection structure of cytochrome b6f complex determined by electron crystallography, J. Mol. Biol., 287, 117, 1999. Chiu, W., Avila-Sakar, A.J., and Schmid, M.F. Electron crystallography of macromolecular periodic arrays on phospholipid monolayers, Adv. Biophys., 34, 161, 1997. Crowther, R.A., Henderson, R., and Smith, J.M. MRC image processing programs, J. Struct. Biol., 116, 9, 1996. Cyrklaff, M. et al. 2-D structure of the Neurospora crassa plasma membrane ATPase as determined by electron cryomicroscopy, Embo J., 14, 1854, 1995. Gonen, T. et al. Lipid-protein interactions in double-layered two-dimensional AQP0 crystals, Nature, 438, 633, 2005. Kühlbrandt, W. Two-dimendional crystallization of membrane proteins: A practical guide, in Membrane Protein Purification and Crystallization, Hunte, C., Von Jagow, G., and Schagger, H., eds., Academic Press, NY, USA, 2003. Levy, D. et al. Two-dimensional crystallization on lipid layer: A successful approach for membrane proteins, J. Struct. Biol., 127, 44, 1999. Plisson, C. et al. Structural characterization of HC-Pro, a plant virus multifunctional protein, J. Biol. Chem., 278, 23753, 2003. Rigaud, J.L. et al. Bio-Beads: An efficient strategy for two-dimensional crystallization of membrane proteins, J. Struct. Biol., 118, 226, 1997. Vonck, J. Parameters affecting specimen flatness of two-dimensional crystals for electron crystallography, Ultramicroscopy, 85, 123, 2000. Walz, T. and Grigorieff, N. Electron crystallography of two-dimensional crystals of membrane proteins, J. Struct. Biol., 121, 142, 1998.

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Cryo-Negative Staining

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CONTENTS

1.

PRINCIPLES OF CRYO-NEGATIVE STAINING....................................... 223

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 223

3.

MATERIALS AND PREPARATION ............................................................. 224 3.1. 3.2. 3.3.

4.

PROTOCOL ...................................................................................................... 226 4.1. 4.2. 4.3. 4.4. 4.5. 4.6. 4.7.

5.

Principles .................................................................................................. 224 Useful Stains............................................................................................. 224 Materials/Small Items/Products/Solutions................................................ 225 3.3.1. Materials ..................................................................................... 225 3.3.2. Small items ................................................................................. 225 3.3.3. Products ...................................................................................... 225 3.3.4. Solutions ..................................................................................... 225

Prepare the Staining Solution ................................................................... 226 Pipet the Sample on the Grid .................................................................... 226 Prestaining Step ........................................................................................ 227 Staining (Dialysis) .................................................................................... 228 Sample mounting...................................................................................... 228 Vitrification .............................................................................................. 229 Transfer..................................................................................................... 230

ADVANTAGES/DISADVANTAGES.............................................................. 231 5.1. 5.2.

Advantages ............................................................................................... 231 Disadvantages........................................................................................... 232

6.

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 233

7.

OBSERVED RESULTS .................................................................................... 234

8.

REFERENCES .................................................................................................. 236

 

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1. PRINCIPLES OF CRYO-NEGATIVE STAINING The only additional step in cryo-negative  The staining solution used is ammonium staining compared to thin-film vitrification molybdate (see below). (see Chapter 3) is a short time of contact between the staining solution and the sample. What happens to the sample during the short time of contact? The biological sample is maintained in a low-salt, physiological buffer. During this step, the ammonium molybdate mixes with the sample buffer. The difference in salt concentration of the two solutions equilibrates so that the biological particles become suspended in the high-salt medium without being diluted in the staining droplet.

 When prepared with this staining technique, the resulting density of the vitrified ammonium molybdate is higher than the density of the protein (without taking into account hydration water).That is why the biological samples appear white in a dark background in the electron micrographs of a cryo-negatively stained preparation.

Figure 9.1 Schematic view of the steps in sample preparation. (Reprinted from Adrian et al. 19981 with permission from Micron.)

2. SUMMARY OF THE DIFFERENT STEPS 1. 2. 3. 4. 5. 6. 7.

Preparing the staining solution Pipetting the sample on the grid Prestaining step Staining (dialysis) Sample mounting Vitrification Transfer

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3. MATERIALS AND PREPARATION 3.1. Principles In the case of cryo-negative staining, the staining solution is used to increase the contrast and decrease the electron beam  Contrast is measured in terms of signalsensitivity of partially hydrated biological to-noise ratio. samples. Unlike conventional air-drying negative staining, the samples are still in solution, where the aqueous buffer surrounding the biological particles is replaced by a higher density, heavy metal salt. The staining solution, composed of ca. 1.2 M salt (saturated solution of ammonium molybdate), has a higher density than the protein. This results in a contrast inversion as observed by electron microscopy.

 The final state of hydration of the sample depends on blotting time with filter paper.  The time taken for blotting depends on the relative humidity of the surrounding sample environment.

Figure 9.2 The chaperonin GroEL visualized by cryo-EM. (Left: 1.4 m defocus) as compared to cryo-negative staining (right: 0.5 m defocus). (Reprinted from De Carlo et al. 20023 with permission from Elsevier.) Bar = 50nm Between the staining and the vitrification  The gain in contrast strongly depends step, the sample IS NOT DRIED AT ALL. on the thickness of the vitreous ice layer and on the distribution of the stain surrounding the biological particles. 

3.2. Useful Stains Ammonium molybdate so far is the only heavy metal salt that can be used in a reproducible way to successfully achieve cryo-negative staining. Other typical heavy metal salts often used in conventional air-drying negative staining, such as uranyl acetate, phosphotungstic acid (PTA), do not produce a marked increase in contrast.

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 In some cases, a contrast increase obtained after mixing a 1 to 2% PTA solution with the sample, prior to vitrification, has been reported in the literature.2

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When used at 1 to 2% (w/v), no significant contrast increase is observed. When used at a higher concentration, the salt precipitates and no useful results can be obtained.

 Another technique also known as the “sandwich method” uses uranyl formate (0.5 to 2%) in addition to glycerol (>10 to 20%) prior to plunging in liquid nitrogen.4

 Ammonium molybdate tetrahydrate  Can be purchased from different (H24Mo7N6O24.4H2O; MM = 1235.86) companies.  Has an acidic pH (4.5) if not buffered.  The volumetric density of the final staining solution, after buffering with sodium hydroxide (NaOH,) is 2.35 g/cm3. 

3.3. Materials/Small Items/Products/Solutions 3.3.1. Materials  Balance.  Magnetic mixer and heater.  Pyrex container (100 mL).

 Used to dissolve 40g of NaOH 1 M in 100mL water.  For storage of the NaOH 10 M solution.

3.3.2. Small items  Bulbs, pasteur pipettes.  Electron microscopy forceps.  EM grid.  Filter paper.  Glass/plastic Petri dish.  Gloves, mask.  Micro-pipettes.  Parafilm foil.

 Does not need to be sterile.  Needs to be cleaned before and after usage.  Holey or continuous carbon already mounted.  No need of specific type.  Does not need to be sterile.  Indispensable (NaOH is highly concentrated and ammonium molybdate is toxic).

3.3.3. Products  Ammonium molybdate tetrahydrate.  NaOH

3.3.4. Solutions  Water.

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 From Fluka, Buchs, Switzerland or Sigma, Saint Louis, Missouri, USA.  1 M stock solution.

 Do not use deionized water.

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4. PROTOCOL 4.1. Prepare the Staining Solution 1. Weigh 1.0 to 1.2 g of ammonium   Fluka, Sigma molybdate tetrahydrate. 2. Add 0.9 mL water. 3. Shake.

 It is a saturated solution, some powder will remain and form a slurry.

4. Add 0.1 mL NaOH 10 M. 5. Shake.        Figure 9.3 Keep the ammonium molybdate solution in a vertical position (slurry on the bottom) and use only the supernatant. 

4.2. Pipet the Sample on the Grid 1. The EM grid can be previously glowdischarged if needed.



2. The carbon film can be continuous or holey depending on your needs. 3. Avoid plastic (e.g., Formvar) on grids. If still present, dissolve plastic with ethyl-acetate/chloroform prior to experiment.

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   See Chapter 3. 

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4. Typically use 3 to 5 µL of sample on  the EM grid, let the sample adsorb for  a 10 to 30 seconds if it is a continuous  carbon film.



       Figure 9.4 Make sure the sample droplet is evenly distributed on the grid.   

4.3. Prestaining Step 1. Prepare a Petri dish (plastic or glass)  with cover. 2. Put a square piece of Parafilm paper in the Petri dish. 3. Deposit a 80 to 100 L droplet of the staining solution on parafilm paper.         Figure 9.5 Use a new stain droplet each time.    

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4.4. Staining (Dialysis) 1. Place the grid on the staining solution  Place sample face down, toward the droplet. stain.           Figure 9.6. Sample facing the stain droplet.  2. Remove the grid after 10 to 60  seconds, depending on the sample  sensitivity to highly concentrated  salts. Longer times of contact allow better stain distribution.         Figure 9.7 Make sure the grid does not sink. 

4.5. Sample mounting Mount the specimen on the plunge-freezing  apparatus as fast as you can to avoid further water evaporation, or use commercially available units that allow environmentcontrolled vitrification.  E.g., Vitrobot, FEI Company, see Chapter 4.

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                  Figure 9.8 Tweezers mounted on the plunger.  

4.6. Vitrification 1. Apply filter paper to remove excess liquid.



        Figure 9.9 Apply the filter paper directly on the grid (one-sided blotting shown here).   2. Remove filter paper and wait 1 to 3  seconds before plunging the sample in the cryogen.

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         Figure 9.10 Wait 1 to 2 seconds before releasing the plunger.         Figure 9.11 Vitrification occurs as the grid falls gradually into liquid ethane previously cooled at liquid nitrogen temperature.



4.7. Transfer 1. Transfer the grid from liquid ethane to liquid nitrogen. 2. Make the transfer as fast as possible. Avoid warming up the sample. Devitrification occurs at 135°C.         Figure 9.12 Transfer the grid gently to the grid box.

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3. Transfer the grid to the appropriate   storage box.           Figure 9.13 Make sure the grid box lid is tightly closed before storage.   4. Transfer the sample to your cryo-  workstation and prepare to transfer to  your usual specimen holder for cryo-  microscopy (cold stage).  

5. ADVANTAGES/DISADVANTAGES  Cryo-negative staining is a practical method that combines negative staining and electron cryomicroscopy.5,6 The biological samples are imaged with a high signal-to-noise ratio (contrast) provided by the surrounding heavy metal while maintaining the advantages of the frozen-hydrated state (for a comparison, see Chapter 3).

5.1. Advantages  The signal-to-noise ratio (contrast, object visibility) is dramatically increased. 

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 This technique is very useful to increase the signal-to-noise ratio (SNR) of small biological complexes that are hard to visualize in standard cryo-EM because of their relatively small size.

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 The sensitivity of the biological  It has been shown that samples samples exposed to the electron beam preserved in saturated ammonium is decreased. molybdate can withstand a three-fold increase in the exposure to the electron beam (as demonstrated by imaging GroEL at 1.5 nm resolution with a total electron dose of ~ 3600 electrons/nm2; (see Figure 9.16 from De Carlo et al. 2002 3).

Figure 9.14 Fourier shell correlation (FSC) plots. The FSC are represented with the following labels: LowD (low-dose, 1000 e/nm2), HighD (high-dose, 3000 e-/nm2), water (unstained particles), and Stain (cryonegatively stained particles). The corrected 3 FSC criterion is also represented “FSC Crit”). (Reprinted from De Carlo et al. 20023 with permission from Elsevier.)

5.2. Disadvantages  Partial evaporation.

 Removal of excess buffer (partial dehydration) is necessary to obtain a thin film prior to vitrification.

 Sensitivity to salt.

 Some macromolecular assemblies do not withstand the high salt concentration and may fall apart.

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233

6. WHY AND WHEN TO USE A SPECIFIC METHOD As mentioned in this chapter, the main advantage of the technique is an increased signalto-noise ratio when compared to unstained cryo-electron microscopy. Many enzymes of fundamental importance in cellular processes are in the range of 200 kDa, and this represents the limit of molecular cryo-EM in terms of particle size amenable to image processing. With an increased signal-to-noise ratio provided by the ammonium molybdate stain, we were able to reconstruct the 3-D model of a human transcription factor whose size is only 120 kDa7 provided the sample can stand 0.8 M salt due to the ammonium molybdate and does not fall apart; this is a method of choice to get better contrast in cryo-EM. Ammonium molydbate is the only useful stain with this technique. If you need to use uranyl acetate or uranyl formate, still widely and successfully used in many EM labs, the cryo-negative staining sandwich method developed by Golas and colleagues5 will be very useful, especially if the sample is stored in 20 to 30% glycerol and glycerol is needed for complex stability.

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7. OBSERVED RESULTS  Figure 9.15 

 Tomato Bushy Stunt Virus, as observed unstained by cryo-EM (left) or by cryo-negative staining in a saturated solution of ammonium molybdate (right).4 Bar = 50nm (Reprinted with permission from M.A. Hayat. Principles and Techniques of Electron Microscopy: Biological Applications. 4th ed., Cambridge University Press, Cambridge, U.K., 2000.)   

 Figure 9.16

  Cryo-electron micrographs of GroEL  (a–c) Unstained vitrified native GroEL solution.  (d–f) Cryo-negatively stained preparation with saturated ammonium molybdate.

Bar = 50 nm

The total cumulative dose is  1000 electrons/nm2 in (a) and (d)  2000 electrons/nm2 in (b) and (e)  3000 electrons/nm2 in (c) and (f).3  (Reprinted from De Carlo et al. 2002,3 with permission from Elsevier.)

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8. REFERENCES 1.

Adrian, M. et al. Cryo-Negative Staining, Micron, 29, 145, 1998.

2.

Böttcher, C. et al. Structure of influenza haemagglutinin at neutral and at fusogenic pH by electron cryo-microscopy, FEBS Letters, 463, 255, 1999.

3.

De Carlo, S. et al. Cryo-negative staining reduces electron-beam sensitivity of vitrified biological particles, J. Struct. Biol., 138, 216, 2002.

4.

De Carlo, S. Cryo-negative staining: Advantages and applications for threedimensional electron microscopy of biological macromolecules. PhD thesis, University of Lausanne, 2002.

5.

Golas, M.M. et al. Molecular architecture of the multiprotein splicing factor SF3b. Science, 300, 980, 2003.

6.

Harris, J.R. Negative Staining and Cryoelectron Microscopy. BIOS Scientific Publishers Limited, Oxford, UK, 1997.

7.

Jawhari, A. et al. Structure and oligomeric state of human transcription factor TFIIE. EMBO Rep., 7, 500, 2006.

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Vitrification of Dynamic Microtubules

239

CONTENTS

GENERAL INTRODUCTION .................................................................................... 241 1.

PRINCIPLE OF THE METHOD .................................................................... 242

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 243

3.

MATERIALS/PRODUCTS/SAFETY PRECAUTIONS................................ 244 3.1. 3.2. 3.3.

Materials ................................................................................................... 244 Products .................................................................................................... 248 Safety Precautions .................................................................................... 249

4.

PROTOCOL ...................................................................................................... 250

5.

ADVANTAGES/DISADVANTAGES/POSSIBLE IMPROVEMENTS ....... 253 5.1. 5.2. 5.3.

6.

Advantages ............................................................................................... 253 Disadvantages........................................................................................... 254 Possible Improvements............................................................................. 254

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 255 6.1.

6.2.

Self-Assembly Versus Nucleated Assembly ............................................ 255 6.1.1. Microtubule self-assembly.......................................................... 255 6.1.2. Centrosome nucleated assembly ................................................. 255 6.1.3. Axoneme nucleated assembly..................................................... 255 Incubation in Tubes or on Grid................................................................. 255 6.2.1. Incubation in tubes...................................................................... 255 6.2.2. Incubation on grid ....................................................................... 255

7.

OBSERVED RESULTS .................................................................................... 256

8.

REFERENCES .................................................................................................. 258

 

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GENERAL INTRODUCTION

Microtubules are dynamic polymers built from the  tubulin heterodimer and associated proteins collectively called MAPs (for microtubule-associated proteins). Tubulin, from most organisms, is soluble at 4°C and assembles into microtubules when the temperature is raised (e.g., to 37°C) in the presence of guanosine-5´-triphosphate (GTP). Therefore, a preparation of frozen-hydrated microtubules in their dynamic state requires that the temperature and humidity of the specimen be regulated before freezing in liquid ethane. In this chapter, we describe a simple environmental device that has been used to study the structure of microtubule ends during assembly and disassembly.1-4 This device can also be used with any kind of specimen that necessitates regulation of its environmental conditions.

 CT = Centrosome  MT = Microtubule

Figure 10.1 Microtubules assembled from purified tubulin and nucleated by an isolated centrosome, observed by videoenhanced differential interference contrast light microscopy.3 Microtubules displaying dynamic instability are either growing (white arrow) or shrinking (black arrow).

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1. PRINCIPLE OF THE METHOD The aim is to vitrify dynamic microtubules in a state as close as possible to the one they had in solution before freezing. A small aliquot of the suspension is deposited on a holey carbon grid held by tweezers fixed to a guillotine device over a small cup filled with liquid ethane at ~ 180°C. Most of the specimen is removed using a filter paper so that the remaining suspension forms a thin layer of about 50 to 100 nm thickness spanning the holes of the carbon film. The grid is rapidly plunged into liquid ethane by releasing the mobile arm of the guillotine device, and is transferred to the cryo-stage of the electron microscope or stored in liquid nitrogen for further use.

 Without regulation of humidity, the solution evaporates, which induces an increase in the overall salt concentration.  The temperature drops as a consequence of evaporation.5 This can be easily monitored using a thin thermocouple immersed in the specimen while it sits on the electron microscope grid.  To limit the extent of these artifacts, it is necessary to regulate both the temperature and humidity around the specimen before freezing.

 Note that warming only the air around the specimen without saturating the For most structural studies where one wants atmosphere with water vapor will result to have the overall structure of a only in increased evaporation and, thus, macromolecule, the steps described above increased cooling of the specimen. may appear sufficient. However, when one wants to relate structural features to dynamic events, such as conformational changes or a polymerization process, additional care should be taken to preserve the environmental conditions during specimen preparation, because its exposure to the ambient atmosphere may induce several significant artifacts.

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2. SUMMARY OF THE DIFFERENT STEPS

1. Equilibrate the environmental device to the desired temperature.

2. Incubate the specimen on a grid or in a test tube.

3. Blot the specimen.

4. Freeze in liquid ethane.

5. Store in liquid nitrogen or observe in the electron microscope. Figure 10.2 Summary of the different steps to prepare frozen samples under controlled temperature and humidity conditions.

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3. MATERIALS/PRODUCTS/SAFETY PRECAUTIONS 3.1. Materials   Chemical Hood

  Necessary, to reduce airflow and evacuate gaseous ethane and nitrogen if required. No electrical or electronic apparatus that may produce sparks should be placed in this area. An explosion-proof hood is preferred. A = Two-liter water flask warmed by the heating mantle B = Temperature controller C = Guillotine device D = Tubing output near specimen grid E = Polystyrene box with small metallic cup F = Ethane bottle Figure 10.3 Open environmental device.

 Two-liter round-bottom flask with two  Fill 2/3 with distilled water. or three necks (see Figure 10.4D).   Should be equipped with a separate  Heating mantle temperature controller located outside the hood (see Figure 10.7). A = Tubes coming from the air compressors B = External temperature controller probe C = Thermometer to record the temperature inside the flask D = Two-liter round-bottom bottle flask E = Tube between the water flask and the reservoir Figure 10.4 Source of humid and warm air.

  Thermometer 

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 Used to record the temperature inside the flask (see Figure 10.4C).

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 Tubing

 Two pieces ~ 80 cm and 15 cm in length, and ~ 1.5 cm internal diameter, connected by a Y-shaped connector (see Figures 10.4E and 10.5B).  Used to collect condensed water from the tubes. An Erlenmeyer flask may be suitable.

 Reservoir

A = Tube coming from the two-liter water flask B = Tube going to the guillotine device C = Erlenmeyer flask

  Plastic tube 

  Compressed air

Figure 10.5 Reservoir to collect condensed water.    A 10 × 2 cm tube placed at the output of the tubing system. A 15 mL Falcon tube cut at its base will work just as well. A tiny hole can be drilled at its extremity to install a thin thermocouple to monitor the airflow temperature (see Figure 10.6B). A = Thermocouple plunged into the 4 L sample B = Thermocouple that records the output temperature C = Output tube  Figure 10.6 Device output.    Be careful of oil contamination when using oil-lubricated air compressors; use air filters if necessary. Alternatively, use a nitrogen bottle (it might empty quickly) or small compressors used to inject air in aquariums (we use two of them). A pressure regulator might be needed to finely regulate the airflow. A = Air compressors B = External temperature regulation of the heating mantle Figure 10.7 Compressors and heating  mantle.

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 Guillotine device

 This can be purchased from various companies or be homemade. We use a model that has been designed and built at the EMBL workshop in Heidelberg. Modifications of the environmental device presented here might be necessary if other types of guillotines are used. A = Mobile arm B = Gallows C = Tweezers 

 Thin thermocouple



Figure 10.8 Guillotine device.   Used to record the temperature inside the specimen droplet. A 0.25 mm diameter chromel-alumel thermocouple can be used. A second thermocouple can be fixed on the plastic tube to record the output temperature (see Figure 10.6B). A = Tweezers B = Thermocouple C = Four microliter sample on holey carbon-coated grid. Figure 10.9 Sample temperature recording.

 Tweezers

 Grids

 Several pairs of tweezers can be used in a single study. Electronic tweezers covered with plastic except at their tip may be used to reduce water condensation and to facilitate manipulation at liquid nitrogen temperature. We use short tweezers (A) for the plunging and long tweezers (B) for manipulating the grids inside liquid nitrogen.  A = Tweezers used for plunging B = Tweezers used for manipulating specimen grids inside liquid nitrogen Figure 10.10 Tweezers of electronic quality.   Use 100 to 400 mesh-size grids covered with holey carbon films. Holey carbon grids can be purchased or made according to published protocols (see Chapters 3, 7 or Chrétien et al.5). Figure 10.11 Holey carbon grid square observed by light microscopy.

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 Filter paper

 Whatmann n° 1 or n° 4 depending on the viscosity of the suspension. Small rectangular pieces can be prepared in advance (~ 1 × 3 cm) and folded at a right angle to facilitate blotting. Figure 10.12 Filter paper.   Must be cut at the tip if the microtubules become very long, and prewarmed if needed.   Used to store liquid ethane. Must be cooled by liquid nitrogen. Solidification of the ethane may arise at liquid nitrogen temperature. This can be reversed by touching the solid ethane with a metallic object (e.g., a scalpel handle). A = Polystyrene box covered inside with aluminum foil and filled with liquid nitrogen B = Small metallic cup filled with liquid ethane

  Cone tips

 Metallic cup



 Polystyrene boxes (2)

 Small electron microscope boxes

  Liquid nitrogen tank 

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Figure 10.13 Polystyrene box and metallic cup.   One used to cool the ethane cup (see Figure 10.13A) and another to transfer the specimen grid inside small boxes under liquid nitrogen. Can be covered inside with aluminum foil to reduce liquid nitrogen bubbling.   These can be prepared from rectangular boxes commercially available. A round piece is cut so as to include four grid positions. A small Plexiglas lid is designed that covers three positions and a thread is drilled at their center to screw the lid. A = Storage box B = Box (~ 1.3 cm in diameter) C = Plastic screw D = Plexiglas lid Figure 10.14 Storage box.   Used to store the grid boxes. 50 mL Falcon tubes can be used to store several round boxes (drill a hole in the cap to allow air evacuation during cooling).

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3.2. Products  Ethane

To cryofix the samples. Always have a fire extinguisher at hand.   To liquefy ethane. Keep free of ice contamination.

 Liquid nitrogen  Tubulin

 Tubulin is commercially available or can be isolated from a variety of organisms and cell types. The most convenient source of tubulin is mammalian brain (e.g., calf or pig brain) because this organ is rich in microtubules that direct vesicular fluxes within axons and dendrites. Purification of brain tubulin relies on its ability to polymerize into microtubules at 37°C and to be soluble at 4°C. Several rounds of polymerization at 37°C and depolymerization at 4°C followed by ultracentrifugation give rise to a fraction enriched in tubulin and MAPs (2X or 3X tubulin depending on the number of cycles). Tubulin is globally negatively charged near neutral pH and can be further separated from positively charged MAPs by ionexchange (phosphocellulose) chromatography.6 

 Axonemes

Figure 10.15 Tubulin molecule.7  Several protocols can be found in the literature that allow purification of axonemes from various organisms and cell types, such as seaurchin sperm8 or the protozoa Chlamydomonas. In contrast to centrosomes, large quantities of axonemes can be easily obtained at a high concentration. The protocol involves demembranation of the axonemes and removal of the molecular motors (dynein) that induce flagella or ciliary beating. Figure 10.16 Axoneme observed by cryoEM (Chrétien D., unpublished observations).



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

Vitrification of Dynamic Microtubules

 Centrosomes



249  Centrosomes can be purified from different cell types, but a concentration of ~ 1.108/mL after dilution with the tubulin sample should be reached to facilitate their visualization by cryo-EM. A protocol for the purification of centrosomes from the KE-37 lymphoblastic cell line has been described that gives rise to this concentration range.9 The KE-37 cells grow in suspension and display the special feature of a high ratio of nuclear to cytoplasmic volume, which provides centrosome-enriched cytosolic fractions after cell lysis. Centrosomes are further purified on a discontinuous sucrose gradient, but sucrose does not seem to influence the assembly properties of the microtubules nor does it cause major problems with their visualization by cryoEM. Figure 10.17 Centrosome observed by cryo-EM. (Reprinted with permission from Elsevier.9)



3.3. Safety Precautions  Ethane

 Liquid nitrogen

 Tubulin

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 Avoid placing any electrical apparatus inside the hood. A 5 mL cup of liquid ethane represents a volume of ~2 L gaseous ethane at room temperature, and may produce a large explosion. A fire extinguisher must be placed close to the hood where the guillotine device is installed.   Follow all safety instructions when handling LN2. In particular, wear goggles and trousers during specimen preparation.   Tubulin is isolated from mammalian brain, such as cow, so the risk of prions being present in such samples cannot be totally neglected. Be careful to avoid piercing your fingers with tweezers that have been in contact with the tubulin preparations.

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4. PROTOCOL  Small variations in temperature (+/-1°C) can be recorded. For tubulin, it is safer to  Let the device equilibrate before work at an average temperature of 35°C to avoid denaturation. starting an experiment The water flask must be heated above – 30 min  Check that the temperature inside the this temperature (e.g., 45°C) because droplet (on a blank specimen) and cooling of the air will arise during its travel into the tubing system. beside it are identical. 1. Environmental device preparation

2. Preparation microtubules

of

self-assembled  Microtubule self-assembly requires two main steps: Nucleation and elongation.

 Prepare tubulin at a concentration above ~ 20 M in the presence of GTP. – 10 min at 4°C  Incubate in a test tube or directly on the grid (see below, Steps 5 and 5’). – Variable time at 35 °C

3. Preparation of centrosome-nucleated microtubules  Mix centrosome and tubulin solutions to get a final centrosome concentration of ~ 1.108/mL.

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Nucleation is an energetically unfavorable step and requires a critical concentration (~ 20 M) that must be reached before elongation can proceed.   Various buffers can be used to assemble tubulin, but they share some common features. Microtubule assembly is efficient at pHs just below neutrality (6.8 ~ 6.9). Buffers below or above this pH range may induce the formation of other polymeric forms (sheets, ribbons, double-walled microtubules). Magnesium ions and GTP should be present unless other polymerization-promoting agents are added to the reaction mixture; such as the antimitotic drug Taxol®. Calcium ions are very efficient at depolymerizing microtubules and ethylene glycol tetra-acidic acid (EGTA) should always be present in the assembly buffer (note that calcium can be released from the filter papers used for blotting the specimens). Some compounds such as glycerol can also be used to facilitate microtubule assembly, but these can be difficult to use with cryo-EM of vitrified samples because their density may match that of proteins and, hence, decrease the contrast present in the images.  Centrosomes nucleate microtubules at ~ 5 M tubulin. Most microtubules will start to assemble synchronously, which facilitates observation of their extremities by cryo-EM.

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251

 The elongation rate is concentration dependent (~ 0.2 mM-1min-1) and, thus, this parameter can be adjusted to control the length of the microtubules at a given assembly time.  Incubate in a test tube or directly on  Same protocol as with axonemes. the grid (see below, Steps 5 and 5’). – Variable time at 35°C – 10 min at 4°C

4. Holey carbon grids  Choose grids with large holes (~ 10 m) to observe microtubule asters.  Glow discharge lightly for a short time (light pink discharge). – 10 to 30 sec

 See Chapters 3, 7 and Chrétien et al.5  Grids with mesh sizes of 200 or 300 may be preferred.  This avoids droplet spreading on both sides of the grid, which can be critical if long incubation times are required. 

5a. Incubation in tubes  Pipette a 4 L specimen and apply on the grid held by the guillotine device.      Blot and release the mobile arm of the guillotine device.

 Cut off cone tips to avoid breakage of the microtubules if they become too long.  Preincubate cone tips in buffer at the desired temperature.  The temperature inside the specimen can be rapidly checked using the small thermocouple if desired.  Microtubule suspensions can become very viscous if one works at high tubulin concentrations or in the presence of polymerization-promoting factors, such as MAPs. A rapid filter paper, such as Whatmann n° 4 (or equivalent), might be needed to blot the specimen.   With some practice, time periods as short as a few seconds after the beginning of incubation can be reached.  For diluted suspensions, such as centrosome-tubulin mixtures, a slow-rate filter paper (e.g., Whatmann n° 1) can be used.  In practice, time periods up to 3 min of incubation at 35°C can be performed on the grid. Beyond this time period, water tends to condense onto the tip of the tweezers and to fall inside the specimen, resulting in its dilution. To overcome this effect, it may be necessary to also warm the tweezers (see Section 5.3).

5b. Incubation on the grid  Same steps as above.

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5. Specimen storage  Studying a polymerization process  Transfer specimen grids into small involves preparation of several grids at round boxes (see Figure 10.14). different assembly times, and eventually in different assembly conditions (e.g., different tubulin concentrations). Hence, it is convenient to store the specimen grids in a liquid nitrogen tank until their observation by cryo-EM.  Care should be taken to monitor the liquid nitrogen level from time to time and to avoid water contamination of the nitrogen that may induce crystal formation on the grid.  6. Electron microscope observation  Under these high defocus conditions, the microtubules are easily visualized and the under low dose conditions quality of the ice can be monitored  Search specimen at low magnification (presence of ice contaminants, cubic or (2000 to 3000 ×), with the beam hexagonal ice, ethane contamination (see spread as much as possible and under Chapter 7).  high defocus (10 to 15 m).  Purified centrosomes without microtubules or after short assembly times may be difficult to find because they tend to localize at the edges of the carbon holes where the ice layer is thicker. This becomes less critical after longer times of assembly because the microtubules help to localize the centrosomes inside the holes. Selfassembled microtubules tend to span the holes of the carbon, and visualization of  their extremities is more difficult.  Blank the electron beam from the  For those interested in fine structural specimen. details (e.g., for three-dimensional  Take pictures. reconstructions), a magnification of ~ 60,000 × may be required. However, for those who wish to take pictures of the whole microtubule asters with a chance of visualizing several ends in the same image, magnifications as low as 20,000 and ~ 1.5 m defocus can be used.  

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5. ADVANTAGES/DISADVANTAGES/POSSIBLE IMPROVEMENTS 5.1. Advantages  Open design 

 Modularity

 Economical

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 More sophisticated devices than the one presented here can be used to control the environmental conditions around the specimen when it sits on the electron microscope grid. However, these devices are closed and require an automatic system to blot the specimen (see Chapter 4). These are certainly very well suited for specimens of constant viscosity, but seem to us more difficult to use with a polymer such as microtubules whose viscosity changes with time and assembly conditions. We find that it is easier to blot the specimen manually, which provides — with a little practice — a good feeling of the viscosity of the suspension.   Can be easily modified to suit one’s needs. We have used this device to observe microtubules polymerized at 35°C,3 and also near room temperature (~ 23°C) with a very light heating of the water balloon.1 Under these conditions, the device avoids evaporation and cooling of the specimen. We found that formation of the thin layer of suspension was facilitated and more reproducible, probably because it remains in equilibrium with the humid atmosphere for a longer time than when exposed to ambient atmosphere. We have also used a similar device, but with a flow of cold air generated by a reservoir installed into a cooler.4 Addition of these two apparatuses may be used to perform fast temperature jumps. 

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5.2. Disadvantages  Water condensation on the tweezers.

 The setup presented here can only be used for short reaction times (i.e., up to three minutes on the grid) because water tends to condense on the tweezers and to dilute the specimen.

 Specimen exposed to ambient  The specimen remains exposed to the atmosphere during plunging. ambient atmosphere during its plunge into liquid nitrogen. Although this is a very fast process (a few tenths of a millisecond), we cannot exclude some molecular rearrangements before it is cryofixed.  However, comparison of the structure  of the ends of growing microtubules with those of cold-induced depolymerized ones revealed that this device is well suited for visualizing slow conformational changes such as those involved in the assembly and disassembly of microtubules. 

5.3. Possible Improvements  Regulation of the tweezers’ tem-  It may be advantageous to design a system that warms the tweezers at the same perature. temperature as that of the specimen to avoid water condensation.   It would be convenient to have special tweezers whose tips would act as thermocouples to monitor the temperature of the specimen when it sits on the grid as well as its temperature history during plunging into liquid ethane.   Automatic regulation of the tem-  We have tried to regulate the temperature of the heating mantle using a perature. temperature controller linked to the thermocouple that records the droplet temperature, but the inertia of the system is such that this induced too large a fluctuation of the output temperature.  Thermocouple.

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6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Self-Assembly Versus Nucleated Assembly 6.1.1. Microtubule self-assembly  

 Assembling microtubules in a test tube is easy as long as the tubulin concentration is above ~ 20 M (~ 2 mg/mL). Ends may be difficult to observe, and the polarity of the microtubules must be determined to differentiate plus and minus ends (see Chrétien et al.10). 

6.1.2. Centrosome nucleated assembly  



 All microtubules start to assemble at the same time.  Only one type of end is observed, because the minus end is anchored at the centrosome, while the plus end is growing away from it.  Centrosome preparation necessitates two to three weeks of cell culture followed by one day of purification. 

6.1.3. Axoneme nucleated assembly  

 Large quantities can be obtained in a single day from sea urchin sperm.   Although the plus end grows more rapidly than the minus end, differentiating them in the cryo-EM images may be difficult.



6.2. Incubation in Tubes or on Grid 6.2.1. Incubation in tubes  

 Easy to perform.  Microtubule suspensions can become very viscous (use fast-rate filter paper).

6.2.2. Incubation on grid 

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 Gives access to the initial reaction times.

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7. OBSERVED RESULTS   Figure 10.18 Microtubules nucleated by a centrosome in the presence of purified tubulin. Note the presence of the long outwardly curved tubulin sheets at the ends of growing microtubules (see Chrétien et al.3).



        Figure 10.19 Microtubules nucleated by an axoneme in the presence of purified tubulin (Chrétien, D., unpublished observations). The arrows indicate the transition between the axoneme (bottom left) and the microtubules nucleated from it (upper right).

   

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   Figure 10.20 Microtubules polymerized in a cell-free extract from Xenopus eggs (Chrétien et al.5). Filaments, vesicles and ribosomes can also be visualized. (Reprinted from Wade and Chrétien11 with permission.)       

Figure 10.21 Microtubules assembled in the presence of the slowly hydrolysable GTP analogue GMPCPP (guanylyl-()methylene-diphosphonate), and depolymerized in the presence of calcium (see MüllerReichert et al.4 for details.)      Figure 10.22 Self-assembly of microtubules in the presence of CLIP-170. Note the presence of curved tubulin-CLIP-170 oligomers in the background and at the extremity of the growing microtubules. (Reprinted from Arnal et al.2 with permission.)  

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8. REFERENCES 1. 2. 3. 4.

5. 6.

7. 8.

9. 10. 11.

Arnal, I. et al. Structural transitions at microtubule ends correlate with their dynamic properties in Xenopus egg extracts, J. Cell Biol., 149, 767, 2000. Arnal, I. et al. CLIP-170/tubulin-curved oligomers coassemble at microtubule ends and promote rescues. Curr. Biol., 14, 2086, 2004. Chrétien, D. et al. Structure of microtubule ends: Two-dimensional sheets close into tubes at variable rates. J. Cell Biol., 129, 1311, 1995. Müller-Reichert, T. et al. Structural changes at microtubule ends accompanying GTP-hydrolysis: Information from a slowly hydrolyzable analogue of GTP, GMPCPP. Proc. Nat. Acad. Sci., 95, 3661, 1998. Chrétien, D. et al. Lattice defects in microtubules: Protofilament numbers vary within individual microtubules. J. Cell Biol., 117, 1031, 1992. Ashford, A.J. et al. Preparation of tubulin from bovine brain, in Cell Biology: A Laboratory Handbook, 2nd ed., Celis, J.S., eds., Academic Press, San Diego, CA, USA, 2, 205, 1998. Nogales, E. et al. Structure of the alpha beta tubulin dimer by electron crystallography. Nature, 391, 199, 1998. Detrich, H.W. et al. Purification, characterization, and assembly properties of tubulin from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. Biochem., 22, 2453, 1983. Chrétien, D. et al. Reconstruction of the centrosome cycle from cryo-electron micrographs. J. Struct. Biol., 120, 117, 1997. Chrétien, D. et al. Determination of microtubule polarity by cryo-electron microscopy. Structure, 4, 1031, 1996. Wade, R.H. and D. Chrétien. Cryoelectron microscopy of microtubules. J. Struct. Biol., 110, 1, 1993.

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CEMOVIS, Cryo-Electron Microscopy of Vitreous Sections

261

CONTENTS

GENERAL INTRODUCTION .................................................................................... 263 1.

PRINCIPLES OF CEMOVIS........................................................................... 264 1.1. 1.2.

1.3.

1.4.

Cryo-Electron Microscopy ....................................................................... 264 Vitrification .............................................................................................. 264 1.2.1. General........................................................................................ 264 1.2.2. Cryoprotection ............................................................................ 265 1.2.3. High pressure .............................................................................. 266 Cryo-Sectioning........................................................................................ 267 1.3.1. Cutting dry .................................................................................. 267 1.3.2. Cutting vitreous material............................................................. 268 Observing in the Cryo-Electron Microscope ............................................ 272 1.4.1. Beam damage.............................................................................. 272 1.4.2. Focus........................................................................................... 274 1.4.3. Volume and not surface .............................................................. 274

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 275

3.

INSTRUMENTS AND INSTALLATION/MATERIALS/CHEMICALS .... 275 3.1. 3.2. 3.3.

4.

Instruments and Installation...................................................................... 275 Materials ................................................................................................... 276 Chemicals ................................................................................................. 276

PROTOCOLS .................................................................................................... 277 4.1.

4.2.

4.3.

4.4.

Preparation of the Material ....................................................................... 277 4.1.1. Cell suspension ........................................................................... 277 4.1.2. Tissues ........................................................................................ 278 Vitrification .............................................................................................. 278 4.2.1. Cell suspension vitrified in a Leica EM-PACT high-pressure freezer ......................................................................................... 278 4.2.2. Organotypic slice of rat brain in high-pressure freezer HPM 10 from BAL-TEC........................................................................... 279 Cryo-Sectioning........................................................................................ 279 4.3.1. Mounting the specimen in the cryo-ultramicrotome ................... 280 4.3.2. Trimming the specimen .............................................................. 280 4.3.3. Cutting sections........................................................................... 281 4.3.4. Flattening the section on the grid................................................ 282 Observing in the Cryo-Electron Microscope ............................................ 282 4.4.1. Transfer....................................................................................... 282 4.4.2. Evaluation of the specimen ......................................................... 283 4.4.3. Observation and image recording ............................................... 283

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ADVANTAGES/DISADVANTAGES.............................................................. 284 5.1. 5.2.

Advantages............................................................................................... 284 Disadvantages .......................................................................................... 284

6.

WHY AND WHEN TO USE CEMOVIS ........................................................ 285

7.

OBSERVED RESULTS.................................................................................... 286

8.

REFERENCES .................................................................................................. 288



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263

GENERAL INTRODUCTION The ultimate method… Can you imagine any better preparation method than CEMOVIS ― cryo-electron microscopy of vitreous sections?

… with restrictions.  Water can only be vitrified in layers in the µm range (see Chapter 1).  The vitrification depth is greater for biological material, but it does not exceed the 10 µm range for most cells and tissues.

Take a sample, cool it so rapidly that it is immobilised before it has time to crystallise, cut it into ultrathin sections and observe, in a cryo-electron microscope, all the finest molecular details of the perfectly preserved specimen. You may even reconstruct the 3-D distribution of the material in the section by means of computerised electron tomography (CET) (see Chapter 12). In addition, the time span from the sample to the image could be as short as one hour rather than days. Yes, in principle, you can’t dream of any thing better than CEMOVIS.

 High-pressure vitrification makes it possible to increase the depth of vitrification 10-fold, but it does not allow vitrification of pure water or a diluted solution (physiological solution).  In general, the extracellular medium must be cryoprotected. 20% dextran ensures that the entire sample has approximately uniform vitrification properties.  After vitrification has been achieved, the temperature must remain below –135°C for all subsequent steps.

The dream of CEMOVIS is old. FernándezMorán12 initiated the effort to make it come true. Vitrification (see Chapter 1) and highpressure freezing (see Chapters 5, 6) were important milestones on the way toward its realisation, which, it can be argued, is now successfully achieved.3,4 It remains to make it generally available. This book may help.

 Vitreous material is a liquid of high viscosity. Cutting-induced deformation is inherent to vitreous specimens. It can only be reduced by cutting conditions that increase fractures, thus favouring crevasses. Cutting artefacts are a serious problem.  The quality of the knife is a critical factor.  It is not a trivial matter manipulating a sample below 140°C without excessive frost contamination.

Maybe, one day, CEMOVIS will replace conventional embedding and room temperature observation. For the moment, however, it requires skill and patience. Consequently, the domain of application of CEMOVIS is where conventional methods fail, namely at high resolution.

 Beam damage is a critical factor. Whereas parsimonious electron irradiation produces only marginal image improvement in conventional observations, an excessive electron dose rapidly causes bubbling and total destruction of vitreous specimens. Even at lower doses, a specimen undergoes significant modification.

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The question is when to choose CEMOVIS? Of course, this depends on the nature of the observed structure ― rigid bodies are better preserved by conventional preparation methods than liquid structures. In general, we suggest that CEMOVIS should be considered for any observation in the 10 nm range or below. The reader may consult other general or recent articles.711,14,16,19,21

1. PRINCIPLES OF CEMOVIS 1.1. Cryo-Electron Microscopy Electrons only travel over micrometres in biological material and centimetres in air. Consequently, at room temperature, the electron microscope column must be under high vacuum and water must be eliminated from the specimen because it evaporates at low pressure. Water can only be conserved in vacuum at a temperature where the evaporation rate becomes negligible. Modern cryo-electron microscopes make such observations possible without loss of image quality and with minimum operational burden.

 The mean free path ― namely the distance between two scattering events in biological matter ― is ca. 0.2 µm for 100 kV electrons. The thickness of a specimen should preferentially be much smaller.  The evaporation rate is less than 1 nm/sec at 100°C. It decreases 10-fold each time the temperature is reduced 10°C thus leading to a negligible evaporation rate of 1 nm/hour at 135°C. It is negligible at 196°C, the temperature of liquid nitrogen.

1.2. Vitrification 1.2.1. General Under normal conditions, water freezes upon cooling and ice formation is associated with large-scale water reorganisation. The induced rearrangement of the biological material may be limited by careful freezing conditions, but it can never be completely avoided.

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 Hexagonal crystals are always very large at the ultrastructural scale. In most cases, they are only one or few per cell. However, under favourable freezing conditions, an ice crystal is not like a compact ice block, but it is ramified like a tree with its branches.

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Cryo-EM became a fashionable method when vitrification of water and diluted aqueous solutions were found to be possible. Vitrification ― namely making water solid while keeping its amorphous state and avoiding any ice formation ― can be achieved under some stringent cooling conditions.

 Vitrification is not crystallisation with very small ice crystals. The vitreous and crystalline states are fundamentally different, nonoverlapping states. Electron diffraction unmistakably shows the difference.

Three parameters are essential for vitrification: cooling rate, cryoprotection, and pressure. Vitrification of pure water at normal pressure is only obtained with an extremely high cooling rate. A slower cooling rate is sufficient when a fraction of the water molecules is mobilised by a cryoprotectant or when cooling is done under high pressure.

 The cooling rate for vitrifying pure water is very uncertain. It lies probably in the range from 106 to 1011°C/sec. For typical cells and tissues cooled under highpressure, the required cooling rate is probably only in the 103 to 104°C/sec range. In such a case, the immobilisation time can be estimated to be in the 10 msec range.

For pure water, vitrification can be  Thin film vitrification (see Chapters 3, 4, achieved on a thin layer of up to ca. 1 µm 7) is very efficient because there is a direct contact between the material to be vitrified thickness. and the liquid cryogen. The vitreous state is metastable. It can only  Pure vitreous water devitrifies within exist at low temperatures. Ice forms when minutes at 135°C. Any operation with vitreous material must be performed below the vitreous state is rewarmed. this temperature. 1.2.2. Cryoprotection The tendency of water to form ice decreases with solute concentration. It can even be practically suppressed with some solutes at high concentration (2.4 M sucrose, glycerol). In general, most solutes act as cryoprotectants. This also holds for the material in a living cell.

 The search for optimal cryoprotectants is a major goal of cryobiology, which has stimulated considerable research during the past 70 years. Much remains to be done, however, for the practice of CEMOVIS.

The vitrification depth that can be reached for typical cells and tissues by plungefreezing or slam-freezing (see Chapter 2) is typically in the 10 µm range.

 A vitrification depth in the 10 µm range is not convenient for most eukaryotic cells and tissues because it hardly corresponds to one cell layer. Good observation conditions are thus limited to the surface layer that, in many cases, is the region damaged during excision.

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Cryoprotection also increases the devitrification temperature of vitrified samples. However, according to Lepault et al.15 and our own experience, the devitrification temperature of typical biological samples remains well below 100°C.

 This observation implies that freezesubstitution, which takes place at 90°C, never occurs in vitreous specimens, but always in samples where water is in a crystalline form. It remains to be seen if cubic ice is frequently the form of water that freeze-substitution is dealing with.

In most tissues or cell suspensions, the extracellular medium is a poor cryoprotectant. It, therefore, has a tendency to crystallise even when vitrification could be achieved in the cell. This is an unacceptable situation because ice formation in the surrounding of a cell causes major osmotic injuries. Therefore, it is important to add a cryoprotectant to the extracellular medium in order to obtain homogeneous vitrification conditions.

 Experience has shown that a ca. 20% sugar solution mimics the vitrification properties of typical cells. This condition causes severe osmotic dehydration of the cell. We have found that the high molecular weight sugar-polymer dextran is a convenient sugar substitute with a negligible osmotic effect. The exact conditions for optimal cryoprotection must be adjusted for every specimen.

1.2.3. High pressure The density of hexagonal ice is ca. 0.92 g/cm3 whereas it is 1 for pure water. In other words, the volume of water increases upon freezing. Consequently, freezing can be retarded if high pressure is applied during cooling. The equilibrium melting temperature of ice can be reduced to as low as 22°C at 2000 bars.

 2000 atmosphere may seem a very high pressure. However, because the compressibility of water is very low, the enthalpy change in the biological sample is small unless some gas bubbles are left.  Using an even higher pressure is detrimental because it favours the formation of a new form of ice with higher density.

Practice supports the rule of thumb that high pressure increases the vitrification depth achievable in cells and tissues by a factor of 10 from the 10 to the 100 µm range. We frequently work with 300 µmthick specimens.

 There are no quantitative experimental data available on how the cooling speed required for vitrification decreases with increasing pressure. The vitrification depth at normal and high pressure could, however, be determined for a number of comparable systems.

High-pressure freezing is performed in commercial apparatus working according to two different principles. In one case, the well-protected sample is placed in a chamber where the cryogen is injected at very high pressure (see Chapter 5). In the other case, the sample is mounted in a small, protected volume (a capillary copper tube or another sandwiched volume). High

 In both cases, a conductive metal layer of 100 to 200 µm separates the sample from the direct action of the cryogen. The maximum cooling speed that can be reached at the surface of the sample is always much smaller than what can be reached by direct cooling. As a consequence, we have never been able to vitrify pure water or diluted aqueous

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pressure is applied in the volume at the very instant it is cooled from outside with a cryogen jet at low (some bars) pressure (see Chapter 6).

Slam-freezing (see Chapter 2) is an interesting method, which should be studied more systematically. It does not provide the advantage of high pressure, but it enables direct contact of the cryogen with the sample; there is no intermediate protective layer between the sample and the cooling material. Furthermore, it does not have the disadvantage of high-pressure freezing, namely the effect of very high pressure applied during the cooling process.

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solutions by high-pressure freezing. In our experience, rare are the biological specimens that can be vitrified with less than 10% added external cryoprotection. We believe that claims of vitrification of much more diluted aqueous solutions that were previously published or announced orally were erroneous.  From the principle of the method, it can be inferred that the cooling speed at the surface of the sample must be comparable to that obtained by plunge-freezing. Consequently, one could guess that it would be possible to vitrify, at least the surface, of water or a diluted solution layer. This has not yet been shown to be the case. The reason could be the retroactive effect of the heat generated by ice formation deeper in the sample.

1.3. Cryo-Sectioning Any user of a sophisticated technology tends to forget the instrument that makes his work possible. Electron microscopists depend on the electron microscope and the various instruments, such as ultramicrotomes used for specimen preparation. Cryo-electron microscopy became possible with the development of reliable electron microscopes capable of dealing with low temperature specimens. CEMOVIS was made possible thanks to the remarkable developments of cryo-ultramicrotomes and diamond knives.17 1.3.1. Cutting dry A good modern cryo-ultramicrotome provides an adequate working environment for cutting vitreous sections. The temperature is well controlled in the working range of 140°C to 170°C. The gas flow in the working area is strong enough to repel frost, though it is smooth enough not to disturb sections. The working place is spacious, well illuminated and observable with high-quality binoculars. In addition, the cryo-ultramicrotome has kept all the cutting properties, which makes it so good for cutting conventional sections at room temperature.

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Cutting conventional plastic sections is eased by the fact that it is possible to float them on water. A liquid performing the same function with vitreous sections has not yet been found.

 The required liquid probably does not exist because it must have mutually exclusive properties. It must remain a liquid at low temperature, i.e., have small cohesive energy and, at the same time, have a high surface tension in order to force the section to float and prevent the liquid from wetting the specimen.

As a consequence, in addition to being very sharp, a knife for cutting vitreous sections must offer a surface on which the section can glide smoothly. A knife with a small cutting angle is preferable as long as it does not compromise the sharpness of the cutting edge.

 The question of whether diamond or glass knives are better has not yet been studied in detail, but for the moment the best results have been obtained with diamond knives. For our purposes, and for the moment, the diamond knives of only one manufacturer are adequate for producing good vitreous sections (see Section 3.1.). The critical point is how the section glides on the knife surface.

Sections cut in the dry state are very  A device providing an adjustable ion sensitive to electrostatic charges, which shower in the vicinity of the knife is an make them fly away or stick to any surface. important aid for controlling the sections. No thin section of good quality (feed of 50 nm) can be obtained using a diamond knife without an ion shower.

1.3.2. Cutting vitreous material Vitreous water is a liquid, but a liquid with very high viscosity. This means that, like honey or butter, a section flows. It flows slowly if the acting force is small as is the case during observation in the EM and it flows rapidly when the force is large as is the case during cutting. Deformation, is therefore, an inherent feature of vitreous sections.

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 The basic features of the sectioning process are probably quite well understood. The consequences of the cutting process are so diverse and so remarkable that they provide rich possibilities for the falsification of any of the theories that have been proposed. The current theory has undergone so many tests that it is probably globally correct, though it obviously needs refinement.5

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 The knife K hits the block at depth f equal to the feed. It exerts a pressure N perpendicular to the knife surface (if the friction along the surface is negligible). N can be decomposed into the compression force C along the cutting direction and the bending force B. The cutting energy is concentrated in the grey region where most of the deformation takes place, transforming the advance ∆x in the block into the displacement ∆x´ of the section on the knife surface. The shear stress increases along N from S to Q where there is no material any more to counterbalance the effect of the cutting force. The stress may exceed the fracture limit of the material, thus producing crevasses (dash line).  A rich collection of cutting artefacts is shown in Figure 11.14 A.

The enormous force density that is locally applied during cutting is amazingly illustrated by the effect of cutting-induced amorphous water (CIA) vitrification.1 It has been shown that, under some uncommon conditions, crystalline ice is transformed into a form of amorphous water during cutting. This effect is known to take place under a pressure of several thousand bars.

The basic deformation that takes place during cutting is compression along the cutting direction compensated by a proportional increase of the section thickness.

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Figure 11.1 Forces and deformations during cutting.  All that glitters is not gold. The fact that a section is vitreous does not prove that the block was vitrified. Material that has undergone CIA, however, has very different cutting properties; in particular, it is more brittle. An experienced CEMOVISt will easily identify CIA by the unusual mechanical properties of the material and the fact that, in spite of vitrification, the biological material has suffered from crystallisation-induced segregation. In our experience, CIA is not observed in thick sections (feed ≥100 nm). Therefore, in case of doubt, vitrification should be tested on such sections.  The compression factor C (length reduction along the cutting direction/original length) is generally between 15 and 60%.

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 According to the above model, cuttinginduced deformation is remarkably homogeneous, namely, it is the same everywhere. A straight line in the block is transformed into a straight line in the section and a plane in a plane. The square marked by the points O and P is transformed in the parallelepiped O’P’. The equations of the transformation are  X´ = xcos  + ysin   Y´ = y t/f in which  is the cutting angle, t is the thickness of the section and f is the feed. Figure 11.2 A square transformed into a parallelepiped. The ideal case of homogeneous deformation holds remarkably well as long as the material is homogeneous. Irregular deformations and fractures, however, are common due to discontinuity in the cutting process or to the non homogeneity of the material.

 The basic goal of a CEMOVISt is to obtain homogeneously compressed sections with no other cutting artefacts, such as crevasses and chatter.

There are indications that an oscillating  The demonstrated advantage of knife may considerably reduce com- oscillating for cutting soft material at room pression.2 temperature has also been observed in vitreous sections, but not yet on a reproductive basis.  The knife edge is very sharp, but not perfectly sharp, thus causing an additional deformation close to the surface of the section. It seems that this effect does not affect more than 1/5 of the thickness of the thinnest sections.  There are also places where the knife edge is damaged (dashed line). At this place the deformation is more severe. A furrow is formed in the bottom face of the section and a corresponding ridge on the block face. These are knife marks.  A more common source of knife marks is particles adhering on the surface of the knife. Figure 11.3 Many knife marks due to irregularities at the knife edge.

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One may try to reduce deformation by applying a higher force during a shorter time period. Unfortunately the cutting force can only increase to the point where shear stress produces a fracture in the material. The result is the very common and nasty artefact of a crevasse.

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 Crevasses are fractures generally arising at the top surface of the section. It is common that a section becomes a field of crevasses. Crevasses are more severe in thick sections and at high cutting speed. Using a small angle knife reduces them.  Chatter is a wavy variation of the section thickness along the cutting direction. Contrary to what may be guessed at first, chatter is not due to a vibration of the knife relative to the block. It is rather a variable compression due to irregular friction on the knife surface. The section moves on the knife like the string of a violin on the bow. According to the model described of the sectioning process, a section of thickness t is formed in absence of friction. Friction F results in the cutting force R and the increased section thickness t´. Chatter is nasty because it is associated with irregular compression and, thus, non homogenous deformation. Chatter depends primarily on the gliding properties of the knife surface. It is reduced at high cutting speed. Figure 11.4 Friction on the knife surface causes chatter.

Biological material itself is a source of irregular deformation. Membranes frequently resist deformation and are a source of fracture. Some regions are more brittle than others and consequently more crevasse-prone. An excellent trimming is key to a tension-free block.

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 A good network of polymer, such as condensed chromatin, is a favourable medium for cryo-sectioning. In this respect, the use of dextran, a sugar polymer, as a cryoprotectant is advantageous, whereas other cryoprotectants, such as glycerol, are detrimental.

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1.4. Observing in the Cryo-Electron Microscope In the electron microscope, a vitreous section differs from a conventional resin section in several aspects. First of all, vitreous biological material is beam-sensitive in a conspicuous way when the section bubbles before disappearing under the beam. Beam damage starts, however, in a much more delicate way when the material of the section flows under the effect of electrical forces generated by the electron beam. The second major difference stems from the fact that, contrary to stained objects, imaging of native biological material nearly exclusively relies on phase contrast, which strongly depends on focus. As was learned from cryo-EM of vitrified suspensions, the choice of a correct focus is of the utmost importance. Finally, contrary to post stained sections, typical for conventional observations in which surface structures are emphasised by reinforced staining, the structures in vitreous sections are equally visible over the entire thickness of the section. Any image must be interpreted as originating from a 3-D object. 1.4.1. Beam damage 1. Bubbling  For a dry specimen, beam damage is reduced 2 to 5-fold13 at liquid nitrogen temperature. In the presence of water, molecular damage is similar, but other effects are conspicuous. The most obvious is bubbling.

 There is a debate about whether beam damage is further significantly reduced at a temperature approaching that of liquid helium (4 K, minus 269oC). At this temperature, however, the greatly reduced electrical conductivity, and some other not yet well understood transformations, make it difficult to take full advantage of an eventual considerable reduction of beam damage. The future will tell if a very low temperature is a useful avenue for cryo-EM.

 Bubbling takes place through excessive accumulation of gas produced by electron beam-induced radio decomposition of biological matter.9 Probably because pores through which the gas could escape are sealed with water, the accumulating gas nucleates into bubbles. The specimen seems to be boiling in ever increasing bubbles, which finally collapse into a large hole marking the ultimate end of any observation.

 With 100 kV electrons, bubbling typically starts between 2000 to 6000 e/nm2. The onset depends on the nature of the organic material. Radiosensitive substances, such as aliphatic hydrocarbons (lipids), bubble first, whereas aromatic-rich compounds (nucleic acids) are more resistant. For example, condensed chromatin resists longer to bubbling than the surrounding structures. Interestingly, this effect is inverted after glutaraldehyde fixation. Bubbling is not only a pain for the observer, it can be used as a focusing aid.

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 Obviously, any high-resolution structural observations cease when bubbling starts. It may happen, however, that the differential onset of bubbling provides valuable information about the nature of material present. Sometimes it also reveals compartments that are not otherwise recognisable. 2. Beam-induced deformation  Because it is a high viscosity liquid, the section undergoes global deformation during irradiation, probably because of the forces generated by non balanced charge accumulations.

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 It is probable that a good part of the gas that produces bubbles is H2 because the effect also exists at temperatures much below the liquefaction temperature of other candidates, such as O2, N2 or CO.

 The effect exists in thin vitrified films but it is more prominent in vitreous sections because they are not always well attached to a rigid support. This long-range deformation is a severe problem in CET where the information from a large number of successive images must be combined into a 3-D model (see Chapter 12).

 Locally the material also is rearranged under the effect of the beam. In particular, any sharp irregularities, such as crevasses or knife marks, seem to be “ironed away” with irradiation. Of course, such a procedure does not repair fine structures.

 The apparent curing of a badly crevassed section by the electron beam is a contraproductive beginner’s mistake. Fine structural details, which were broken by fracture, are rarely repaired when crevasses are ironed away. They leave characteristic discontinuities in any previously welldefined structures, such as membranes or filaments.

 There are also biological structures and particles, which aggregate under the effect of the beam. The effect has been quantitatively documented for domains of condensed chromatin, which contract over a smaller surface during irradiation, thus producing an apparent density increase in these domains.20

 The first visible effect of the electron beam is to reinforce the compaction of various regions, such as chromatin domains, chromatin fragments or other nucleoproteic structures. The effect may also take place in other constituents, but it has not yet been quantitatively documented. It could also be a beginner’s mistake to accept as bona fide compact bodies, originally diffuse structures aggregated under the effect of the electron beam. An image free of knife marks should be considered critically because it generally means that the specimen has been excessively irradiated.

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1.4.2. Focus  Amplitude contrast dominates images of conventionally stained sections. It reveals regions of different densities, such as stained versus unstained regions. It is most clearly visible when the image is recorded close to focus.

 Electrons intercepted in the objective aperture cause amplitude or area contrast. These are mostly scattered by heavy atoms from the stain.

 CEMOVIS demonstrates that the density of native biological material is remarkably constant. The material in the cell is relatively homogenously distributed. Consequently, amplitude contrast does not reveal much. There is little to be seen in a vitreous section recorded at focus.

 Ribosomes are at the limit of visibility with pure amplitude contrast. It comes as a surprise for many that even regions that are reputed to contain dense material, such as condensed chromatin, are only a few percent above the rest of the nucleus.

 Imaging vitreous specimens relies on phase contrast. This mode of image formation is extremely sensitive to small local density difference. The sensitivity of phase contrast imaging is such that single unstained doublestranded DNA molecules floating in water are perfectly visible.6 Phase contrast is focus dependant. Images must be recorded at a defocus corresponding to the expected dimension of the explored structure. In general, several micrographs at different defocus are necessary in order to gain a realistic view of the object.

 Phase contrast is due to a phase shift of the scattered beam induced by short distance density differences. It does not carry long-range information. The focus dependant dimension d, which is best represented on the image, is  dmax = (2∆z)1/2 where  is the electron’s wave length (3.8-3 nm at 100kV) and ∆z is the defocus, whereas there is no information retrieval at:  dmin = (∆z)1/2 For example: Best visibility of chromatin’s granularity (10 nm): ∆z = 13µm closely packed dsDNA (2.5 nm): ∆z = 0.8µm.

1.4.3. Volume and not surface  In conventionally resin-embedded biological samples, post staining reinforces features at the surface of the section. The latter is predominantly visible in the image. The situation is different in vitreous sections of native samples because the whole volume of the section is equally represented on the image. The image is strictly a twodimensional projection of a threedimensional object.21

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 Any image is expected to contain many overlapping structures because the typical fine details that are observed (2 to 10 nm) are much smaller than the section thickness (rarely less than 50 nm).

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 Conclusion 1: CEMOVIS is not limited by the lack of contrast of small, unstained structures, but rather by the plethora of overlapping information projected through the thickness of the section.

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 Conclusion 2: The combination of CEMOVIS and CET (see Chapters 12, 24) leading to a 3-D model of the material distribution in the vitreous section will overcome the limitation due to plethoric information presented in any image.

2. SUMMARY OF THE DIFFERENT STEPS 1. Preparation of the material. 2. Vitrification. 3. Cryo-sectioning. 4. Observing in the cryo-electron microscope.

3. INSTRUMENTS AND INSTALLATION/MATERIALS/CHEMICALS 3.1. Instruments and Installation  High-pressure freezer.

 Leica EM PACT2; Leica, Vienna, Austria: www.leica-microsystems.com  BAL-TEC HPM 010, Balzers, Liechtenstein. Now available from Boeckeler Instruments, Inc., Tucson, Arizona, USA or ABRA-Fluid AG, Widnau, Switzerland: www.abra-fluid.ch  A new modified apparatus HPM 100 is available from Leica.

 Cryo-ultramicrotome.

 Leica EM FC6 (2004); Leica FCS Leica. (We have no experience with instruments from other manufacturers.)  Static Line II, Diatome, Biel, Switzerland: http://www.diatome.ch/en/products/

 Ionizer for cryo-chamber.

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 Diamond cutting and trimming knives;  Diatome, Biel, Switzerland: oscillating knife. http://www.diatome.ch/en/products/  In our hands and until 2005, vitreous sections of comparable quality could not be obtained with diamond knives from other manufactures.  The oscillating knife is promising, but still in development.  Controlled humidity and temperature  Necessary in the cryo-microtome room. and control of O2 in air for safety.

3.2. Materials  Cryo glue.

  

 

 Mixture of 1 vol. ethanol in 3 vol. isopropanol. Mixture ratios in the range from 1:7 to 2:3 are used. 1000 mesh grids with 50 nm carbon  Any producer. film. Thinner grid, smaller holes.  Quantifoil: http://www.quantifoil.com/ Manipulation aids for the  Provided with cryo-microtome and cryoultramicrotome and tools for specimen specimen transfer system. manipulation and storage Mounted eyelash  Own production: An eyelash is glued on a wooden stick. Liquid nitrogen and long term specimen storage place.

3.3. Chemicals  Dextran.  Ethanol and isopropanol for cryo glue.  Methyl cyclohexane for Leica EM PACT.  1-hexadecene for HPM 010.

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 Sigma-Aldrich, St. Louis, MO, USA.  Merck, Eurolab, VWR International, Darmstadt, Germany.  Fluka, Buchs, Switzerland.

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4. PROTOCOLS 4.1. Preparation of the Material The CEMOVIS procedure depends on the material studied and the type of high-pressure apparatus used for vitrifying the sample. In the following description, we consider two characteristic samples. One is bacteria vitrified in a Leica EMPACT high-pressure freezer. The other is an organotypic slice of rat brain vitrified in a BAL-TEC HPM 010 apparatus. In both cases, the external medium must be cryoprotected, but with as little effect as possible on the cells.

4.1.1. Cell suspension



Bacteria grown to observation conditions in growth medium phosphate buffered saline (PBS).

 Centrifugation  1 min at 20,000 g or  15 min at 3000 g Soft pellet gently re-suspended in growth medium supplemented with dextran at final concentration of 20%.

 Second centrifugation  Pellet resuspended in ca. 5 × its volume of medium supplemented with 20% dextran (minimal final volume: 30 µL, see Section 4.2.1).

 Dextran 40 kDa at 20% does not cross the plasma membrane. It has little osmotic effect (20 mOs). A = 20% dextran in PBS Figure 11.5 Cryo-preservation of bacteria.

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4.1.2. Tissues Tissues from the central nervous system are especially difficult to vitrify, probably because they have a higher than average water content. Vitrification of the cells could only be achieved after a slight osmotic dehydration of the tissue by 5% sucrose (total osmolarity: 600 mOs).

 Organotypic slice of rat hypocampus in experimental culture medium; 5 × 2 mm, thickness: 200 µm. Extraction of a 1 mm diameter slice

 The small slice is placed for five minutes in the culture medium supplemented with 20% dextran and 5% sucrose. After this time, the tissue remains functional. Figure 11.6 Extraction of a tissue sample for vitrification

4.2. Vitrification 4.2.1. Cell suspension vitrified in a Leica EM-PACT high-pressure freezer  30 µL of bacteria suspension on a Parafilm.  Suck the suspension into the copper tube with a piston until full. No air bubble must remain in the tube.  Dimensions of the copper tube:  Length: 20 mm  External diameter: 600 µm  Internal diameter: 300 µm The specimen is thus separated from the cryogen by 150 µm of copper.  High-pressure vitrification according to instrument procedure (see Chapter 6).  Pressure should not exceed 2000 bars; higher pressure favours formation of highpressure ice.  After cooling, the tube can be stored at liquid nitrogen temperature or directly mounted in the cryo-ultramicrotome. Figure 11.7 Vitrification in EM-PACT high-pressure freezer.

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4.2.2. Organotypic slice of rat brain in high-pressure freezer HPM 10 from BAL-TEC

 The excised slice in dextran and sucrose is mounted in the HP 010 aluminium holder with a minimum amount of liquid left. The rest of the volume is filled with hexadecane taking care to leave no air bubble; the sandwich is closed.  There are different holder sizes. The specimen is typically separated from the cryogen by 200 µm of aluminium.

 The rest of the procedure follows immediately according to procedure (see Chapter 5).

instrument

Figure 11.8 Vitrification in HPM 010 highpressure freezer. Extraction of the specimen from the holder.  With pins and scalpel at 140°C in the microtome chamber.

4.3. Cryo-Sectioning The work in the cryo-ultramicrotome takes place below 140oC in the atmosphere of the laboratory. Even though the instrument is carefully designed to produce a frost repelling air stream around the working area, the presence of snowflakes may be a serious problem, eventually leading to the deposition of a frost layer, thus impairing any useful work. The cryo-ultramicrotome should be placed in a draft-free room with controlled humidity and temperature. It must be securely ventilated and a safe level of oxygen must be strictly controlled.  Recommended conditions in the cryo-  Danger: Use of liquid nitrogen in a ultramicrotome room area: 20°C and closed room is dangerous. One litre of 30% relative humidity. liquid nitrogen produces 700 litres of nitrogen gas. Suffocation without warning starts when 02 concentrations are slightly reduced (below 19.5%) and it rapidly leads to death.

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4.3.1. Mounting the specimen in the cryo-ultramicrotome  The copper tube from Leica EM PACT2 can be directly mounted in the specimen holder.  The fragment of material extracted from the sandwich of the BAL-TEC HPM 010 freezer is mounted with cryoglue.18 1. Cryoglue is a mixture of ethanol and 2-propanol in a 2:3 ratio. 2. A drop of cryoglue is applied on the specimen head at 140°C. The glue is slightly soft at this temperature. 3. The specimen fragment is forced into the glue and correctly oriented. 4. Temperature is reduced to 160°C. The glue becomes rigid enough for further operations. Figure 11.9 Vitreous specimen mounted in a cryo-ultramicrotome 4.3.2. Trimming the specimen  The specimen surface is prepared flat with a trimming knife. The whole section of the EM PACT2 copper tube is sectioned with ca. 0.05 µm (cryoglued and/or fragile specimens) to 1 µm strokes (clamped copper tubes). Typically, some 200 µm must be removed from the tube; less from a glued specimen.  The first evaluation of the state of the specimen can generally be made at this stage. A well vitrified specimen seen with top illumination is uniformly black in the copper tube. A non vitrified region or inhomogeneous material is shiny or milky. Many bad specimens can already be discarded at this stage.  A square-based pyramid is an excellent shape for final preparation of the specimen. A 45° trimming knife must be used. Suitable dimensions are 100 to 200 µm at the base and 10 to 100 µm high. A rectangular parallelepiped trimmed with a 90° knife may offer advantages, but it can be dangerously prone to fracture. Figure 11.10 Vitreous specimen trimmed in a cryo-ultramicrotome.

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4.3.3. Cutting sections  The conditions for obtaining good sections are a well-prepared, homogeneously vitrified specimen, careful trimming, and an excellent cutting knife. The latter is of utmost importance. Besides being sharp and clean, it must have good gliding properties. The manufacturing procedures are not known publicly. Research on this matter should be encouraged.  In addition, the working room must be reasonably free of snowflake contamination and the forces induced by electric charges must be neutralised. This is best done with the ion shower incorporated into the cryoultramicrotome. Operating conditions must be adjusted according to the situation.  Under good cutting conditions, the sections appear as regular ribbons, which neither stick to the surface of the knife nor fly away. Adjust the ion shower by changing the distance of the ion source to the knife edge and the power of the ionizer.  The ribbons are manipulated with the eyelash. Under well-adjusted conditions, the ribbon just sticks to the eyelash that can be transferred directly onto the prepositioned grid. Two to 10 ribbons can be mounted on each grid.  Some operators hold the growing ribbon with the eyelash and pull it gently. This longer ribbons can be obtained. The incorporation of a micromanipulator into future instruments could help in democratising the method. Figure 11.11 Ribbons of vitreous sections are transferred from knife to grid.

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4.3.4. Flattening the section on the grid  The ribbons on the grid are flattened by pressing gently with a flat surface. This action is necessary to reduce the probability of losing the sections during storage and transfer and to improve the stability of the section during irradiation. A clean, smooth surface and tools, free of snow flakes, are important. Experience dictates the force that should be used for pressing. An exaggerated force destroys the supporting film.

The grids with the ribbons are stored in a specimen box that can be closed and stored for a long period of time in liquid nitrogen.

Figure 11.12 Ribbons of vitreous sections are flattened on the grid.

4.4. Observing in the Cryo-Electron Microscope 4.4.1. Transfer  Transfer takes place as for any vitrified  Under-liquid transfer minimises the risk specimen (see Chapters 3, 4, 7–10, of contamination; above-liquid minimises 12). In the transfer station, the the risk of losing sections. manipulation can be performed under liquid nitrogen or above its surface.  After transfer, the microscope must be  Poor vacuum may contaminate the allowed time for vacuum restoration specimen with a layer of water condensed and temperature stabilisation. from the gas phase in the form of amorphous water or cubic ice.  Non equilibrated temperature causes drift of the specimen, reducing image quality.

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4.4.2. Evaluation of the specimen  Overview

 Low magnification observation of the grid. Localisation and general appearance of the ribbon; level of contamination by snow.  Test of vitrification by electron  No specimen should be studied without diffraction. first checking the state of water. The test is immediate and it prevents many mistakes.  Determination of the thickness of the  This valuable measure can be made rapidly and at low magnification. The sections electron flux, shown in Figure 11.13, of the section (Is) is compared to the flux where there is no section (Io). The thickness is given by the equation in which t is the thickness for a specimen of unit density, and L is the mean free mass thickness — a parameter of the microscope, typically of the order of 200 nm. Even if  is not known precisely, the relative value of t is a precious guide. (Practical details can be found in Dubochet et al.10) Figure 11.13

4.4.3. Observation and image recording A competent cryo-electron microscopist is recognised by the fact that at any given moment he knows the dose already received by the specimen.  Calibration of the electron dose is obtained from direct current determination on the screen, from optical density of photographic film exposed under controlled conditions or from charge coupled device (CCD) calibration data.  A normal illumination on the screen (one second exposure) at 10’000 magnification typically corresponds to ca. 100 electrons/nm2 in a 1 second exposure.  As compared to conventional specimens, images of vitreous sections must be recorded at higher defocus. The optimal defocus value depends on the studied dimension.  Computerised electron tomography, CET

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 For a given illumination on the screen, the dose on the specimen increases with the square of the magnification (a single micrograph at 20,000 × produces the same dose as 100 micrographs at 2000 ×).

 The quantitative values are given by the equations in Section 1.4.2.

 Recording the long-tilt series needed for CET requires a good section, adequate hardware and software and expertise (see Chapter 12).

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5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  Close to native specimen with all the  Is it really so, that apart from viscosity, water immobilised in a liquid state. there is no difference between liquid and vitreous water? It may be so in practice, but fundamentally it is probably not the case (see Chapter 1).  No stain.  The image is a true representation of material present in its natural state.  No chemical fixation.  Chemical fixation is not the worst among conventional artefacts. It produces fine-scale aggregation.  No dehydration.  Dehydration is arguably the most severe artefact of a conventional preparation. It acts at a molecular and at a cellular scale.  No aggregation artefact.  The result of the two previous advantages.  High resolution.  Atomic preservation in the section; 2 nm achievable in the image under favourable orientation. In combination with CET, a 2 nm, 3-D map is a reasonable goal for the years to come.  Potentially very rapid.  Is less-than-an-hour EM possible? Extraction of the sample and vitrification: 4 min; mounting, trimming and sectioning: 30 min; coffee: 5 min; observing, recording and transferring micrographs to the operating surgeon: 20 min.

5.2. Disadvantages  Technically demanding (material and  Yes. But does any good work require skill). less than top quality?  Yes. But they can frequently be  Cutting artefacts.  mastered.  Low contrast.  No. This is not a limitation. Only the limitation on signal-to-noise ratio is important.  Beam damage  Yes. But it can be minimised close to the fundamental limit it imposes on signal-tonoise ratio.  Plethora of information within the  Yes. But it can be overcome by:  Cutting thinner sections thickness of the section.  CET

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 Difficulty in placing fiducial marks for  Yes. But solutions are in progress by:  the adjunction of markers on the CET. vitreous section (see Chapter 12)  fiducial-free tomographic alignment  Not adequate for post immunolabel-  Yes. But staining and labelling can ling. sometimes be performed before specimen preparation and freeze-substitution is a valuable alternative (see Chapter 13).

6. WHY AND WHEN TO USE CEMOVIS CEMOVIS should be used whenever possible because it is the best method for preserving whole cells and tissues in their native state. In particular, it should be used when “liquid” structures are observed ― i.e., when the observed structure has no solid substrate or scaffold ― and when high resolution is aimed for. The contraindications for using CEMOVIS are: (1) the technical difficulty of the method and (2) the beam sensitivity of the vitreous sections. Neither would be fully relevant if freeze-substitution did not exist, but the latter method provides a valuable alternative in many cases (see Chapter 13). If freeze-substitution and CEMOVIS are compared, we note that both preparation methods are identical up to the point where the specimens are vitrified. The roads then diverge. In the first case, the sample is freeze-substituted before being cut and then observed at room temperature, whereas with CEMOVIS the preparation procedure continues at a low temperature. Freeze-substitution, therefore, should be considered as the method of choice for rigid specimens and when high resolution observation is not the major goal. It is then possible to obtain relatively beam-resistant sections particularly suitable for CET (see Chapter 24) allowing a resolution that compares favourably with conventional resin-embedded specimens. It remains, however, that any “liquid” structure, any feature that is partially or totally free to float in the water medium of the native state, will never be faithfully represented in dehydrated specimens. Experience with CEMOVIS shows that the effect of dehydration has probably been grossly underestimated in structural biology and this is why CEMOVIS should be used, whenever possible.

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7. OBSERVED RESULTS  Figure on the Chapter’s title page

 CEMOVIS: This elegant axoneme — probably a terminal portion — was observed in an organotypic slice of rat brain.21 See text for methodological details. Bar = 100 nm

 Figure 11.14

 Three views with CEMOVIS. A) Yeast cells in 20% dextran. Sectioning artefacts. The progression of the knife in the vitreous sample from top-left to bottom-right has deformed the cells into ellipses, produced knife-marks (arrows), numerous crevasses and the undulation of chatter (bottom left half). B) Gram-positive bacteria (Enterococcus faecalis). A good section. C) Organotypic slice of rat brain.  Axons (A)  Presynaptic vesicles (V)  Microtubules in the plane of the section (µa) and perpendicular to it (µb). Bar = 1 µm See text for details.

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8. REFERENCES 1. 2. 3. 4. 5. 6.

7.

8. 9.

10. 11.

12.

13. 14.

15. 16.

Al-Amoudi, A. et al. Amorphous solid water produced by cryo-sectioning of crystalline ice at 113K. J. Microsc., 207, 146, 2002. Al-Amoudi, A. et al. An oscillating cryo-knife reduces cutting-induced deformation of vitreous ultrathin sections. J. Microsc., 212, 26, 2003. Al-Amoudi, A. et al. Cryo-electron microscopy of vitreous sections of native biological cells and tissues. J. Struct. Biol., 148, 131, 2004. Al-Amoudi, A. et al. Cryo-electron microscopy of vitreous sections. EMBO J., 23, 3583, 2004. Al-Amoudi, A. et al. Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy. J. Struct. Biol., 150, 109, 2005. Bednar, J. et al. Determination of DNA persistence length by cryo-electron microscopy. Separation of the static and dynamic contributions to the apparent persistence length of DNA. J. Mol. Biol, 254, 579, 1995. Bouchet-Marquis, C. et al. Cryoelectron microscopy of vitrified sections: A new challenge for the analysis of functional nuclear architecture. Histochem. Cell. Biol., 125, 43, 2006. Bouchet-Marquis, C. et al. Visualization of cell microtubules in their native state. Biol. Cell, 99, 45. 2007. Dubochet, J. et al. Cryoelectron microscopy of vitrified specimens, in Cryotechniques in Biological Electron Microscopy. Steinbrecht, R. A. and Zierold, K., eds., Springer, Berlin, Germany, 1987, 114. Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys., 21, 129, 1988. Dubochet, J. et al. How to "read" a vitreous section, in Cryotechniques in Biological Electron Microscopy, McIntosh, J.R., ed., Elsevier, Amsterdam, The Netherlands, 2007, 386–403. Fernández-Morán, H. Low-temperature preparation techniques for electron microscopy of biological specimens based on rapid freezing with liquid helium II. Ann. New York Acad. Sci., 85, 689, 1960. International Study Group. Cryo-protection in electron microscopy. J. Microsc., 141, 385, 1986. Hsieh, C.-E. et al. Towards high-resolution three-dimensional imaging of native mammalian tissue: Electron tomography of frozen-hydrated rat liver sections. J. Struct. Biol., 153, 1, 2006. Lepault, J. et al. Freezing of aqueous specimens: An x-ray diffraction study. J. Microsc., 187, 158, 1997. Matias, V.R.F. and Beveridge, T.J. Native cell wall organization shown by cryoelectron microscopy confirms the existence of a periplasmic space in Staphylococcus aureus. J. Bacteriol., 188, 1011, 2006.

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17. 18. 19.

20. 21.

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Michel, M. et al. Diamonds are a cryo-sectioners best friend. J. Microsc., 166, 43, 1992. Richter, K. A cryoglue to mount vitreous biological specimens for cryoultramicrotomy at 110K. J. Microsc., 173, 143, 1994. Sartori, N. and Salamin-Michel, L. Cryotransmission electron microscopy of thin vitrified sections, in A Laboratory Handbook, Celis, E., ed., Academic Press, London, U.K., 1994, 323. Sartori Blanc, N. et al. Electron beam-induced changes in vitreous sections of biological samples. J. Microsc., 192, 194, 1998. Zuber, B. et al. The mammalian central nervous synaptic cleft contains a high density of periodically organized complexes. Proc. Natl. Acad. Sci. USA, 102, 19192, 2005.

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CONTENTS

GENERAL INTRODUCTION .................................................................................... 295 1.

PRINCIPLES OF CRYO- ELECTRON TOMOGRAPHY........................... 296 1.1. 1.2. 1.3.

1.4.

Specimen Preservation ............................................................................. 296 Acquisition and Mutual Alignment of Projection Images ........................ 297 Reconstruction.......................................................................................... 299 1.3.1. Principles of three-dimensional reconstruction according to Radon29 1.3.2. Reconstruction algorithms .......................................................... 300 1.3.3. Distortions................................................................................... 300 Image Segmentation and Isosurface Rendering........................................ 301 1.4.1. Denoising .................................................................................... 302 1.4.2. Surface rendering........................................................................ 302

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 304

3.

EQUIPMENT/PRODUCTS/REAGENTS....................................................... 305 3.1. 3.2. 3.3.

4.

PROTOCOLS .................................................................................................... 307 4.1. 4.2.

5.

Preparation of Protein-Stabilised Colloidal Gold ..................................... 307 Application of Quantum Dots to the Surfaces of Vitreous Cryosections . 308

ADVANTAGES/DISADVANTAGES.............................................................. 310 5.1. 5.2.

6.

Equipment................................................................................................. 305 Products .................................................................................................... 306 Reagents ................................................................................................... 306

Advantages of Cryo- Electron Tomography............................................. 310 Technical Limitations in Cryo- Electron Tomography............................. 310

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 311 6.1.

6.2.

Vitrification Methods ............................................................................... 311 6.1.1. Plunge-freezing........................................................................... 311 6.1.2. High-pressure freezing................................................................ 312 Alignment and Reconstruction Methods .................................................. 312 6.2.1. Fiducial marker alignment .......................................................... 312 6.2.2. Cross-correlation alignment........................................................ 312 6.2.3. Choosing a reconstruction algorithm .......................................... 313

7.

OBSERVED RESULTS .................................................................................... 314

8.

REFERENCES .................................................................................................. 316

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

Title page illustration: Courtesy of Dr. Stephan Nickell and with permission from Nature Reviews Molecular Cell Biology.1

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GENERAL INTRODUCTION Cryo- electron tomography is a visualisation technique that aims to combine the resolving power conferred by the short wavelength of electrons with optimal structural preservation and three-dimensional image reconstruction.2 The resolution achieved using this technique currently approaches four nanometres, which is sufficient to resolve macromolecular complexes larger than ca. 400 kDa. The advantages of cryo- electron tomography are obvious, even at this rather modest resolution: It provides the link between high-resolution structural imaging performed on isolated macromolecules and their imaging within the cellular context. The ultimate goal is to create a pseudoatomic atlas of a cell, with high-resolution structures obtained by x-ray crystallography or single particle electron microscopy being substituted for structures in tomographic maps, creating synthetic tomograms that show the absolute positions and relationships between macromolecules within the cell.1 To take full advantage of this emerging technology, steps must be taken to ensure that the specimen remains stable under vacuum and during irradiation with electrons, but also that it closely resembles its native state. These prerequisites are achieved by converting the specimen into a specific frozen-hydrated (vitreous) state3 and by carefully limiting the electron dose encountered by the specimen so as to preclude radiation-induced damage. Vitrification and the low-dose acquisition procedure distinguish cryo- electron tomography from electron tomography of chemically fixed and stained, plastic-embedded material. This is not to suggest that embedding plastics are not affected by the electron beam; on the contrary, they shrink markedly and are typically pre-exposed until the initial, rapid shrinkage has stabilised. Finally, heavy metal stains responsible for amplitude contrast in conventional electron microscopy are also omitted such that the densities of the specimen are represented, rather than indirect visualisation of stain. Contrast is enhanced by recording images at a specified underfocus value, creating so-called phase contrast. The further from focus, the more that resolution is sacrificed. Therefore, interpretation is hindered by the fact that signal is difficult to discern from background noise. Electron tomography is commonly performed using intermediate accelerating voltages, typically 300 kV or 400 kV. The wavelength of a 300 kV electron is ca. 2 pm, i.e. 2 × 10-12 m. Cryo- electron tomography is commonly performed on macromolecules, organelles or cells contained within thin films of ice created by blotting with filter paper and shock-freezing in liquid ethane (see Chapter 3). With few exceptions, such specimens can be captured without significant artefacts. When specimen thickness exceeds more than ca. 500 nm, as is the case for many prokaryotes and nearly all eukaryotes, vitreous slices must be obtained (see Chapter 11), analogous to sectioned plastic material. This currently represents a major bottleneck for cryo- electron tomography of vitrified biological specimens because the options for correcting cuttinginduced artefacts are severely hampered by the need to maintain vitreous specimens at temperatures below 137°C to avoid a deleterious phase change. This chapter is concerned with the workflow in cryo- electron tomography, from preparing the specimen so that it can be frozen without the formation of ice “crystals” to computational rendering of a three-dimensional volume that allows for detailed interpretation.

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1. PRINCIPLES OF CRYO- ELECTRON TOMOGRAPHY 1.1. Specimen Preservation It is common knowledge that biological specimens typically comprise more than 70% water. At first glance, this would seem to contradict the fundamental requirements of an electron microscope: Water boils spontaneously at the low pressures required to allow electrons to travel unimpeded through space. The most accurate preservation of molecular structure as well as specimen stability within the vacuum column of the microscope, thus, is achieved by rapid freezing, hence the prefix “cryo”, meaning “cryogenic”. With a sufficiently fast cooling rate, crystallisation of water is inhibited, and the solid water formed is amorphous (featureless). This is commonly described as vitreous or “glass-like” water.

In addition to the frozen sample’s negligible rate of evaporation in vacuum, cryofixation for electron tomography serves additional purposes: 1. Within the cell interior, biological nanomachines (proteins and protein complexes) must be imaged within a tiny fraction of a second so as not to be blurred by Brownian or ATPdependent motion if they are to be amenable to unambiguous structural recognition. 2. Three-dimensional structure is preserved at least to the level that can potentially be visualised by electrons (i.e. molecules).

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 Specimen preservation for cryoelectron tomography is essentially identical to that used for cryo- electron microscopy. A comprehensive description is provided in Chapter 7.  There are two practical ways to achieve vitrification: “plunge” freezing in liquid ethane (see Chapters 3, 4, 7) and highpressure freezing followed by vitreous cryosectioning (see Chapters 5, 6, 11).  Cryo- electron tomography is sometimes abbreviated to cryo- ET or CET. Although there is no such thing as a cryoelectron (hence the space, as well as the hyphen to indicate the prefix), there is also no such thing as a cryo- tomogram, nor does electron microscopy refer to microscopy of electrons. The debate is rather pointless; names of techniques are invariably compromised by the attempt at being concise.   Every suspended “particle” has an energy of approximately 1.5 kT, where k is the Boltzmann constant, and T is the temperature in Kelvin (see Chapter 1): Of course, vitrification precludes observations of dynamic events, placing emphasis on the need for novel correlative light and electron microscopy approaches.  In fact, structural preservation resulting from vitrification is superior to the practical limit of detection by electrons. It is sufficient for furnishing structures by means of electron crystallography.

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3. The native state is maintained without  After vitrification, image acquisition as well as any preparative steps (usually aggregation. limited to sectioning) must take place at 4. Improved resistance to irradiation with temperatures below 137°C, the devitrification temperature. electrons.

1.2. Acquisition and Mutual Alignment of Projection Images  Acquisition A tomogram is reconstructed from a series of two-dimensional projection images (see Figure 12.1).

  Various acquisition schemes are available in terms of constant exposure time or variations to account for increasing specimen thickness, e.g., linear exposure and Saxton schemes.5    Figure 12.1 Acquisition of a series of twodimensional projection images from a tilted specimen in the transmission electron microscope. Simulated, selected projection images of a macromolecular complex — the 20S proteasome — are shown. 

Imaging with electrons has a price: Biomolecules comprise mainly light atoms (C, H, O, N, P) and thus have little contrast to electrons. The most logical way to increase signal is to apply a larger electron dose; however, biological specimens embedded in a thin film of vitreous ice tolerate only about 5000 electrons nm-2, implying that the cumulative dose for a tiltseries must be kept below this level and distributed between the individual 2-D images, which in the case of cryo- ET typically numbers 60 to 80. This is known as “dose-fractionation.” Accordingly, each projection image has a very low overall contrast, and the final “signal” represents the combination of signal from all projections.

     Or by using the artefact-prone, indirect method: impregnating the specimen with heavy metal stains: Note that there have been attempts to combine the virtues of cryogenic preservation and heavy metal staining (see Chapter 9).

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 The signal of individual projection images must be sufficient to ensure accurate tracking of the specimen during acquisition.  

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The completeness of information is approximated by the Crowther criterion4 defined by the angular range sampled and a finite number of equally spaced projection images. The full angular range (180°) is generally not applicable in electron tomography because the grid holder geometry restricts the tilt range to ±70° and perhaps, more importantly, a tilted specimen with a flat (slab) geometry also becomes prohibitively thick with respect to the beam. For practical reasons (mitigation of beam damage and thus limiting the electron dose and, to a lesser extent, speeding up acquisition), a compromise must be made: The number of images is made constant and these are distributed over the maximum possible angular range, thereby defining the tilt increment. An energy filter operating in “zero-loss” mode removes contributions from inelastically scattered electrons. This diminishes blurring, especially in the case of thicker specimens.  Detectors Digital recording devices were critical for enabling automated data acquisition for electron tomography. Because images are acquired in real time, tracking and focus should be taken into account for subsequent images of a tilt series, leading to routine data acquisition under low-dose conditions. Pixel-based detectors, such as chargedcoupled device (CCD) cameras are rapidly approaching the quality of silver halide film, and single-electron detectors that do not suffer from “leakage” of signal into neighbouring pixels should provide the single most important foreseeable improvement in resolution for electron tomography.1

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 Actually, the Crowther criterion was described earlier by Bracewell and Riddle.6

 Novel sample geometries and tilting devices have the potential to solve both problems. 

 Essentially chromatic aberrations.  The mean free path for electrons in ice is approximately 350 nm, implying that an energy filter will provide an advantage even for a thin specimen that exceeds this apparent thickness during tilting.

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 Alignment Projection images must be aligned to a common coordinate system to account for rotations and unintentional changes in focus. This is most easily achieved by introducing an appropriate amount of 10 nm colloidal gold particles into the sample prior to vitrification. A minimum of three wellspaced particles (and preferably five to account for rotations) visible in all images of a series is usually sufficient to perform a satisfactory alignment. Quality control is possible by analysing the root mean square (RMS) errors and the trajectories (profiles) of gold particles for a tilt series. Analysis of  An RMS error of less than two pixels is marker profiles can help to eliminate an generally considered to be acceptable. outlier (spurious marker) or to show if a more serious problem exists, e.g., unexpected movement of the specimen.

1.3. Reconstruction 1.3.1. Principles of three-dimensional reconstruction according to Radon The mathematical description for producing a three-dimensional reconstruction from a series of two-dimensional images was described in 1917 by Radon.7,8 The reconstruction is achieved by backprojecting the images in reciprocal or Fourier space (see Figure 12.2). A weighting function (hence “weighted” back-projection) is used to account for variations in low- and high-frequency information. Without the weighting factor, reconstructions appear dull and featureless. 

 Even for most German speakers with a background in mathematics, the original version is reputedly difficult to read. Two English-language translations are available, those by Parks9 and by Lohner.10 

Figure 12.2. Computational synthesis of a three-dimensional image volume using back-projection. For clarity, only selected, simulated projection images are shown. 

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1.3.2. Reconstruction algorithms In addition to weighted back-projection, a number of more sophisticated reconstruction algorithms have been rediscovered recently for electron tomography. These include the iterative reconstruction algorithms ART (algebraic reconstruction technique) and SIRT (simultaneous iterative reconstruction technique). Both begin with an initial back-projection followed by multiple cycles of image comparison until a given stopping criterion is fulfilled. ART compares one slice to other slices, while SIRT compares each slice to the entire volume. 

 Weighted back-projection will continue to be applied for tomographic reconstruction because it is extremely rapid. If the results of weighted back-projection look satisfactory, the user can start a more timeconsuming iterative reconstruction.

1.3.3. Distortions Distortions in electron tomograms arise principally from the incomplete sampling of information. The specimen holder restricts the maximum tilt range to ±70° instead of the full tilt range of ±90° required for “isotropic” (balanced) resolution. This is known as the “missing wedge” in reference to its appearance in Fourier space. It gives rise to the perception that spherical objects are elongated.

 Even if holders did not restrict the angular tilt range, progressive thickening of the specimen relative to the electron beam during tilting to extreme angle prevents a full angular scan for most conventional sample geometries (plunge-frozen or cryosectioned materials have a “slab” geometry). For an untilted specimen with thickness 500 nm, tilting to 70° results in an apparent thickness of well over 1 µm.

Acquisition of a complementary tomogram  Distortions in accurately representing the obtained by tilting about an orthogonal axis 3-D information are covered in the results in more complete and more isotropic following section. resolution, the smaller “missing pyramid”. A second potential source of distortions is the fact that some features appear to be continuous with one another due to smearing, when in fact they might be discontinuous. This is a consequence of the finite tilt increment. More detailed reconstructions would result from smaller tilt increments. 

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1.4. Image Segmentation and Isosurface Rendering Homo sapiens is a “visual-centric” species,11 with a disproportional dependence on the sense of sight, and a unique ability to perceive three-dimensional space and colour. A three-dimensional volume that can be magnified, rotated and viewed from any direction thus is indispensable as a research tool. The aim of segmentation and isosurface rendering is the objective decomposition of a complex, three-dimensional object into its individual components for the purposes of improved comprehension12 (see Figure 12.3). 

Although computer-based segmentation is in principle more objective, manual segmentation is often the only way to delineate some portions of a cryotomogram. This is because human perception is superior to that of available segmentation algorithms. An a priori knowledge of an object’s identity, e.g., an actin filament, might be obtained by independent means, such as diffraction patterns indicating the characteristic spacing of actin monomers; however, the human mind is prone to biasing the perception of whether two features are truly interconnected.

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 Post selection operations, such as Gaussian smoothing or the removal of outlying pixels, may improve the aesthetic appearance of surface renderings, but possibly at the expense of accuracy and objectivity.   Interactive visualisation techniques — manually rotating and zooming with a computer or movies that animate aspects of this process — offer the possibility to examine a 3-D object to scale and without distortion. Furthermore, human vision is certainly capable of seeing potential connections between objects, but to some extent, it relies on motion for 3-D perception.  Figure 12.3. Selected, surface-rendered features of a cryo- electron tomogram displayed as an offset above an orthoslice (digital slice) of the tomogram. To aid in segmentation, the tomogram was denoised using nonlinear anisotropic diffusion (see following section) .13       

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1.4.1. Denoising In cryo- electron tomography, it is common practice to apply some form of denoising in  order to facilitate segmentation.14 Because denoising procedures typically reduce noise at the expense of some signal, this practice is not carried out prior to performing a pattern recognition procedure, such as template matching. Different levels of denoising can be used to enhance the delineation of various features having specific shapes, e.g., microtubules. For this purpose, a broad mask is applied for a conservative segmentation that includes a number of adjacent pixels on either side of the object. The mask is then applied to the non denoised data and thresholded to limit bleeding of the selection into other features. Where averaging of symmetrical or repetitive structures is not applicable, as is the case for the many uniquely shaped (pleomorphic) cellular features, classical, real-space digital filters, such as Gaussian smoothing, can be useful. Filtering means that the grey-scale value of a pixel is replaced by a new value based on its current value and the values of neighbouring pixels. More advanced digital filters, such as non-linear, anisotropic diffusion have been applied recently.  1.4.2. Surface rendering Surface rendering is used to improve the perception of the 3-D data. It is used to highlight, for example, the continuity of membranes, and the spatial relationships of macromolecular complexes to neighbouring organelles or molecules. A threedimensional image volume necessitates creativity in image display — a 3-D volume cannot be portrayed on a flat medium such as this sheet of paper without causing distortions to either shape (lack of conformity) or size. This is most easily comprehended by examining 2-D representations of the surface of a spherical object, such as the globe (see Figure 12.4). Map projections are typically visualized

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              The Earth is actually slightly elliptical, but the principle described here is still valid.

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geometrically as if they are transferred onto a cylinder, cone or plane. Reliefs of the Earth’s surface are described as conformal, equal area, or neither — they cannot be conformal and equal area. Cartographers have attempted to overcome this problem for centuries. All representations of this kind are compromises, but they may be useful for certain purposes. The Mercator projection, only recently superseded for navigation by satellite-based global positioning systems (GPS), is extremely useful for navigation because distortion is constant along any parallel. In other words, at any given point, east–west scale is the same as the north–south scale.

 “Conformal” refers to a projection for which all angles at each point are preserved.  “Equal-area” projections show all areas in proportion to their true areas, but shapes become distorted. 



 Despite its distortions, the Mercator projection is the only map projection that shows true bearings for navigation.

 Distortion map (Tissot Indicatrix) for the Mercator projection. All circles are perfect spheres, indicating that there is no angular distortion, but the areas vary. The consequence is that a land mass, such as Greenland, appears to be the size of Africa, when it is, in fact, only a fraction of the size.   Figure 12.4 Distortions induced by projecting a sphere onto a cylinder: the Mercator projection.

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Discontinuous projections (see Figure 12.5) are useful for emphasising certain features at the expense of others, e.g., land masses and oceans, and potentially restricting the gross distortions to the regions that are not emphasised.

    Selected references:  John B. Garver Jr., New Perspective on the World, National Geographic Magazine, 1988, pp. 911–913.  John P. Snyder, Flattening The Earth – 2000 Years of Map Projections, The University of Chicago Press, Chicago, USA, 1993, pp. 214216.  Figure 12.5 Mollweide projection, which biases the perception by emphasising continuities between land masses.

2. SUMMARY OF THE DIFFERENT STEPS 1. Culture of cells in suspension or on TEM grids. 2. Addition of colloidal suspension cells.

gold

to

3. Vitrification by plunge freezing in liquid ethane or using a highpressure freezing instrument. 4. Vitreous sectioning (if high-pressure frozen).  4a = Deposition of inorganic, colloidal particles onto vitreous section 5. Transfer to electron microscope in cryo- holder. 6. Mapping of specimen grids using low magnification; determination of suitable areas for tomography. 7. Basic transmission electron microscope (TEM) alignments, setting of eucentric height, setting of tomography parameters (tracking area and dose, focus area and dose, exposure weightings). Figure 12.6

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3. EQUIPMENT/PRODUCTS/REAGENTS 3.1. Equipment  Plunge-freezing apparatus

 High-pressure freezer

 Grid boxes and opening tool  Self-closing forceps

 Plasma device for glow-discharge

 Liquid nitrogen Dewars for storage  Cryo- ultramicrotome  Diamond knives

 Cryo- electron microscope

 Cryo- tilt holder

 Light microscope and cell incubator

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 Leica Microsystems, Vienna, Austria, or homemade; controlled temperature and humidity using the Vitrobot, FEI Electron Optics, Eindhoven, The Netherlands (see Chapter 4).  Leica; BAL-TEC HPM 010 now available from Boeckeler Instruments, Inc., Tucson, Arizona, USA; M. Wohlwend GmbH, Sennwald, Switzerland (see Chapters 5, 6).  Self-made or commercial supplier.  Dumont #5, Dumont S.A., Switzerland, preferably Teflon®-coated or insulated with additional foam; more sophisticated and costly forceps are available: Dumont #545.  For rendering carbon-coated TEM grids hydrophilic and for removing organic contaminants, e.g., Harrick Plasma, Ithaca, New York, USA.  For example, Taylor-Wharton, Husum, Germany; Theodore, Alabama, USA; Air Liquide S.A., Düsseldorf, Germany.  For example, Leica Ultracut UC6 + FC6 (see Chapter 11.)  For example, Diatome AG, Biel, Switzerland; Drukker International B. V., Cuijk, The Netherlands; Delaware Diamond Knives, Wilmington, Delaware, USA.  An energy filter is indispensable for thicker specimens, where “thicker” refers to the relationship between the mean free path of electrons in ice and the maximum specimen thickness encountered during tilting.  For example, Gatan Model 626, Gatan, Pleasanton, California, USA; dedicated cryo-microscopes, such as the Polara and the Krios, have built-in capabilities, FEI Company, Eindhoven, The Netherlands.  Quality control, correlative microscopy.

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3.2. Products  TEM grids

 Various commercial suppliers; more sophisticated carbon coatings from Quantifoil Micro Tools GmbH, Jena, Germany or C-flat, Protochips, Inc., Raleigh, North Carolina, USA.  Tomography acquisition and  Commercial products, such as reconstruction software Explore3D, FEI Electron Optics, Eindhoven, The Netherlands or opensource, dedicated tomography packages, e.g., TOM Toolbox, www.biochem.mpg.de/tom  Segmentation and rendering software  Amira, Visage Imaging, inc., Karlsbad, California, USA or Imaris, Bitplane AG, Zürich, Switzerland; enhanced capabilities are provided with 3DSMax or Maya, Autodesk, Munich, Germany. 

3.3. Reagents  Liquid nitrogen

 In Europe: Messer Griesheim, Linde, Westfalen. Must be kept dry.  Ethane gas  BOC gases, part of Linde AG, Munich, Germany. w  20% and 40% ( /v) Dextran in  Neutral polysaccharide from physiological buffer or growth Leuconostoc mesenteroides. Sigma D-4876, medium Mr 100,000 to 200,000 dissolves quite rapidly.  Colloidal gold  Self-made as described in Section 4.1, or e.g., Sigma G-1527, Sigma Chemicals, St. Louis, Missouri, USA; note that the commercial product contains sodium azide, a potent respiratory poison. In either case, it is preferable to conjugate with protein A or bovine serum albumin to prevent autoaggregation. In the absence of NaN3, shelflife of protein-stabilised gold is limited to several weeks at room temperature, and dissociation may also limit the usefulness of older preparations. Safety information Extra caution is required when using liquid nitrogen (suffocation risk, cold injury) and ethane (explosion risk). Read the Materials Safety Data Sheets for all reagents before use. Disclaimer The authors do not have financial interests in any of the above-mentioned companies or products.

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4. PROTOCOLS 4.1. Preparation of Protein-Stabilised Colloidal Gold It is common practice in cryo- electron  tomography to add colloidal gold particles to cell suspensions shortly prior to plunge freezing (see Figure 12.7).    Figure 12.7 Projection image of a bacterium embedded in vitreous ice. The preparation contains colloidal gold particles to enable subsequent alignment. For details, see Section 7, Observed Results, Figure 12.3. Bar = 200 nm Colloidal gold with a size of 10 to 12 nm is  most commonly prepared by the citrate reduction method followed by conjugation with either Protein A (from Staphylococcus aureus) or albumin. Although there are other ways of preparing colloidal gold, this method is easy and reliable.  Citrate reduction method, based on the 1926 Nobel Prize lecture of R. A. Zsigmondy, for 10 to 12 nm gold colloids.15

     1. Solution A: Add 1 mL 1% tetrachloro  aurate to 80 mL distilled water in a  strongly stirred beaker.  2. Solution B: Dilute 4.5 mL 1% trisodium citrate dihydrate to 20 mL with distilled water and add 40 µL 1% tannic acid (e.g., Mallinckrodt #1764).

 The ratio of gold to tannic acid governs the resulting colloid size. 40 µL of tannic acid should give approximately 12 nm colloids.

3. Warm solutions A and B separately to  The resulting solution has a pH of about 60°C. Mix solutions A and B rapidly, 5.0. which should become black and then turn red after 30 minutes.

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Advantage over commercial preparation: no  sodium azide and, therefore no immediate toxicity when added to cells.  Stabilisation of gold colloids with  bovine serum albumin (BSA) 1. Bring the pH of the gold colloid  suspension to pH 6 using dilute base (NaOH, KOH) while stirring. 2. Dissolve BSA at a concentration of 2 mg/mL in a pH 6, low-molarity buffer, e.g., 1 to 2 mM phosphate buffer. Recheck the pH and adjust to 6 if necessary.

 Protein A from Staphylococcus aureus can be used instead of BSA; the stock solution should be made at 1 mg/mL, and the amount to be added to the gold solution is about 5 µg/mL, i.e., about five times less Protein A is needed than for BSA. The exact amount to be added can be determined by a microtitration assay. Usually, coupling with Protein A is followed by the addition of BSA to a concentration of 0.2% to ensure maximum stability.

3. Add 10 µL of the BSA solution per mL of gold suspension while stirring. Allow to stand for 10 minutes, and then adjust the pH depending on the intended application.

      



4.2. Application of Quantum Dots to the Surfaces of Vitreous Cryosections This procedure was introduced by Masich et al.16 It is based on Tokuyasu’s observation that isopentane is liquid at 150°C, and that commercially available inorganic semiconductor particles (lead sulfide quantum dots) are soluble in toluene, which in turn is miscible with isopentane. An abbreviated version is described here.

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 Analysis of marker alignment profiles suggests that particles associated with surface artefacts (crevasses) move during the course of image acquisition, resulting in poor alignment. It is possible to incorporate fiducial markers within suspensions of microorganisms prior to vitrification and sectioning; however, introduction of foreign particles to the cell interior is invasive unless the aim is to study endocytosis.  

Cryo- Electron Tomography

The main advantage of this method is that it is universally applicable, i.e., independent of specimen type (cryosections of eukaryotic or prokaryotic cells) and thus non invasive. Additional special equipment: steel or aluminium blocks containing two wells, one for the quantum dot suspension and one for liquid ethane. The well for the isopentanebased quantum dot suspension should be countersunk to form a funnel at the top of the well. 1. Cut vitreous sections and apply them to TEM grids as usual.

309  Disadvantages: The method is based on adsorption of inorganic particles onto the section surface, the same region which suffers from the most artefacts and thus changes most rapidly during acquisition. This is, of course, a fault of the section, not the marker deposition method. Unlike colloidal gold, quantum dots are perfect crystals. This means that the contrast changes abruptly for different tilt angles, making detection difficult for thicker specimens containing large variations in contrast. A further problem is that this particular type of quantum dot (PbS in toluene) is especially large (15 to 17 nm) which is inaccurate for a tilt series taken using high magnifications.

2. Cool the metal block using liquid nitrogen and allow the nitrogen to evaporate from the wells in the cryoultramicrotome chamber. 3. Fill one well with ethane gas, which will liquefy within the cooled well. Immerse the block in liquid nitrogen. The ethane will solidify to a white mass. Transfer the block to the chamber of the cryo-microtome. The ethane will quickly become a liquid as the block warms to 150°C in the microtome chamber. If the ethane fails to liquefy, move the block to the surface of the knife holder. Fill the other well with a suspension of lead sulfide quantum dots dissolved in toluene and diluted 50:1 (v/v) with isopentane. 4. Dip the grid containing the sections into the quantum dot suspension, rinse briefly in liquid ethane, and then blot the excess ethane by wicking onto a piece of filter paper in the chamber. The ethane must be seen to have wet the paper. Store the grids in liquid nitrogen for microscopy.

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 The back of the chamber might be significantly cooler than the setting indicated on the microtome display panel.  The ethane will liquefy quickly, but it does not evaporate very fast at this temperature; there is no need to rush the experiment.

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5. ADVANTAGES/DISADVANTAGES 5.1. Advantages of Cryo- Electron Tomography  Intuitive, 3-D depiction.  Maintenance of specimen hydration.  No staining.  No chemical fixation.  Direct recognition of large complexes.  Spatial resolution of 4 to 5 nm.  Safe.

 Clear visualisation concerning the spatial relationships of a macromolecular complex to other macromolecules and organelles.  Essential for maintaining the spatial coordinates of “soluble” cytoplasmic components.  Direct visualisation of native structure rather than an uninterpretable combination of stain (dominant) and specimen.  Physical fixation (cryo- fixation) serves the purpose of immobilisation without the aggregation artefacts; faster.  Indirect recognition by immunolabelling is restricted to the section surface (2-D).  An advantage relative to light microscopy.  Fewer and less hazardous reagents reduce the potential for generating artefacts in specimen and user.

5.2. Technical Limitations in Cryo- Electron Tomography  Some specimens, e.g., brain tissue,  Vitrification is essentially an all-ormay be difficult to vitrify.17 nothing phenomenon and depends on the ratio of water to polymer.  Limited contrast of specimen.  Reliance mainly on phase (cf. amplitude) contrast; low signal-to-noise ratio makes interpretation and segmentation difficult.  Low tolerance to radiation damage.  Limit to the tolerable dose = limit to the number of images = finite tilt increment = incomplete information.  “Working blind” — difficulty in surveying the specimen without spending the restricted dose on secondary tasks.  Optimal alignment often possible only  Low signal-to-noise level means that by means of fiducial markers. cross-correlation approaches are either unreliable or less accurate; introduction of markers should be done in a non invasive manner.

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 Limited tilt range, typically ± 60°.

 Incomplete information, manifested as distortions.

 Kinetic studies difficult.

 Requirement to capture specific events, and to relate tomograms to events immediately prior to vitrification (correlative microscopy); this is, of course, a characteristic of all electron microscopy, not just cryo- ET.

 Thinning of vitreous specimens is  Choices are few and imperfect; the difficult. vitreous character of the specimen must not be compromised.  Dedicated cryo- stages/microscopes  The price for trying to maintain biology necessary. within a vacuum column: “suspended animation.”

6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Vitrification Methods 6.1.1. Plunge-freezing



 Depending largely on polymer concentration and as a corollary, on water content, plunge-freezing can be used to vitrify biological specimens contained within thin films to a depth of 3 to 20 µm. Such specimens include purified suspensions of macromolecules or suspensions of unicellular microorganisms.

 Specimens thicker than ca. 800 nm are not electron-transparent. The limiting factor for tomography, therefore, is specimen thickness (cell + overlying ice) rather than vitrification depth.



 Because the depth of vitrification  Physical ablation results in the achieved by plunge-freezing is under- irreversible loss of a significant portion of a utilised and bypasses the artefacts of specimen. the cryo- sectioning process, it may be possible in the future to take greater advantage of the effective depth of vitrification by means of specimen thinning techniques, such as focussed ion beam milling.  

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6.1.2. High-pressure freezing  High-pressure freezing is the method  High-pressure freezing is the method of of choice for specimens that are not choice for mammalian cells and tissue electron transparent, i.e., > ca. 1 µm, biopsies. with the implication that it can only be used in conjunction with appropriate sectioning techniques. The depth of vitrification is at least 200 µm, allowing vitrification of cells and tissues that are not amenable to plunge freezing.  

6.2. Alignment and Reconstruction Methods 6.2.1. Fiducial marker alignment  This method is generally used in combination with plunge-freezing (see Section 5.1) for suspensions of small, unicellular organisms or macromolecules where the gold does not penetrate the specimen and thus can be used for non invasive alignment.

 Incorporation of particles within the ice and thus within the specimen volume should result in the most satisfactory results because multiple planes of the specimen are taken into account during alignment, and the markers are less likely to move as may occur for surface-adsorbed particles.

 With a little more effort, the variant method described for vitreous cryosections in Section 4.2 is suitable for any specimen because it is applied post vitrification.  6.2.2. Cross-correlation alignment  This method is generally used when fiducial marker alignment is not possible. An example is the lack of sufficient gold particles in vitreous cryosections, especially in the case of eukaryotic cells where the extracellular matrix may not be captured within the imaged area.

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 Cross-correlation or general featurebased alignment may be used in combination with marker alignment, although this is generally not applicable for cryo- tomography; either marker alignment is satisfactory or there are no markers available. The “features” of unstained specimens are generally not obvious at all tilt angles.

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 Cross-correlation often fails at high tilt angles because the signal-to-noise ratio is insufficient to generate a reliable cross-correlation peak.

6.2.3. Choosing a reconstruction algorithm  Weighted back-projection (WBP) is a mathematically sound and computationally inexpensive means of reconstructing an image volume. Iterative algorithms18 are computationally expensive, but may give more realistic-looking reconstructions.

 WBP reconstructions are rich in contrast, but this is partly a result of halo-like brightness around dark objects. Depending on once point of view, this is either aesthetically pleasing or artificial.

 SIRT is preferable to ART for cryoelectron tomography projections because it is less prone to fail when the images are dominated by noise. SIRT reconstructions are not dramatically different to those produced by weighted back-projection, but the images have a natural appearance in that they lack artificial edge enhancements, and back-projection “rays” are suppressed.

 Computation time will eventually become irrelevant. WBP is a convenient way of seeing the reconstruction within a few moments.

 If the goal is to perform templatematching, it is essential to use a method, which does not contain constraints, e.g., WBP.



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7. OBSERVED RESULTS  Figure on the Chapter’s title page

 Three-dimensional reconstruction of a hydrated biological specimen from a series of two-dimensional projection images (schematic).

 Figure 12.8 (see colour inserting  Multiscale imaging of hydrated following page???.) mammalian cells. A: Phase contrast light micrograph B: Cryo- EM projection image C: Segmented cryo-electron tomogram from a plunge-frozen mouse adenocarcinoma cell grown on an EM specimen grid Note that the central regions are inaccessible unless sectioned. Bar for A = 20 µm Bar for B = 500 nm  Additional colour information:  Blue: Cell membrane  Green: Microtubules  Yellow: Transport vesicles  Red: Macromolecular complexes  Segmentation and surface rendering by M. Gruska and L. Andrees, MPI for Biochemistry.  Cells were provided by W. G. Müller and J. McNally, National Institutes of Health.  Figure 12.9

 Low-dose projection image of a portion of a bacterium, Magnetospirillum gryphiswaldense, showing the 10 nm colloidal gold particles used for alignment. Magnetosomes are iron oxide particles used for orientation according to the Earth’s magnetic field. For details, see Scheffel et al.19. Bar = 200 nm

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8. REFERENCES 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18. 19.

Nickell, S. et al. A visual approach to proteomics, Nat. Rev. Mol. Cell Biol., 7, 225, 2006. Baumeister, W. Mapping molecular landscapes inside cells, Biol. Chem., 385, 865, 2005. Dubochet, J. and Sartori Blanc, N. The cell in absence of aggregation artifacts, Micron, 32, 91, 2001. Crowther, R.A., DeRosier, D.J., and Klug, A. The reconstruction of a threedimensional structure from its projections and its applications to electron microscopy, P. Roy. Soc. Lond. A Mat., 317, 319, 1970. Saxton, W.O., Baumeister, W., and Hahn, M. Three-dimensional reconstruction of imperfect two-dimensional crystals, Ultramicroscopy, 13, 57, 1984. Bracewell, R.N. and Riddle, A.C. Inversion of fan-beam scans in radio astronomy, Astrophys. J., 150, 427, 1927. Radon, J. Über die Bestimmung von Funktionen durch ihre Integralwerte längs gewisser Mannigfaltikeiten. Berichte über die Verhandlung der Königlich Sächsischen Gesellschaft der Wissenschaften zu Leibzig, in Mathematische Physische Klasse, 1917, 262. Radon, J. On the determination of functions from their integral values along certain manifolds, IEEE Trans. Med. Imaging, MI-5, 170, 1986. Parks, P.C. IEEE Trans. Med. Imaging, MI-5, 170, 1986. Radon, J. On the determination of functions from their integrals along certain manifolds, in The Radon Transform and Some of Its Applications, Deans, S.R., ed., Wiley-Interscience, New York, USA, 1983, Appendix A. Anon Seeing is believing, Nat. Methods, 2, 889, 2005. Frangakis, A.S. and Hegerl, R. Segmentation of three-dimensional electron tomographic images, in Electron Tomography: Methods for Three-Dimensional Visualization of Structures in the Cell, Frank, J., ed., Springer-Verlag, Heidelberg, Germany, 2006, 353. Frangakis, A.S., Stoschek, A., and Hegerl, R. Wavelet transform filtering and nonlinear anisotropic diffusion assessed for signal reconstruction performance on multidimensional biomedical data, IEEE Trans. Biomed. Eng., 48, 213, 2001. Hegerl, R. and Frangakis, A.S. Denoising of electron tomograms, in Electron Tomography: Methods for Three-Dimensional Visualization of Structures in the Cell, Frank, J., ed., Springer-Verlag, Heidelberg, Germany, 2006, 331. Slot, J.W. and Geuze, H.J. Sizing of protein A-colloidal gold probes for immunoelectron microscopy, J. Cell Biol., 90, 533, 1981. Masich, S. et al. A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography, J. Struct. Biol., 156, 461, 2006. Zuber, B. et al. The mammalian central nervous synaptic cleft contains a high density of periodically organized complexes, Proc. Natl. Acad. Sci. USA, 102, 19192, 2005. Gilbert, P. Iterative methods for the three-dimensional reconstruction of an object from projections, J. Theor. Biol., 36, 105, 1972. Scheffel, A. et al. An acidic protein aligns magnetosomes along a filamentous structure in magnetotactic bacteria, Nature, 440, 110, 2006.

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Part III Low Temperature Embedding

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CONTENTS

GENERAL INTRODUCTION .................................................................................... 323 1.

PRINCIPLES OF FREEZE-SUBSTITUTION............................................... 325

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 326

3.

MATERIALS/PRODUCTS/SOLUTIONS ...................................................... 327 3.1. 3.2. 3.3.

4.

PROTOCOLS .................................................................................................... 331 4.1. 4.2. 4.3. 4.4. 4.5. 4.6. 4.7. 4.8.

5.

Sample Preparation................................................................................... 331 Preparation of Substitution Apparatus...................................................... 331 Freeze-Substitution................................................................................... 331 Preparation for Resin Embedding............................................................. 332 Low-Temperature Embedding in Methacrylates ...................................... 332 Low-Temperature Polymerisation ............................................................ 333 Curing of Methacrylates ........................................................................... 333 Embedding in LR White and Epoxy Resins ............................................. 333

ADVANTAGES/DISADVANTAGES.............................................................. 334 5.1. 5.2.

6.

Materials ................................................................................................... 327 Products .................................................................................................... 327 Solutions ................................................................................................... 329 3.3.1. Substitution media ...................................................................... 329 3.3.2. Embedding media ....................................................................... 330

Advantages ............................................................................................... 334 Disadvantages........................................................................................... 334

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 335 6.1. 6.2. 6.3.

Choice of Substitution Medium................................................................ 335 Choice of Resin ........................................................................................ 335 Osmium Tetroxide and Low-Temperature Embedding ............................ 335

7.

OBSERVED RESULTS .................................................................................... 336

8.

REFERENCES .................................................................................................. 340



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GENERAL INTRODUCTION Our life is confined to water. All cellular processes need water to work. As soon as water is removed, not only do the processes cease and life stops, but also the cellular ultrastructure is altered. Therefore, the most straightforward method to study cells and cellular structure and architecture by microscopy is to visualise them in their water environment. For light microscopy, live cell imaging has become a well-established method. For higher resolution in an electron microscope, however, the vacuum required to allow electrons travel does not allow visualisation of living cells in liquid water. The closest approach is to embed the biological material in a thin layer of ice and image it in a cryo-electron microscope (see Chapters 1, 11).1,2 This method has its limitations: A thin sample is needed and ice or ice-embedded biology is very beam sensitive. Recently, marvellous results were obtained by cryo-electron tomography (see Chapter 12)3,4 and even non invasive, immunolabelling by template matching is within reach5 and with it, imaging life and identification of the molecular machinery in situ. Up to now this fantastic technique has been limited to very thin samples, which can be cryo-fixed on a specimen grid by, e.g., plunge-freezing.3 A more useful method would be to cryofix a thick specimen, a piece of tissue, and make thick cryo-sections. Cryo-fixation of reasonably thick samples, (2 mm diameter, 100 to 300 μm thickness) can be achieved by high-pressure freezing.6-8 From these samples ultrathin cryo-sections can be cut with a cryo-ultramicrotome9, but not thick sections10 employed in electron tomography. Despite the limitation of section thickness, electron tomography on ultrathin cryo-sections is very useful to acquire high-resolution data (see Chapter 12), but cannot visualise the distribution of larger entities in their cellular context. A solution for this problem is looming on the horizon. With a new instrument, a focused ion beam (FIB) scanning electron microscope, a semithin (ca. 300 nm) section can be cut with a gallium ion beam under observation with an electron beam.11 The section can be lifted out and transferred into a cryo-transmission electron microscope. This method is still emerging and, until this new method is established for routine applications, we use a hybrid technique, freezesubstitution, which can bridge the gap between cryo- and conventional electron microscopy. To ensure high quality preservation of biological samples, we choose cryo-fixation with high-pressure freezing in combination with freeze-substitution and resin embedding.12-14 The freeze-substitution technique comprises dehydration of a cryo-fixed sample at 90oC by dissolving the ice with an organic solvent and, if wished, chemical fixation during the substitution process. The term freeze-substitution often also implies subsequent resin embedding.

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The methodology of freeze-substitution already has a long history. It was first described by Simpson in 1941.15 Thereafter, it was further developed and improved.16-20 Today, it has become a standard technique (for review, see12). Probably the most convincing data to demonstrate the supremacy of cryo-fixation and freeze-substitution has been presented by Studer et al.22 In chemically fixed nodules of soybean, the Rhyzobium bacteria have no contact with the cell membranes of the nodules, whereas there is a tight connection when visualised by cryo-fixation and freeze-substitution. In fact, this direct contact is a prerequisite for their function because a mutant, which has no contact to the nodule membranes, cannot convert atmospheric nitrogen into NH4+. Another great advantage of the freeze-substitution technique is that it can be combined with on-section immunogold labelling12,23 for localisation studies with reliable morphology. Several modifications of the freeze substitution procedure have been devised to overcome two major draw-backs: 1) the heavy metal salts used for contrasting penetrate poorly into the section, and 2) in well preserved, i.e., optimally cryo-fixed cells, internal membranes are difficult to visualise. These limitations have been addressed by changing the substitution method. Three-dimensional contrast can be achieved by adding uranyl acetate and osmium tetroxide to the substitution medium.12 Membrane contrast can also be enhanced by adding contrasting agents,24 but more reliably by adding a small amount of water to the substitution medium.25 The amount of water to be added is dependent on the sample and varies between 1 and 5%. In this chapter, I would like to present and discuss our recently developed protocols to improve sample preparation by freeze-substitution, especially with respect to their application in cellular electron tomography14,26-27 (also see Chapter 24).

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1. PRINCIPLES OF FREEZE-SUBSTITUTION The biological sample is cryofixed by  Optimum structural preservation, close means of one of the methods suited for to the native state of the biological cryo-fixation (see Part I, Cryo-Fixation specimen. Methods), e.g., by high-pressure freezing. The layer of optimum cryo-fixation is dependent on the method used and on the content of free water within the specimen. For high-pressure freezing, the thickness of well-frozen samples may vary between 100 μm to about 300 μm. At about –90°C, the cryofixed samples are put into an organic substitution medium, based on, e.g., acetone, ethanol or methanol, containing chemical fixatives, such as uranyl acetate, osmium tetroxide and aldehydes. The samples are kept at –90°C for 8 hours up to as much as 80 hours.

 At –90°C, the ice will be dissolved by the organic solvent, the sample is dehydrated. The solvent and the chemical fixatives will surround the biological structures. The dehydration time is dependent on the specimen, e.g., cells with thick cell walls, such as yeast or plant cells, will need more time for the exchange of water than cultured cells. In addition, slow dehydration better preserves the natural distribution of ions and reduces shrinkage artefacts (see Chapter 15).

After dehydration, the temperature is raised to the desired embedding temperature, e.g., –40°C to –30°C for Lowicryl HM20 or K11M, but also for Epon and LR White.

 During the warming-up process the chemical fixatives start to react: uranyl acetate as soon as the negative charges of the nucleic acids and phosphate groups become accessible, osmium tetroxide at around –70°C28 and glutaraldehyde between –40°C and –30°C.12

For certain protocols, the temperature is  To improve membrane contrast.24 raised up to 0°C for 1 hour before embedding. Embedding in the Lowicryls takes place  K4M at –30°C; HM20 from –30°C to between –60°C and –30°C depending on –50°C; K11M to –60°C; and HM23 may be the formulation of the resin. used down to –70°C.

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The first dilutions of Epon and LR White can also be infiltrated at around –30°C, but the further embedding steps are done at room temperature and curing at +50°C.

 A 30% solution of Epon or LR White is liquid at –30°C. Epon/acetone mixtures are liquid at –30°C up to 60% Epon, however, not Epon/ethanol mixtures! In case of LR White, mixtures with ethanol can and should be used.

The methacrylate-embedded samples are suited for on-section immunolabelling studies, whereas the Epon-embedded samples are best suited for morphological analysis including electron tomography.

2. SUMMARY OF THE DIFFERENT STEPS 1. Cryo-fixation

 Best preservation of the cellular and molecular architecture by immediate arrest.  Note, that during freeze-substitution the dehydration and chemical fixation step are inverted. Figure 13.1 Schematic representation of the freeze-substitution process.

 During warming up, amorphous water can crystallise into cubic ice; ice is the Chemical fixation thermodynamically more stable form of water. This process is also called deResin infiltration and vitrification and in pure water depolymerisation vitrification takes place at around –135°C (see Chapters 1, 11). At the temperature Sectioning freeze-substitution starts, amorphous ice should not exist anymore, this would imply Immunolabelling that cubic ice has no influence on the morphology at the level of resolution of Analysis by light microscopy and biological samples. electron microscopy

2. Dehydration 3. 4.

5. 6. 7.

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3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Freeze-substitution apparatus. Insets for the freeze-substitution apparatus with small holes in the bottom to hold glass vials and microtubes.  Microtubes 500 μL and 1500 μL.

 Glass vials. 

 Perforated insets.  Forceps.  Styrofoam boxes.  Ice.  UV lamp ~ 360 nm.  Oven.

 Self-made21 Leica EM AFS or EM AFS2, Leica, Microsystems, Vienna, Austria. RMC FS-7500, Boeckeler Instruments Inc. Tucson, Arizona, USA.  The screw and seal tubes are very well suited for freeze-substitution (1.5 mL) tubes #72.609; Sarstedt AG & Co, Nümbrecht, Germany.  The tubes used for methacrylate embedding need to be absolutely air tight. Here we use 0.5 mL Sarsteadt #72.699.  Used to prepare the solutions to be precooled in the substitution unit.  Borosilicate glass counting vials (20 mL) from Wheaton, Millville, New Jersey, USA, with urea screw cap #986542 (Closure Liner: Metal Foil) or 986546 (Closure Liner: Poly-Seal Cone) or Wheaton Liquid Scintillation Vials from Electron Microscopy Sciences, Hatfield, Pennsylvania, USA, #72632 or PerkinElmer, Waltham, Massachusetts, USA, #6000096.  Leica plastic capsules D5 x H 15 mm #16702738; Leica, Microsystems, Vienna, Austria.  For example, Dumont (#2a, #3, #4, #4N, #7), Dumont & Fils, Switzerland.  Use different sizes for the preparation of the sample and media.  Used for incubation at 0°C.  Used for low temperature embedding in methacrylates.  Used for embedding in LR White or epoxy resins.

3.2. Products  Acetone.

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 Dry acetone (Merck #1.00299.0500, Merck KGaA, Frankfurter Strasse 250, D64293 Darmstadt, Germany): The open bottle is stored under nitrogen gas in a closed container above silica gel.

328  Methanol.

 Ethanol.

 Sodium hydroxide (NaOH).  Glutaraldehyde.

 Osmium tetroxide.

 Uranyl acetate.

 Double distilled water.

 Lowicryl resin:  HM20, HM23, K4M, K11M

 London Resins:  LR White, LR Gold

 Epon.

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 Dry methanol (Merck #1.06012.0500), the open bottle is stored under nitrogen gas in a closed container above silica gel.  Dry ethanol (Merck #1.00990.0500), the open bottle is stored under nitrogen gas in a closed container above silica gel.  Merck.  Glutaraldyde is available as a 10% solution in water-free acetone (EMS #16530), ethanol (EMS #6531) or methanol (EMS #16532) or a 50% solution in water.  10 × 1 g ampoules #19110 or 10 × 0.1 g ampoules #19134; EMS, Hatfield, Pennsylvania, USA.  EMS, Hatfield, Pennsylvania, USA or SPI Supplies, West Chester, Pennsylvania, USA.  Quartz double distilled water is preferred over Millipore filtered water, but both can be used.  The HM resins are hydrophobic, whereas the KM resins have some hydrophilic properties, e.g., EMS or Polysciences Europe GmbH, Eppelheim, Germany.  There are premixed versions of the Lowicryl resins. We, however, prefer to mix the solutions ourselves to avoid aging and prepolymerisation during storage.  LR White is for heat polymerisation and LR Gold may be UV polymerised down to ca. 10°C. By changing the initiator, using dibenzoyl peroxide, LR White is also suited for low temperature embedding down to about 5°C; EMS. Caution: LR White does not always contain the catalyst needed for polymerisation. It can also happen that mixtures of LR White and ethanol polymerise prematurely. Therefore, it is advisable to test every new batch before use.  For example, #45359, Fluka, SigmaAldrich Chemie GmbH, CH-9471 Buchs, St. Gallen, Switzerland.

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 Spurr’s resin.



329  Due to its toxicity, carcinogenicity and the high vapour pressure, we prefer not to use Spurr’s. In our hands, Epon embedding works even with samples difficult to infiltrate, such as yeast.  #14300, EMS or #R1032, Agar Scientific Ltd, Stansted, UK.

3.3. Solutions 3.3.1. Substitution media  High contrast FS medium17  3% (v/v) Glutaraldehyde  1% (w/v) Osmium tetroxide 0.5% (w/v) Uranyl acetate in methanol

 Standard FS medium19,29  1 to 5% (w/v) osmium tetroxide in acetone

 Uranyl acetate is prepared as a 20% (w/v) stock solution in methanol. Equal amounts of 6% (v/v) glutaraldehyde, 1% (w/v) uranyl acetate in methanol and 2% (w/v) osmium tetroxide in methanol are cooled above liquid nitrogen close to the freezing point of the solutions. Then the glutaraldehyde/uranyl acetate solution is poured into the osmium tetroxide solution and the container is well shaken. This medium can be stored in Teflon® bottles under liquid nitrogen. As long as the solution has a greenish yellow colour, the quality of the medium is good. Do not use it anymore when it turns golden yellow. Always keep close to the freezing point when handling (Dr. M. Müller, personal communication).  This is the classical freeze-substitution medium.  We usually use 2% osmium tetroxide.

    

 Either of the chemical fixatives or any combination of them may be used.  To enhance membrane contrast, depending on the sample, 1 to 5% water can be added.

Membrane contrast medium 0.5% (v/v) glutaraldehyde 1% (w/v) osmium tetroxide 0.2% (w/v) uranyl acetate x% (v/v) water25 in acetone

 Wild FS medium24  Used to enhance resolution of the  0.25% glutaraldehyde (50% aqueous membrane bilayer. stock solution)  0.5% osmium tetroxide in acetone

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 In combination with rapid freeze Hawes FS medium30 a) morphology substitution and embedding in Lowicryl  2% uranyl acetate in acetone (from a HM20 at 50oC. 20% methanol stock solution) b) immunolabelling  0.2% uranyl acetate in acetone (from a 20% methanol stock solution)  Pure solvent for FS20  Acetone, or  Ethanol, or  Methanol.

 In combination with low-temperature embedding, this medium is suited for immunolabelling of aldehyde-sensitive proteins.

 Low concentration of aldehyde FS  Either of these chemical fixatives is medium12 used.  2% (w/v) formaldehyde, or  In combination with low-temperature  0.05% (v/v) glutaraldehyde, or embedding, this medium is suited for immunolabelling.  0.5 % (w/v) uranyl acetate in methanol  Paraformaldehyde can be dissolved in methanol by adding sodium hydroxide pellets.  Methanol may be replaced by acetone. In this case, uranyl acetate can only be dissolved at the maximum concentration of 0.2% (w/v).  This medium is used for optimum  Epoxy fixation FS medium31  20% Araldite/Epon embedding mix- preservation of the cellular ultrastructure. The intracellular membranes are only ture in acetone visible in negative contrast.  This medium is also suited for immunolabelling. 3.3.2. Embedding media

 The embedding media are prepared  Epon, Spurr’s, Lowicryl and London according to the manufacturer’s instructResin embedding tions.  Dr. M. Müller, personal communication.  Araldite/Epon embedding mixture31  49% (w/w) Araldite/Epon stock solution:  41% (w/w) Epoxy-812 substitute  Fluka #45345  54% (w/w) Durcupan A/M epoxy  Fluka #D0291 resin  5% Dibutyl phthalate  Fluka #80100  49% (w/w) Hardener DDSA

 Fluka #45346

 2% (w/w) Accelerator DMP-30

 Fluka #45348

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4. PROTOCOLS 4.1. Sample Preparation  Sample the biological specimen as carefully as possible.  Cryofix with one of the cryo-fixation machines.  The frozen samples may be stored under liquid nitrogen until use.

 Extreme care should be taken so that the natural environment is altered as little as possible (temperature, pH, osmolarity, etc.).  For excision of tissue samples, it is advised to use the biopsy systems of Hohenberg et al.32 (Transfer System; Leica Microsystems, Vienna, Austria, see Chapter 6).

4.2. Preparation of Substitution Apparatus  Fill the substitution apparatus with liquid nitrogen.  Choose the desired settings for the different steps (time and temperature).  Place all the holders needed for the microtubes and the glass vials with the substitution medium into the apparatus.  Start the cooling.

 Our standard settings are 8 hours at 90°C, 8 hours at 60°C and 8 hours at 30°C.17  If desired, e.g., for plant cells or fungi or for more careful substitution to also retain ions (see Chapter 15), the initial step can be prolonged up to 80 hours.  Recently Hawes30 et al. got very promising results with much shorter time settings.

4.3. Freeze-Substitution  Wait until the apparatus has equilibrated at 90°C.  Put the frozen samples into a 1.5 mL microtube containing a perforated inset.  Add cold, 90°C substitution medium into the tube.  Start the program.

 Caution: Often the temperature display of the substitution apparatus is not reliable. The apparatus should be calibrated by measuring the actual temperature in the tubes.  HPF samples and those sandwiched between two copper plates (e.g., propane jet) should be opened under liquid nitrogen.  When using 1-hexadecene in combin-ation with HPF, it needs to be brushed off under liquid nitrogen, otherwise the substitution process could be hampered.33

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 Alternatively, the medium is filled in the microtubes and then frozen in liquid nitrogen. The sample is put into the tube on top of the frozen medium. The tubes are put into the device and, during melting, the sample will slowly sink into the substitution medium. This method may not be useful for substitution media containing water.

4.4. Preparation for Resin Embedding  After the substitution process, the  At 30°C, osmium tetroxide and or uranyl samples are kept at 30°C. acetate can be removed.  The tubes can also be brought to 0°C in an  They are washed with pure acetone, ice/water bath for one hour to increase methanol or ethanol. membrane contrast before they are put back to 30°C for resin infiltration.24  For low-temperature embedding the samples are kept at 30°C.  Note: Acetone may interfere with the polymerisation of the methacrylates. In such  Or they are brought to room tem- a case it is advised to wash with ethanol or perature for embedding in LR White methanol before embedding, when acetone or epoxy resin. was used as the substitution medium.  When the epoxy fixation FS medium is used, embedding is continued without washing.

4.5. Low-Temperature Embedding in Methacrylates  Embed in the methacrylates at 30°C,  We usually use 1/3, 2/3 resin/alcohol, then or lower depending on the Lowicryl 100% resin 2 to 4 hours, 100% resin overformulation used, following the night and 100% resin 2 to 4 hours. manufacturer’s instructions.  For samples with thick cell walls, such as yeast or plants, more and longer infiltration steps may be used, e.g., 5 to 7 days in a series of dilutions of ethanol and methacrylate (1/100, 1/50, 1/10, 1/5, 1/2, 2/1, 5/1, and 6 changes of pure resin).23

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4.6. Low-Temperature Polymerisation  Methacrylates for low-temperature embedding are polymerised for 24 hours in the substitution apparatus at 30°C with indirect UV illumination.

 Indirect illumination is important for more even polymerisation.12,20  The tubes should be cooled in an alcohol bath to guarantee optimum heat transfer and avoid bubbling during polymerisation.12,20

4.7. Curing of Methacrylates  The low-temperature embedded  Fully polymerised K4M and HM20 have a samples need to be cured at room yellowish and reddish colour, respectively. temperature, either outdoors in the sun or under a UV lamp in a fume hood.

4.8. Embedding in LR White and Epoxy Resins  The samples are infiltrated with 30%  The first 30% step for LR White or epoxy Epon in acetone or 30% LR White in may be done at 30°C, and then the sample is ethanol at 30°C for 2 to 5 hours. warmed to room temperature in the closed tube to avoid water condensation.  Then the tubes are brought to room  Epon/acetone mixtures are liquid at 30°C temperature and infiltration is up to 60% Epon. continued as for routine embedding.  Polymerisation takes place at 50°C for  For samples with thick cell walls, such as at least 24 hours. yeast or plants, more and longer infiltration steps may be used, e.g., 5 to 7 days in a series of dilutions of acetone and Epon (1/100, 1/50, 1/10, 1/5, 1/2, 2/1, 5/1, and 6 changes of pure resin).23  Alternatively, Spurr’s low viscosity resin may be used.34

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5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  The optimum preservation of the  cellular architecture achieved by cryofixation can be combined with resin embedding.  Methacrylate sections may be used for  Methacrylate sections can also be used immunolabelling studies. for correlative light and electron microscopy (see Chapter 21).  Epon embedded samples are best  Epon is more stable in the electron beam suited for reliable morphological than methacrylates. studies.  Thick Epon sections can easily be  prepared and are suitable for electron  tomography (see Chapter 24).   

5.2. Disadvantages  Methacrylate sections are generally  less sensitive for immunolabelling studies than Tokuyasu cryo-sections (see Chapter 19).  In contrast to frozen-hydrated sections (CEMOVIS, see Chapter 11,) the biological structures are still exposed to organic solvents and chemical fixatives, which might alter the cellular architecture.

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 Collapse of delicate, highly hydrated nanostructures.        

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6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Choice of Substitution Medium      

High contrast medium Standard FS medium Membrane contrast medium Wild FS medium Hawes FS medium (2% UA) Epoxy fixation FS medium

 These media are used for morphological analysis and electron tomography.  Used for morphological analysis and electron tomography.

 Used for morphological analysis and electron tomography.  With caution it can also be used for immunolabelling.  Hawes FS medium (0.2% UA)  In combination with methacrylate  Low concentration of aldehyde FS embedding these media are best suited for medium immunolabelling.  Pure solvent for FS 

6.2. Choice of Resin  Freeze-substitution in pure solvent  followed by low-temperature embedding in methacrylate is used for immunolabelling of aldehyde-sensitive proteins.  (Low-temperature) embedding in methacrylate is used for immunolabelling.  Embedding in epoxy resin is used for morphological analysis, in particular for electron tomography. 

6.3. Osmium Tetroxide and Low-Temperature Embedding  In contrast to the general belief, biological samples fixed with osmium tetroxide during freeze-substitution can be embedded in methacrylates, e.g., Lowicryl, at low temperature with UV polymerisation.  Osmium tetroxide used during freezesubstitution affects antigenicity to a much lesser extent than when used at higher temperatures.

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 As long as the substitution is stopped below or at 30°C and the temperature is not raised during embedding, the sample has only a yellowish colour and does not get black. The UV light can easily penetrate the sample and the resin polymerises.20  At temperatures below 30°C, osmium tetroxide reacts in the fixing manner as described in the textbooks and does not get black,28 whereas at temperatures above 0°C, osmium tetroxide reacts more like a protease with all the consequences expected for protein epitopes.35,36

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7. OBSERVED RESULTS  Figure on the Chapter’s title page

 Clamydomonas reinhardii cells were pelleted and picked up with a copper grid and cryofixed by plunging into liquid propane. Freeze-substitution was done in ethanol containing 0.5% uranyl acetate and 0.1% glutaraldehyde, 8 hours at –90°C; 8 hours at 60°C; 8 hours at –35°C using a homemade freeze-substitution apparatus. The samples were embedded at 35°C in Lowicryl HM20. (B.M. Humbel, unpublished results, 1989.) Full width corresponds to 2 µm.

 Figure 13.2

 Escherichia coli cells grown at 37oC were cryofixed by high-pressure freezing (Leica EM HPF, Leica Microsystems, Vienna, Austria; now M. Wohlwend, Sennwald, Switzerland). Substitution was done in acetone containing 2% osmium tetroxide, 0.2% uranyl acetate and 1% H2O; 48 hours at 90°C; warmed with a slope of 2°C/h to 60°C, 8 hours at 60°C; warmed with a slope of 2oC/h to 30°C, 8 hours at 30°C in a AFS substitution apparatus (Leica EM AFS, Leica Microsystems, Vienna, Austria). After one hour at 0°C,24 the E. coli cells were embedded in Epon. (E. van Donselaar, B.M. Humbel, unpublished results, 2006.) Bar = 500 nm

 Figure 13.3

 Saccharomyces cerevisiae cells were grown to stationary phase. Then they were cryofixed by high-pressure freezing (Leica EM HPF, Leica Microsystems, Vienna, Austria; now M. Wohlwend, Sennwald, Switzerland) and freeze-substituted in acetone containing 0.5% glutaraldehyde and 0.2% uranyl acetate and lowtemperature embedded in Lowicryl HM20. (E. van Donselaar, B.M. Humbel, unpublished results, 2006.) Bar = 500 nm

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 Figure 13.4

 Cartilage of mouse hip was cryofixed by high-pressure freezing (Leica EM PACT, Leica Microsystems, Vienna, Austria; see Chapter 6) and freeze-substituted in acetone containing 0.5% glutaraldehyde and 0.2% uranyl acetate and 1% H2O; 48 hours at 90°C; warmed with a slope of 2°C/h to 60°C, 8 hours at 60°C; warmed with a slope of 2°C/h to 30°C, 8 hours at 30°C in a AFS2 substitution unit using the automatic reagent handling system (Leica EM AFS2/Leica EM FSP, Leica Microsystems, Vienna, Austria). The sample was low-temperature embedded in HM20. (E. Van Donselaar, J.W. Slot, B.M. Humbel, unpublished results, 2005.) Bar = 5 µm

 Figure 13.5

 NRK cells grown on Aclar® disks were cryofixed by high-pressure freezing (Leica EM HPF, Leica Microsystems, Vienna, Austria; now M. Wohlwend, Sennwald, Switzerland) and freeze-substituted in acetone with 2% osmium tetroxide; 48 hours at 90°C; warmed with a slope of 2oC/h to 60°C, 8 hours at 60°C; warmed with a slope of 2°C/h to 30°C, 8 hours at 30°C in a AFS substitution apparatus (Leica EM AFS, Leica Microsystems, Vienna, Austria). After one hour at 0°C,24 the sample was embedded in Epon.37 (N. Jiménez, E. van Donselaar, B.M. Humbel, unpublished results, 2005.)

Bar = 500 nm

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8. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.

14.

15. 16. 17. 18.

Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens, Q. Rev. Biophys., 21, 129, 1988. Fernández-Morán, H. Low-temperature preparation techniques for electron microscopy of biological specimens based on rapid freezing with liquid Helium II, Ann. NY Acad. Sci., 85, 689, 1960. Medalia, O. et al. Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography, Science, 298, 1209, 2002. Leis, A.P. et al. Cryo-electron tomography of biological specimens, IEEE Signal Proc. Mag., 23, 95, 2006. Frangakis, A.S. et al. Identification of macromolecular complexes in cryoelectron tomograms of phantom cells, Proc. Nat. Acad. Sci. USA, 99, 14153, 2002. Studer, D. et al. A new approach for cryofixation by high-pressure freezing, J. Microsc., 203, 285, 2001. Müller, M. and Moor, H. Cryofixation of thick specimens by high pressure freezing, in Science of Biological Specimen Preparation 1983, Revel, J.P., Barnard, T., and Haggis, G.H., eds., SEM Inc., AMF O'Hare, IL, USA, 1984, 131. Riehle, U. Über die Vitrifizierung von verdünnter wässriger Lösungen. ETH Diss Nr 4271. Federal Institute of Technology (ETH), Zürich, Switzerland, 1968. Al-Amoudi, A., Norlen, L.P.O., and Dubochet, J. Cryo-electron microscopy of vitreous sections of native biological cells and tissues, J. Struct. Biol., 148, 131, 2004. Al-Amoudi, A., Studer, D., and Dubochet, J. Cutting artefacts and cutting process in vitreous sections for cryo-electron microscopy, J. Struct. Biol., 150, 109, 2005. Marko, M. et al. Focused-ion-beam thinning of frozen hydrated biological specimens for cryoelectron microscopy, Nat. Methods, 4, 215, 2007. Humbel, B.M. and Schwarz, H. Freeze-substitution for immunochemistry, in Immuno-Gold Labeling in Cell Biology, Verkleij, A.J. and Leunissen, J.L.M., eds., CRC Press, Boca Raton, FL, USA, 1989, 115. Schwarz, H., Hohenberg, H., and Humbel, B.M. Freeze-substitution in virus research: A preview., in Immunoelectron Microscopy in Virus Diagnosis and Research, Hyatt, A.D. and Eaton, B.T., eds., CRC Press Inc., Boca Raton, FL, USA, 1993, 97. Geerts, W.J.C. et al. Electron microscopy tomography and localization of proteins and macromolecular complexes in cells, in Protein-Protein Interactions. A Molecular Cloning Manual, Golemis, E.A. and Adams, P.D., eds., Cold Spring Harbor Laboratories Press, NY, USA, 2006, 715. Simpson, W.L. An experimental analysis of the Altmann technic of freezingdrying, Anat. Rec., 80, 173, 1941. Fernández-Morán, H. Low-temperature preparation techniques for electron microscopy of biological specimens based on rapid freezing with liquid Helium II, Science, 129, 1284, 1959. Müller, M., Marti, T., and Kriz, S. Improved structural preservation by freeze substitution, in Proc. 7th Eur. Congr. Electron Microsc., Brederoo, P. and de Priester, W., eds., The Hague, The Netherlands, 1980, 720. Steinbrecht, R.A. and Müller, M. Freeze-substitution and freeze-drying, in Cryotechniques in Biological Electron Microscopy, Steinbrecht, R.A. and Zierold, K., eds., Springer-Verlag, Berlin, Heidelberg, Germany, 1987, 149.

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19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.

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Van Harreveld, A., Crowell, J., and Malhotra, S.K. A study of extracellular space in central nervous tissue by freeze-substitution, J. Cell Biol., 25, 117, 1965. Humbel, B., Marti, T., and Müller, M. Improved structural preservation by combining freeze substitution and low temperature embedding, Beitr. Elektronenmikroskop. Direktabb. Oberfl., 16, 585, 1983. Humbel, B. and Müller, M. Freeze substitution and low temperature embedding, in The Science of Biological Specimen Preparation 1985, Müller, M., et al., eds., SEM Inc., AMF O'Hare, IL, USA, 1986, 175. Studer, D., Hennecke, H., and Müller, M. High-pressure freezing of soybean nodules leads to an improved preservation of ultrastructure, Planta, 188, 155, 1992. Humbel, B.M. et al. In situ localization of β-glucans in the cell wall of Schizosaccharomyces pombe, Yeast, 18, 433, 2001. Wild, P. et al. Enhanced resolution of membranes in cultured cells by cryoimmobilization and freeze-substitution, Microsc. Res. Tech., 53, 313, 2001. Walther, P. and Ziegler, A. Freeze substitution of high-pressure frozen samples: The visibility of biological membranes is improved when the substitution medium contains water, J. Microsc., 208, 3, 2002. Marsh, B.J. et al. Organellar relationships in the Golgi region of the pancreatic beta cell line, HIT-T15, visualized by high resolution electron tomography, Proc. Nat. Acad. Sci. USA, 98, 2399, 2001. Murk, J.-L.A.N. et al. 3-D Structure of multilaminar lysosomes in antigen presenting cells reveals trapping of MHC II on the internal membranes, Traffic, 5, 936, 2004. White, D.L. et al. The chemical nature of osmium tetroxide fixation and staining of membranes by x-ray photoelectron spectroscopy, Biochim. Biophys. Acta, 436, 577, 1976. Van Harreveld, A. and Crowell, J. Electron microscopy after rapid freezing on a metal surface and substitution fixation, Anat. Rec., 149, 381, 1964. Hawes, P. et al. Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells, J. Microsc., 226, 182, 2007. Matsko, N. and Müller, M. Epoxy resin as fixative during freeze-substitution, J. Struct. Biol., 152, 92, 2005. Hohenberg, H., Tobler, M., and Müller, M. High-pressure freezing of tissue obtained by fine-needle biopsy, J. Microsc., 183, 133, 1996. Hohenberg, H., Mannweiler, K., and Müller, M. High-pressure freezing of cell suspensions in cellulose capillary tubes, J. Microsc., 175, 34, 1994. Spurr, A.R. A low-viscosity epoxy resin embedding medium for electron microscopy, J. Ultrastruct. Res., 26, 31, 1969. Behrman, E.J. The chemistry of osmium tetroxide fixation, in The Science of Biological Specimen Preparation 1983, Revel, J.P., Barnard, T., and Haggis, G.H., eds., SEM Inc., AMF O'Hare, IL, USA, 1984, 1. Maupin, P. and Pollard, T.D. Actin filament destruction by osmium tetroxide, J. Cell Biol., 77, 837, 1978. Jiménez, N. et al. Aclar discs: A versatile substrate for routine high-pressure freezing of mammalian cell monolayers, J. Microsc., 221, 216, 2006.

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CONTENTS

GENERAL INTRODUCTION .................................................................................... 347 1.

PRINCIPLES OF THIS METHOD ................................................................. 348 1.1. 1.2. 1.3. 1.4. 1.5. 1.6.

Cryo-Fixation ........................................................................................... 348 Dehydration and Chemical Fixation ......................................................... 348 Rehydration and Postfixation ................................................................... 348 Tokuyasu Cryo-Sectioning ....................................................................... 348 Immunolabelling....................................................................................... 348 Staining and Methyl Cellulose/Moviol Embedding ................................. 349

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 349

3.

MATERIALS/PRODUCTS/SOLUTIONS ...................................................... 350 3.1. 3.2. 3.3.

4.

PROTOCOLS .................................................................................................... 354 4.1. 4.2. 4.3.

5.

Materials ................................................................................................... 350 Products .................................................................................................... 350 Solutions ................................................................................................... 352

Cultured Mammalian Cells and Tissue, Bacteria and Yeast..................... 354 Plants, Nematodes, Drosophila................................................................. 355 Preparation for Tokuyasu Cryo-Sectioning .............................................. 356

ADVANTAGES/DISADVANTAGES.............................................................. 357 5.1. 5.2.

Advantages ............................................................................................... 357 Disadvantages........................................................................................... 358

6.

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 358

7.

OBSERVED RESULTS .................................................................................... 360

8.

REFERENCES .................................................................................................. 364



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GENERAL INTRODUCTION Cryo-fixation has proven to be the best method for the preservation of cellular ultrastructure.1-10 On the other hand, the open structures obtained with Tokuyasu cryosections in most cases are ideally suitable for efficient immunolabelling.11-14 The advantages of Tokuyasu cryo-sections are probably related to the absence of dehydration so that molecules remain in their natural hydrophilic surrounding. Moreover, this method usually involves weak fixation with low concentrations of aldehydes resulting in a partial loss of proteins and an increase in the three-dimensional access of antibodies. For many years, a method was sought that combines cryo-fixation with efficient immunolabelling. Up until recently, only freeze-substitution and embedding in methacrylate resins (see Chapter 13) or freeze-drying (see Chapter 15) could be used for immuno-electron microscopy.15,16 Although cellular morphology is good or even excellent using the latter methods, the section surface only provides a two-dimensional access for antibodies, which makes it a difficult task to localise sparse antigens. In the last years two groups, Slot and colleagues17 and Stierhof and Schwarz,18 have developed a technique that combines high-pressure freezing with the Tokuyasu immunolabelling technique. They applied this new method to tissue and cultured mammalian cells, but the real power of this method is to successfully prepare difficult-to-chemically-fix material, such as yeast, bacteria, plants, nematodes and insects. In all cases, the cryo-fixation, freeze-substitution and rehydration method resulted in good cellular ultrastructure and a high labelling efficiency, which is comparable to that of the conventional Tokuyasu technique.            

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1. PRINCIPLES OF THIS METHOD 1.1. Cryo-Fixation  The sample is cryofixed by high-  See Chapters 5, 6. pressure freezing.

1.2. Dehydration and Chemical Fixation  The cryofixed specimen is dehydrated  See Chapter 13. and chemically fixed during freeze-  Uranyl acetate (UA), glutaraldehyde (GA), and osmium tetroxide (OsO4) may be substitution. used as fixatives.

1.3. Rehydration and Postfixation  After freeze-substitution a rehydration  After completing the freeze-substitution process is carried out on ice at about process, samples have to be rehydrated in order to enter the conventional Tokuyasu 0oC to 4oC. cryo-sectioning procedure.  During rehydration, samples are  Fixation during rehydration is necessary additionally fixed with glutaraldehyde because the chemical fixation during freeze-substitution turned out to be at 0oC to 4oC. insufficient.17,18

1.4. Tokuyasu Cryo-Sectioning  In the last step, the sample is  See Chapter 19. impregnated with a cryoprotectant (e.g., 2.3 M sucrose), frozen on a specimen pin of the ultramicrotome and sectioned at around 120oC to  For PVP-sucrose at around – 115oC. 140oC. The sections are picked up, thawed, and mounted on grids.

1.5. Immunolabelling  On-section immunogold labelling or  See Chapters 19, 21, 23. immunofluorescence labelling.

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1.6. Staining and Methyl Cellulose/Moviol Embedding  UA staining and stabilisation of the sections with methyl cellulose (EM) or with Moviol (LM).

 See Chapters 19, 21, 23.

2. SUMMARY OF THE DIFFERENT STEPS

1. See Chapters 5, 6. 2. See Chapters 5, 6.

3. See Chapter 13.

5. See Chapter 19.

6. See Chapter 19.

7. See Chapters 19, 21, 23.

8. See Chapter 19, 21.   

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Figure 14.1 Flow chart showing the rehydration hybrid technique. The conventional Tokuyasu cryo-section labelling technique is in grey.  

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3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Freeze-substitution apparatus with  Self-made.19 inset to hold glass vials and micro- Leica AFS or AFS2, Leica, Microsystems, tubes. Vienna, Austria. RMC FS-7500, Boeckeler Instruments Inc. Tucson, Arizona, USA (no longer sold).  Microtubes.

 0.5 and 1.5 mL Sarstedt AG & Co, Nümbrecht, Germany.

 Glass vials.

 Used to prepare the solutions.

 Perforated insets.

 Leica plastic capsules D5 × H 15 mm # 16702738; Leica, Microsystems, Vienna, Austria.

 Forceps with insulation coating.

 For example, Dumont e.g., (#2a, #3, #4, #4N, #7), Dumont & fils, Switzerland.

 Rotating wheel.  Stubs/pins.

 Used for sucrose impregnation at 4oC.  Copper or aluminium rivets, used as specimen holders.

3.2. Products  Acetone.

 Dry acetone (Merck #1.00299.0500, Merck KGaA, Darmstadt, Germany); the open bottle is stored under nitrogen gas in a closed container above silica gel or molecular sieve (3Å) is added to the solution.

 Ethanol.

 Dry ethanol (Merck #1.00990.0500); the open bottle is stored under nitrogen gas in a closed container above silica gel or a molecular sieve (3Å) is added to the solution.

 PIPES.

 1,4-Piperazine-bis(ethanesulfonic acid).

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 HEPES.

 2-[4-(2-Hydroxyethyl)-1-piperazine] ethanesulfonic acid.

 Magnesium chloride.  EGTA.

 MgCl2.  Ethyleneglycol-bis(β-aminoethyl)-N,N, N´,N´-tetracetic acid

 Sucrose.  Gelatine.  Glutaraldehyde (GA).

 GA is available as a 10% solution in water-free acetone (EMS # 16530), ethanol (EMS # 16531) or methanol (EMS # 16532).  50% or 25% solution in water (Sigma, #G-5882; Sigma-Aldrich: Fluka, Buchs, Switzerland) (keep frozen).  8% solution, Polysciences, # 00216A.

 Glycine.  Methanol.

 Dry methanol (Merck # 1.06012.0500); the open bottle is stored under nitrogen gas in a closed container above silica gel or a molecular sieve (3Å) is added to the solution.

 Osmium tetroxide (OsO4).

 EMS, Hatfield, Pennsylvenia, USA.

 Uranyl acetate (UA).

 EMS, Hatfield, Pennsylvenia, USA or SPI Supplies, West Chester, Pennsylvenia,, USA.

 H2O.

 Double distilled or MilliQ water.

 Polyvinylpyrrolidone 10,000).

(PVP,

MW  Sigma PVP10-100G.

 Sodium chloride.

 NaCl.

 Disodium hydrogenphosphate.

 Na2HPO4.

 Sodium dihydrogen phosphate.

 NaH2 PO4.

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 Silica gel.  Molecular sieve (3 Å).  Methyl cellulose.

 Sigma M-6385.



3.3. Solutions    

PBS buffer (pH 7.4) 137 mM sodium chloride NaCl 2.7 mM potassium chloride KCl 8.1 mM disodium hydrogenphosphate Na2HPO4  1.5 mM sodium dihydrogen phosphate NaH2 PO4

 Phosphate buffered saline, a standard buffer.  Note: Phosphate buffers are incomepatible with uranyl acetate based substitution media.

    

 Cytoskeleton buffer.20  Note: PHEM is compatible with uranyl acetate-based substitution media.

PHEM buffer (pH 6.9) 60 mM PIPES 25 mM HEPES 10 mM EGTA 2 mM MgCl2

 Membrane contrast medium17  Acetone + 15% (v/v) H2O21

+ 0.10.2% (w/v) UA

+ 0.5% (v/v) GA, or 12% (w/v) OsO4  Membrane contrast medium18  Acetone + 24% (v/v) H2O21 + 0.10.2% (w/v) UA + 0.5% (v/v) GA + 0.1% (w/v) OsO4

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 Medium for bacteria, yeast, and cultured mammalian cells and tissue.  Used to enhance membrane contrast, depen-ding on the sample 1 to 5% water can be added.  Directly dissolved in dry acetone.  Note: Uranyl ions can impair immunolabelling.  GA from 10% stock in acetone.  Medium for plants, nematodes and Drosophila.  Used to enhance membrane contrast, depending on the sample, 2 to 4% water can be added.  From 20% UA stock in methanol.22  From 10% stock in acetone.  When OsO4 is omitted, we use 0.5% UA.

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 Postfixation.  0.25% GA in H2O18  Rehydration solution17  95 to70%: Acetone in H2O + 0.5% GA  50 to 30%: Acetone in PHEM buffer  Note: To make a glutaraldehyde solution + 0.5% GA in 30% acetone in PHEM, 8% aqueous  0.5% GA in PHEM buffer glutaraldehyde must be used, otherwise the acetone-based GA precipitates.  Rehydration solution18  80 to 10%: Acetone in H2O + 0.5 to 0.25 % GA  Then H2O + 0.25% GA  10%12% (w/v) gelatine in PHEM.

 Suspend in phosphate buffered saline (PBS), warm up to about 60°C, stir until the gelatine powder has dissolved.

 2.3 M sucrose in PHEM buffer.

 Weigh 78.73 g sucrose in a 100 mL volumetric flask. Add PHEM buffer pH: 7.4 while stirring and wait until the sucrose has dissolved. Remove the stir bar and top up with the buffer to the mark. The mixture is stored frozen in 10 to 20 mL aliquots.

 Polyvinylpyrrolidone (PVP)-sucrose23  20% PVP  1.84 M sucrose in phosphate carbonate buffer

 Infiltration time is longer compared to pure sucrose.  To make 100 mL, mix 20 g PVP and 4 mL of 1.1 M disodium carbonate (Na2CO3), in 0.1 M disodium hydrogen phosphate (Na2HPO4) and 80 mL of 2.3 M sucrose in 0.1 M Na2HPO4.

 Inactivation buffer for fixative (GA) 50 mM glycine in H2O

 Weigh 38 mg glycine in 10 mL H2O.

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4. PROTOCOLS 4.1. Cultured Mammalian Cells and Tissue, Bacteria and Yeast  See Chapters 5, 6.  Embedding of the cell pellet in 2% gelatine before cryo-fixation may prevent loss of cells during freeze-substitution.

1. Cryo-fixation

2. Freeze-Substitution  FS medium  Acetone  + 1 to 5% H2O  + 0.1 to 0.2% UA + 0.5% GA Instead of GA, 1 to 2% OsO4 can be used or GA and OsO4 combined.  4 rinses  In freeze-substitution solution without UA  Warming up  Incubation

 Addition of water may improve the visualisation of membrane bilayers.21 90°C, 48 h 60°C, 8 h  Increase with 2°C/h to 60°C. 30°C, 8 h  Increase with 2°C/h to 30°C.

30°C  This step is only done when the 4 x 10 min substitution medium contains UA.

3. Rehydration and postfixation  Rehydration  95% acetone in H2O + 0.5% GA  90% acetone in H2O + 0.5% GA  80% acetone in H2O + 0.5% GA  70% acetone in H2O + 0.5% GA  50% acetone in PHEM buffer + 0.5% GA  30% acetone in PHEM buffer + 0.5% GA

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0°C 0°C, 1 h 0°C  In case of OsO4 fixation, rehydration is 10 min done with acetone only. 0°C 10 min 0°C 10 min 0°C 10 min 0°C 10 min 0°C Note: 8% aqueous glutaraldehyde must be 10 min used!

Cryo-Fixation, Freeze-Substitution, Rehydration and Tokuyasu Cryo-Sectioning

 PHEM buffer + 0.5% GA  PHEM buffer + 0.5% GA  PHEM buffer  PHEM buffer

355

0°C, 10 min 0°C, 10 min 0°C, 10 min 0°C, 10 min

 Preparation for Tokuyasu cryo-sec-  See Section 4.3. tioning.

4.2. Plants, Nematodes, Drosophila  For details, see Chapters 5, 6.

1. Cryo-fixation 2. Freeze-Substitution  FS medium Acetone  + 2% H2O  + 0.1% OsO4  + 0.1-0.2% UA  + 0.5% GA

 Water is required for improved visualisation of the structure of membrane bilayers (plants, Drosophila; for nematodes, 4% H2O may be better).18 90°C, 48 to 72 h 60°C, 8 h  Increase with 10°C/h to 60°C. 35°C, 8 h  Increase with 10°C/h to 35°C.  When OsO4 is omitted, we use 0.5% UA.

 Washing Acetone  + 2% H2O  + 0.5% GA  Warming up

4 to 5 × 30 to 45 min 35°C 20°C, 10 to 60 min

 Warming up

0°C

3. Rehydration and postfixation  Rehydration  80% Acetone in H2O + 0.5% GA  50% Acetone in H2O + 0.38% GA

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 Note: If a buffer is used instead of H2O, 5 to 10 min it should be compatible with UA, e.g., 0°C PHEM buffer, to prevent precipitation of uranyl ions. 5 to 10 min 0°C

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 25% Acetone in H2O + 0.25% GA  10% Acetone in H2O + 0.25% GA  H2O + 0.25% GA

 Postfixation  H2O + 0.25% GA

5 to 10 min 0°C 5 to 10 min 0°C 5 min 0°C  Necessary for stabilisation of ultra30 to 90 min structure 0°C  Postfixation time can vary, depending on sample type and size, fixation cocktail during freeze-substitution and on the antigen to be localised.18

 Inactivation of residual fixative mole-  Optional. cules (GA)  H2O 5 min, 0°C  50 mM glycine in 2 × 10 min H2O 0°C  Preparation sectioning

for

Tokuyasu

cryo-  See Section 4.3.

4.3. Preparation for Tokuyasu Cryo-Sectioning 1. Gelatine embedding

 To improve sectioning properties.

 Cell pellets and cells grown on filters  The gelatine density should match the or beads17,24 are embedded in gelatine sample density; we usually use 10 to 12%. in PHEM buffer and the gelatine is  The pellet should be loose. In dense solidified on ice. pellets, the cells are closely packed and  Small blocks of not more than 1 mm3 individual cells are difficult to photograph. are cut with razor blades from the Often dense packing also impairs proper gelatine-embedded cells. sectioning.

2. Sucrose impregnation

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 To prevent freezing artefacts and improve sectioning properties.

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 The small blocks of tissue or gelatine embedded cells are put into a microtube containing 2.3 M sucrose in PHEM buffer. The tube is mounted on a rotating wheel and the tubes are slowly rotated.  Impregnation is done for 4 to 20 hours in a cold room at 4°C.

357

 After successful impregnation, the blocks float in the solution or sink to the bottom. Under no circumstances should they swim.  Infiltration in several steps, e.g., 0.7 M, 1.4 M, 2.3 M sucrose, may reduce possible osmotic effects, such as vacuole collapse and shrinkage of highly vacuolated plant tissue.  Alternatively, PVP-sucrose may be used if gelatine embedding was omitted. PVP does not permeate cells and may reduce differences in matrix densities that can impair the sectioning process.23

 For details, see Chapter 19. 3. Mounting the sample  The gelatine or tissue blocks are mounted on stubs, frozen and inserted in the specimen holder of the cryoultramicrotome. 4. Tokuyasu cryo-sectioning.

 For details, see Chapter 19.

5. Immunolabelling.

 For details, see Chapters 21, 23.

6. Staining and methyl cellulose or  For details, see Chapters 19, 21, 23. Moviol embedding.

5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  The rehydration hybrid technique  See Chapters 2, 5, 6, 13, 19. combines the excellent preservation of  This method can also be used for in situ cellular ultrastructure by cryo-fixation hybridisation (see Chapter 18). with the high labelling efficiency of thawed cryo-section labelling according to Tokuyasu.  Chemical fixation during freeze-  Suitable for fixation-sensitive antigens. substitution causes fewer artefacts than when carried out at temperatures  above 0°C. 

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 Osmium tetroxide and uranyl acetate  The chemical reaction of osmium may be used as primary fixatives tetroxide at low temperature is different than at ambient temperature. At low during freeze-substitution temperatures, it works as a fixative and displays little if any proteolytic activity (See Chapter 13).

5.2. Disadvantages  During freeze-substitution, the spec-  In contrast to the original Tokuyasu imen is exposed to inorganic solvents. technique.  Limited sample size.

 To prevent ice crystal damage during cryo-fixation.

 More time consuming when compared  Due to the additional freeze-substitution to the original Tokuyasu procedure. and rehydration steps.  Requires freezing apparatus (e.g., a HPF).    Possible artefacts due to rehydration  (e.g., extraction).    Sometimes inhomogeneous appearance of cytoplasm.

6. WHY AND WHEN TO USE A SPECIFIC METHOD 1. If cellular ultrastructure cannot be preserved when using chemical fixation needed for the Tokuyasu cryosectioning technique. 2. If conventional fixation at ambient temperature is not useful.

 For example, in the case of bacteria, yeast, plants, nematodes and insects.  Due to inactivation of antigens by fixation at ambient temperature.  Due to artefacts caused by slow fixative penetration (due to large sample size or dense cell walls, cuticles, etc.).

3. If cryo-fixation is necessary but resin  For example, in case of sparse antigens. section labelling does not work.

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7. OBSERVED RESULTS  Figure on the Chapter’s title page

 Immunogold localisation of the vacuolar ATPase in a thawed cryo-section of an Arabidopsis thaliana pollen grain after high-pressure freezing, freeze-substitution, rehydration and further processing for Tokuyasu cryo-section labelling.

 Figure 14.2

 Mouse hip cartilage was cryo-fixed by high-pressure freezing. Freeze-substitution and rehydration was done as described in Section 4.1. The substitution medium contained 0.5% glutaraldehyde, 0.2% uranyl acetate and 1% H2O. The rehydrated sample was further processed for Tokuyasu cryo-sectioning (see Section 4.3.) and the sections labelled for COPII. Bar = 500 nm

 Figure 14.3

 Saccharomyces cerevisiae was cryofixed by high-pressure freezing. Freezesubstitution and rehydration was done as described in Section 4.1. The substitution medium contained 2% osmium tetroxide, 0.2% uranyl acetate and 5% H2O in acetone. The rehydrated sample was further processed for Tokuyasu cryo-sectioning (see Section 4.3.). Bar = 500 nm

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 Figure 14.4

 Immunogold labelling of a thawed cryosection of an Arabidopsis thaliana embryo after high-pressure freezing, freezesubstitution, rehydration and further processing for Tokuyasu cryo-section labelling. Freeze-substitution was carried out in acetone containing 0.5% glutaraldehyde, 0.2% uranyl acetate, 0.1% osmium tetroxide and 2% H2O, rehydration was done in the presence of 0.5% glutaraldehyde, and postfixation with 0.25% glutaraldehyde in H2O was carried out for 60 min at 0°C. Ultrathin thawed cryo-sections were labelled with rabbit antibodies and silverenhanced goat antirabbit IgG coupled to Nanogold.  DV = Dense vesicle  G = Golgi stack  SV = Storage vacuole Bar = 250 nm

 Figure 14.5

 Immunogold labelling of a thawed cryosection of the nematode Pristionchus pacificus, after high-pressure freezing, freeze-substitution, rehydration and Tokuyasu cryo-section labelling. Freezesubstitution was carried out in acetone containing 0.5% glutaraldehyde, 0.2% uranyl acetate, 0.1% osmium tetroxide and 4% H2O. Rehydration was done in the presence of 0.5% glutaraldehyde, and postfixation with 0.25% glutaraldehyde in H2O was carried out for 90 min at 0°C. A microtubule bundle close to the pharynx region was labelled with mouse antitubulin antibodies and silver-enhanced goat antimouse IgG coupled to Nanogold.®  N = Nucleus Bar = 1 µm

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8. REFERENCES 1. 2. 3.

4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17. 18.

Dubochet, J. et al. Cryo-electron microscopy of vitrified biological specimens, Trends Biochem. Sci., 6, 143, 1985. Fernández-Morán, H. Low-temperature preparation techniques for electron microscopy of biological specimens based on rapid freezing with liquid Helium II, Ann. NY Acad. Sci., 85, 689, 1960. Knoll, G., Braun, C., and Plattner, H. Quenched flow analysis of exocytosis in Paramecium cells: Time course, changes in membrane structure, and calcium requirements revealed after rapid mixing and rapid freezing of intact cells, J. Cell Biol., 113, 1295, 1991. McDonald, K.L. High pressure freezing for preservation of high resolution fine structure and antigenicity for immunolabelling, Methods Mol. Biol., 117, 77, 1999. Menco, B.P.M. A survey of ultra-rapid cryofixation methods with particular emphasis on applications to freeze-fracturing, freeze-etching, and freezesubstitution, J. Electron Microsc. Tech., 4, 177, 1986. Moor, H. Theory and practice of high pressure freezing, in Cryotechniques in Biological Electron Micoscopy, Steinbrecht, R.A. and Zierold, K., eds., SpringerVerlag, Berlin, Heidelberg, Germany, 1987, 175. Müller, M. The integrating power of cryofixation-based electron microscopy in biology, Acta Microsc., 1, 37, 1992. Müller, M., Meister, N., and Moor, H. Freezing in a propane jet and its application in freeze-fracturing, Mikroskopie, 36, 129, 1980. Studer, D., Hennecke, H., and Müller, M. High-pressure freezing of soybean nodules leads to an improved preservation of ultrastructure, Planta, 188, 155, 1992. Szczesny, P.J., Walther, P., and Müller, M. Light damage in rod outer segments: The effects of fixation on ultrastructural alterations, Curr. Eye Res., 15, 807, 1996. Liou, W., Geuze, H.J., and Slot, J.W. Improving structural integrity of cryosections for immunogold labeling, Histochem. Cell Biol., 106, 41, 1996. Tokuyasu, K.T. A technique for ultracryotomy of cell suspensions and tissues, J. Cell Biol., 57, 551, 1973. Tokuyasu, K.T. A study of positive staining of ultrathin frozen sections, J. Ultrastruct. Res., 63, 287, 1978. Tokuyasu, K.T. Cryosections for immunohistochemistry, J. Electron Microsc., 35, 1977, 1986. Humbel, B.M. and Schwarz, H. Freeze-substitution for immunochemistry, in Immuno-Gold Labeling in Cell Biology, Verkleij, A.J. and Leunissen, J.L.M., eds., CRC Press, Boca Raton, FL, USA, 1989, 115. Schwarz, H., Hohenberg, H., and Humbel, B.M. Freeze-substitution in virus research: A preview., in Immunoelectron Microscopy in Virus Diagnosis and Research, Hyatt, A.D. and Eaton, B.T., eds., CRC Press Inc., Boca Raton, FL, USA, 1993, 97. Van Donselaar, E. et al. Immunogold labeling of cryo-sections from high-pressure frozen cells, Traffic, 8, 471, 2007. Ripper, D., Schwarz, H., and Stierhof, Y.-D. Cryo-section immunolabelling of difficult to preserve specimens: Advantages of cryofixation, freeze-substitution and rehydration, Biol. Cell, 100, 109, 2008.

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19. 20. 21. 22. 23. 24.

365

Humbel, B. and Müller, M. Freeze substitution and low temperature embedding, in The Science of Biological Specimen Preparation 1985, Müller, M., et al., eds., SEM Inc., AMF O'Hare, IL, USA, 1986, 175. Schliwa, M., van Blerkom, J., and Porter, K.R. Stabilization of the cytoplasmic ground substance in detergent-opened cells and a structural and biochemical analysis of its composition, Proc. Nat. Acad. Sci. USA, 78, 4329, 1981. Walther, P. and Ziegler, A. Freeze substitution of high-pressure frozen samples: The visibility of biological membranes is improved when the substitution medium contains water, J. Microsc., 208, 3, 2002. Hawes, P. et al. Rapid freeze-substitution preserves membranes in high-pressure frozen tissue culture cells, J. Microsc., 226, 182, 2007. Tokuyasu, K.T. Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryoultramicrotomy, Histochem. J., 21, 163, 1989. Zeuschner, D. et al. Immuno-electron tomography of ER exit sites reveals the existence of free COPII-coated transport carriers, Nat. Cell Biol., 8, 377, 2006.

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CONTENTS

GENERAL INTRODUCTION .................................................................................... 371 1.

PRINCIPLES OF THE METHOD .................................................................. 371 1.1. 1.2. 1.3. 1.4. 1.5. 1.6. 1.7.

1.8.

Check Freeze-Drying Apparatus CFD...................................................... 371 Cryo-Fixation ........................................................................................... 371 Preparation of Small Samples on the Object Table .................................. 371 Automatic Freeze-Drying (FD) ................................................................ 372 Optional: Osmication of Freeze-Dried Specimens ................................... 372 Infiltration with Resin............................................................................... 373 Polymerization.......................................................................................... 374 1.7.1. UV polymerization at low temperature....................................... 374 1.7.2. Heat polymerization.................................................................... 374 Ultramicrotomy and Analysis of Ultrathin Sections................................. 374

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 375

3.

MATERIALS/PRODUCTS/SOLUTIONS ...................................................... 375 3.1. 3.2. 3.3.

Materials ................................................................................................... 375 Products .................................................................................................... 375 Solutions ................................................................................................... 376

4.

PROTOCOLS .................................................................................................... 376

5.

ADVANTAGES/DISADVANTAGES.............................................................. 379 5.1. 5.2.

Advantages ............................................................................................... 379 Disadvantages........................................................................................... 379

6.

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 380

7.

OBSERVED RESULTS .................................................................................... 382

8.

REFERENCES .................................................................................................. 387



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GENERAL INTRODUCTION Freeze-drying (FD) is a technique in which the frozen water of a cryofixed specimen is removed by sublimation at low temperature in a vacuum chamber. Any contact with an organic solvent is avoided during the dehydration process. The dry specimen may be resin embedded at low or high temperature and used for conventional thin sectioning at room temperature. The sections may be used for electron microscopic detection of ultrastructural features of native biological material that are not preserved in specimens obtained by other embedding and sectioning techniques. Note that the quality of structure preservation after freeze-drying is highly dependent on the quality of cryo-fixation and on the physiological state of the biological material. Even mildly chemically fixed (dead) cells are differently freeze-dried than undisturbed living cells.5 The technique as performed with the Leica EM cryo-sorption freeze-drying (CFD) System is presented.

1. PRINCIPLES OF THE METHOD 1.1. Check Freeze-Drying Apparatus CFD Prepare CFD according to the instructions  This step requires about two hours and of the manufacturer. It is placed in the neck can be done one day before starting freezeof a Dewar flask filled with liquid nitrogen drying. (LN2) and evacuated to a pressure of at least 10-3 mbar.

1.2. Cryo-Fixation Cryo-fix biological material, e.g., by  See Chapters 2-6 in part I, Cryo-Fixation metal-mirror freezing (MMF) or by high- Methods pressure freezing (HPF).

1.3. Preparation of Small Samples on the Object Table Under the control of a stereo light microscope, prepare and transfer small frozen samples to the cold object table (OT) of the CFD apparatus.

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 At least one side of a specimen should not exceed 0.3 mm.  Small samples are important for minimizing freeze-drying times, for uniform drying at low temperatures and for easy resin infiltration.

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 Small specimens (S) are prepared on a metal plate (MP) using a precooled scalpel (SC) and transferred into small metal (MC) or plastic containers (PC).  Plastic containers (PC) are used if drying, infiltration and UV polymerization are carried out without any further manual transfer of specimens until final embedding. Figure 15.1 Object table (OT) cooled by liquid nitrogen in a Styrofoam box (SB).

1.4. Automatic Freeze-Drying (FD) 1 Vent the vacuum chamber of the CFD by pure nitrogen gas (GN2) (TF button of the CFD) and open the chamber. 2 Transfer the object table into the CFD and fix it with a built-in cold finger (cold trap). 3 Close the vacuum chamber and evacuate it. 4 Start automatic temperature controlled freeze-drying.

 The open vacuum chamber is always flooded with GN2.  During transfer, water molecules from the air may condense at the cold surface of the object table. This condensed water will sublimate during freeze-drying.  The vacuum chamber of the CFD is evacuated in two steps: 1. To a pressure of about 15 mbar by means of a membrane pump and 2. To a pressure of about 10-4 mbar by a built-in cryo-sorption pump. The extremely low water vapor pressure necessary for successful freeze-drying at low temperature is provided by built-in cold traps.2,8

1.5. Optional: Osmication of Freeze-Dried Specimens Osmication is performed outside the CFD  Osmicated specimens are usually not suited for low-temperature embedding under a fume hood: (LTE) in Lowicryl because of insufficient 1. Warm up object table a few degrees penetration of UV light through black above room temperature (RT) inside specimens. Osmication of freeze-dried the vacuum chamber. specimens may be performed before 2. Vent vacuum chamber, remove metal embedding in epoxy resins. plate (MP) (see Figure 15.1), close object table with a glass plate (GP) and transfer object table to a fume hood (see Figure 15.2).

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 Osmication directly after freeze-drying is not necessary to obtain excellent structure preservation (See Section 1.8. and 7, Observed Results).  The temperature of the specimens 4. Remove the container with OsO4 and must be higher than RT in Step 3 in order put drops of pure resin, e.g., Spurr’s to avoid adsorption of water to the resin, into container with osmicated hygroscopic freeze-dried specimens. specimens.

3. Open object table, place a small container with an osmium tetroxide (OsO4) crystal on the table and close it for some time, e.g., our hour.

COS = Container with an OsO4 crystal GP = Glass plate OT = Object table

Figure 15.2 Object table (OT) tightly closed with a glass plate (GP).

1.6. Infiltration with Resin 1. Vent and open vacuum chamber, remove metal plate (MP) (see Figure 15.1) from object table (OT) inside the chamber. 2. By using a syringe with a metal needle, introduce small amounts of embedding medium, e.g., 0.1 mL, into the containers with the dried specimens. 3. Vacuum infiltration of small freezedried specimens is not recommended for most biological material.5

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 The resin or a component of the resin with which infiltration starts must be liquid at the given object table temperature. For example, pure Lowicryl HM 20 can be used at an object table temperature of – 40oC. When final embedding is carried out with Spurr’s resin or EPON, the infiltration may start at an object table temperature of –20oC with a 1:1 DER-ERL mixture (two components of Spurr’s resin or a 1:1 propylenoxide/Epon mixture, respectively).  By introducing 0.1 mL of embedding medium, the liquid will be precooled during flow through the needle of the syringe inside the chamber, which is constantly flooded with cold GN2.

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1.7. Polymerization 1.7.1. UV polymerization at low temperature 1. Completely fill plastic containers (PC)  Plastic containers are used for LTE; on the object table inside the CFD metal containers are used for heat chamber with resin (e.g., Lowicryl polymerization (see Section 1.7.2). HM 20).  Different Lowicryls can be used for 2. Close object table by a UV translucent embedding and UV polymerization at low glass plate (GP), see Figure 15.2. temperature. (See Chapter 13, Figures 15.3.9 and 15.3.10, and References 2,5,6) 3. Place a UV lamp above the glass plate thereby closing the vacuum chamber  A Lowicryl K11M/HM20 mixture, (not evacuated). supplemented with 0.3% uranyl acetate, may be used to stabilize the specimen 4. After a few hours the infiltration of the during low temperature embedding, (see specimens may be complete and the Section 4, protocol P10; Reference 6 and UV lamp is switched on. Section 7, Figure 15.3.10). 1.7.2. Heat polymerization Warm up object table with metal container a few degrees above RT (see Section 1.5.) and remove it from the CFD. 1. Transfer small metal container into larger containers with pure resin.

 Transfer of the small specimens has to be carried out under the control of a stereo light microscope.

 The embedding moulds should have a glossy bottom in order to see the very small specimens in the polymerized blocks under 2. After sufficient time of infiltration (a a stereo light microscope. A small piece of few hours) transfer specimens into aluminum foil may be placed into embedding moulds with fresh pure embedding moulds with a rough surface. resin and polymerize at about 60°C. The foil may be removed after polymerization.

1.8. Ultramicrotomy and Analysis of Ultrathin Sections See Section 7, Observed Results.

 For analysis of mobile ions, such as sodium Na+ and potassium K+, prepare dry-cut sections (see Section 7, Figure 15.3.2 and References 2-4).  Lowicryl embedded specimens may be very labile and difficult to cut (see Section 7, Figure 15.3.9 and References 5,6). In this case the preparation can be stabilized by exposing the entire polymerized blocks to OsO4 vapor for, e.g., 30 min (see Section 4, protocol P9).

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2. SUMMARY OF THE DIFFERENT STEPS 1. Cryo-fixation.



2. Transfer of small frozen samples on cold object table. 3. Transfer of cold object table into freeze-drying apparatus CFD and automatic temperature controlled freeze-drying (FD). 4. Infiltration of freeze-dried specimens with resin. 5. Polymerization. 

3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Equipment for cryo-fixation 

    

 See Chapters in ACryo-Fixation Methods. Freeze-drying equipment, e.g., Leica  Other freeze-drying units suited for longEM CFD (Leica Microsystems, term freeze-drying at low temperatures Vienna, Austria) with UV lamp. down to 140ºC are no longer7 commercially available.  Homemade freeze-drying equipments, including adapted freeze-etch units, are mentioned in Reference 10. Stereo light microscope   LN2 Dewar  Oven for heat polymerization Scalpel   Tweezers

3.2. Products  Liquid nitrogen (LN2).  Resins for electron microscopy:  Spurr’s resin is mostly used due to its  Epoxy resins, e.g., Epon, Araldite, low viscosity that enables easy infiltration. Spurr’s resin. For resins used, see Section 7, Observed Results.  Acrylic resins, e.g., Lowicryl HM 20, HM23, K4M, K11M

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 Uranyl acetate.  Osmium tetroxide.

  OsO4. 



3.3. Solutions  Spurr’s resin.  Epon.  Lowicryl  HM20  K11M

 The embedding media are prepared according to the manufacturer’s instructtions.  See Chapter 13.

4. PROTOCOLS A general protocol for freeze-drying (FD)  These parameters are, e.g.: of biological material cannot be given  Quality of cryo-fixation because of the many parameters that  Heat transfer between specimen and determine the freeze-drying process which object table in the vacuum chamber are different for different biological  Size of specimen material in different physiological states.  Degree of hydration of the specimen  Distribution and structure of macromolecules  Distribution of ions  Interaction of water with macroIn general, long freeze-drying times molecules and ions. between 100ºC and 80ºC and long  freeze-drying times between 80ºC and 50ºC are essential in order to avoid severe shrinkage artefacts.2 Freeze-drying Protocols (P1P8) used for  The water of chemically fixed biological the different biological materials shown material is less attracted by cellular proteins under Observed Results are listed below. than the water of living cells. Therefore, freeze-drying of chemically fixed material Note: Infiltration with resin (see Section is faster than freeze-drying of native 1.6) as a rule starts at lower temperatures material.5 than the final temperatures of the freezedrying protocols. Specimens embedded in Lowicryls were infiltrated at 25°C for one day and polymerized for one day at 25°C. Protocol P1. FD  3 days at 80ºC  6 days at 60ºC

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 Striated frog muscle, (see Section 7, Figures 15.3.1, 15.3.2, 15.3.4) freeze-dried in a homemade, cryo-sorption freeze-drying unit.

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Protocol P2. FD  Temperature increase 1ºC h-1 from  Rat kidney, see Section 7, Figure 125ºC to 50ºC (75 h) 15.3.5 AB.  90 h at 50ºC  1ºC h-1 from 50ºC to +25ºC (25 h)  24 h at +25ºC  Total time: 214 h ~ 9 days

Protocol P3. FD  Temperature increase 0.2ºC h-1 from  Rat liver, see Section 7, Figure 15.3.6 90ºC to 30ºC (300 h) and the figure on the Chapter’s title page.  Temperature increase 1ºC h-1 from 20ºC to 10ºC (20 h)  10 h at 10ºC  Total time: 330 h ~ 14 days

Protocol P4. FD  Temperature increase 25ºC h-1 from  Dendritic cells, see Section 7, Figure 140ºC to 90ºC (2 h) 15.3.7 and Reference 9.  Temperature increase 0.4ºC h-1 from 90ºC to 50ºC (100 h)  Temperature increase 1ºC h-1 from 50ºC to 0ºC (50 h)  24 h 0°C  Total time: 176 h ~ 7 days

Protocol P5. FD  Temperature increase 50ºC h-1 from  Human platelets, see Section 7, Figure 140ºC to 90ºC (1 h) 15.3.8 and Reference 5.  Temperature increase 0.27ºC h-1 from 90ºC to 50ºC (150 h)  Temperature increase 1ºC h-1 from 50ºC to 0ºC (50 h)  24 h 0°C  Total time: 225 h ~ 9.5 days

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Protocol P6. FD  24 h 100ºC  Human lymphocytes, see Section 7,  Temperature increase 0.9ºC h-1 from Figure 15.3.9 and Reference 6. 100ºC to 10ºC (100 h)  10 h 10°C  After freeze-drying and LTE in Lowicryl  Total time: 134 h ~ 5.6 days HM20, the polymerized block has been stabilized by OsO4 vapor, see P9. Protocol P7. FD  Temperature increase 10ºC h-1 from 160ºC to 90ºC (7 h)  32 h 90ºC, temperature increase 0.3ºC h-1 from 90ºC to 0ºC (300 h)  48 h 0°C  Total time: 387 h ~ 16 days

 Jurkat cells, see Section 7, Figure 15.3.10 and Reference 5. During embedding in Lowicryl, the freeze-dried cells have been stabilized by uranyl acetate, see P10.

Protocol P8. FD  Temperature increase 1ºC h-1 from  This freeze-drying procedure has been 140ºC to 70ºC (7 h) called Molecular Distillation Drying.7 -1  Temperature increase 10ºC h from After freeze-drying and exposure to OsO4, vapor, specimens were embedded in 70ºC to +25ºC (9.5 h)  48 h at +25°C Spurr’s resin. The rather short period of  Total time: 127.5 h ~ 5.3 days freeze-drying at low temperature has been criticised.5,8 Protocol P9. Osmication after freezedrying and LTE in Lowicryl  Place a polymerized block for about 30 min into a 0.5 mL Eppendorf capsule together with a small amount (< 0.1 g) of OsO4 crystals.

 As a rule the block is first sectioned until the embedded biological material appears at the surface. After exposure to OsO4 vapor, the block appears dark brown and turns black after a few hours without further exposure.6

Protocol P10. Fixation with uranyl acetate during LTE  Dissolve overnight 0.4% (w/v) uranyl acetate in Lowicryl K11M.  Mix 3 parts of this solution with 1 part of pure HM 20.  Use this mixture with 0.3% uranyl acetate for complete LTE.

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 Uranyl acetate could not be dissolved in pure HM20.  See Section 7, Figure 15.3.10 and Reference 6.

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5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  No chemical fixatives are necessary.  Exceptions are described.  No organic solvent during dehydration.  Only minute amounts of embedding  media are needed.  Retention of mobile macromolecules  See References 2-5. and mobile ions (Na+, K+).  Preservation of selective ion adsorp-  See Section 7, Figure 15.3.4 and tion to cellular proteins. Reference 4.  Preservation of cellular ultrastructure  Precondition: excellent cryo-fixation, see and molecular conformation of Section 7, Figures 15.3.6, 15.3.10 and proteins. Reference5  Preservation of antigenicity.

 See References 5,9.

5.2. Disadvantages  Highly dependent on quality of cryo-  Not necessarily a disadvantage. fixation.  Handling of very small specimens.



 Time consuming.

 One to two weeks for small samples.

 Sophisticated freeze-drying equipment  The apparatus may be available through is necessary. a special order to Leica or possibly other companies or homemade.   A general protocol for every kind of  Optimization of protocols is required. specimen is not available.

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6. WHY AND WHEN TO USE A SPECIFIC METHOD The advantages of the freeze-drying technique for electron microscopic studies are given in Section 5.1 and exemplified by Observed Results (see Section 7). From the results obtained so far, it appears that the interactions between the main components of living cells (namely water, proteins and ions) are best preserved during and after proper freeze-drying because the biological material can be dehydrated by sublimation without changing the life-like conformation of proteins as it takes place during freezesubstitution with an organic solvent.  As a consequence, mobile ions, such as potassium K+, remain associated at or near their original protein sites and layers of water molecules remain associated at cytoplasmic protein sites thereby preventing condensation artifacts. This is concluded from the findings: 1. That even after long freeze-drying at rather high temperatures, a considerable amount of water remains in cryo-fixed native biological material.5 2. That the proteins of the cytoplasm are very homogeneously distributed.

             See Detailed Discussion in Reference 5.

The discovery of the unknown ultrastructure of cytoplasmic proteins of living cells and of their interactions with water and ions is a demanding goal that may be approached by using and further developing the freezedrying technique for electron microscopy.

   See Reference 5. 

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  See Discussions in References3-5

 See Section 7, Figures 15.3.6, 15.3.8. 

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7. OBSERVED RESULTS  Figure on the Chapter’s title page

 See Figure 15.3.6.

 Figure 15.3.1 Chemically unfixed frog Sartorius muscle, cryofixed on a LN2 cooled copper block, freeze-dried, embedded in Spurr’s resin, and section stained with uranyl acetate and lead citrate. (From Reference 2, reprinted with permission.)

 The muscle has been freeze-dried for three days at 80˚C followed by six days at 60˚C (see Section 4, Protocol P1.) Shorter drying times produced severe structural artifacts.2

 Figure 15.3.2 Dry-cut sections of a thallium Tl+-loaded (A), and of normal potassium K+-containing (B), cryofixed, freeze-dried and embedded muscle as in Figure 15.3.1. (From Reference 1, reprinted with permission.)

 Cellular K+ has been reversibly replaced with Tl+ in living muscle. The Tl+ contrast in (A) represents adsorption sites of K+ onto cellular proteins. For confirmation of this claim by independent microanalytical methods, see References 1,2,4.

 Figure 15.3.3 Cryo-section (0.1 µm thick) after freeze-drying for 33 h at 100˚C. Cryo-transfer of the dry cold section to a Zeiss EM 902. (From Reference 8, reprinted with permission.)

 Severe shrinkage even of ultrathin cryosections of chemically unfixed biological material during freeze-drying can only be avoided by long drying times (e.g., 33 hours) at low temperature.2,8

 Figure 15.3.4 Muscle sections stained with a solution containing 100 mM lithium chloride LiCl and 10 mM caesium chloride CsCl. (A) after freeze-drying and embedding in Spurr’s resin (see Figure 15.3.1.), (B) after glutaraldehyde fixation and embedding. (From Reference 3, reprinted with permission.)   Figure 15.3.5 Rat kidney, cryo-fixed (Leica MM80), freeze-dried and embedded in Spurr’s resin. For freezedrying times, see Section 4, Protocol P2. Sections stained with uranyl acetate and lead citrate. (A) No chemical fixation, N: nucleus; (B) with osmium tetroxide vapor fixation after drying (see Protocol P9), M: mitochondria. (From Reference 5, reprinted with permission). 

 In (A) the electron dense Cs+ is adsorbed with a high selectivity to certain cell proteins. Selective adsorption of alkali metal ions like K+ or Cs+ to cellular proteins can only be demonstrated in freeze-dried preparations, but not in chemically fixed preparations (no staining of section (B) nor in protein solutions4).

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 Without osmication, the membranes appear in negative contrast. They are not visible at the low magnification (A). Black membranes are shown in (B).

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 Figure 15.3.6 Rat liver, cryo-fixed (Leica MM80), freeze-dried and embedded in Spurr’s resin (no chemical fixative). Section stained with uranyl acetate and lead citrate. For freeze-drying times, see Section 4 (Protocol P3). aER: agranular endoplasmic reticulum, rER: rough endoplasmic reticulum, M: mitochondrion, P: peroxisome. The area around the asterisk (*) in the mitochondrion (M) is shown at higher magnification in the inset: Membranes are oriented perpendicular to the plane of the section. The two cristae membranes (CM) are thicker than the outer and the inner membrane (MM) of the mitochondrion. (From Reference 5, reprinted with permission.)

 The section was taken from the area of best cryo-fixation, at a distance less than 0.5 µm from the freezing plane. Membranes are clearly seen in negative contrast.

 Figure 15.3.7 Highly magnified MHC  Note satisfying immunolabeling of class II compartment from a human freeze-dried specimens embedded in Spurr’s immature dendritic cell, cryofixed resin.5,8 (Leica MM80), freeze-dried, see Section 4 (Protocol P4), embedded in Spurr’s resin. Immunolabeling (10 nm gold) for the MHC class II protein. (from Reference 9, reprinted with permission.)  Figure 15.3.8 Human platelet, cryofixed (Leica MM80), freeze-dried (see Section 4 (Protocol P5) and resin embedded in Spurr’s resin. Immunolabeling (10 nm gold) for CLP 36, a PDZ-LIM domain protein associated with α-actinin. G: secretory storage organelles, DTS: dense tubular system = agranular endoplasmic reticulum. (From Reference 5, reprinted with permission.)

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 The platelet stems from an area distant from the freezing plane less than 5 µm. Membranes appear in well-defined negative contrast (as in Figure 15.3.6). The very homogeneous aspect of the cytoplasm is typical for excellent cryo-fixation.

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 Figure 15.3.9 Ultrathin section of human lymphocytes after freezedrying; see Section 4 (Protocol P6) and low temperature embedding in Lowicryl HM20 at 25°C. Polymerized block exposed to OsO4 vapor (see Protocol P9). Uranyl acetate and lead citrate staining. (From Reference 6, reprinted with permission.)

 Compression and extraction artifacts may occur during wet cutting of well-fixed, freeze-dried and Lowicryl embedded specimens. After exposure of the polymerized block for 30 min to OsO4 vapor, the whole resinbiological material complex is stabilized and the ultrastructure is well preserved.6

 Figure 15.3.10 Jurkat cells, highpressure frozen in cellulose microcapillaries, freeze-dried, see Section 4 (Protocol P7) and resin embedded at 25°C in a K11M/HM20 Lowicryl mixture, supplemented with 0.3% uranyl acetate (see Protocol P10). Uranyl acetate and lead citrate staining. The asterisk (*) represents the microcapillary that was used. (From Reference 5, reprinted with permission).

 The freeze-dried cells are stabilized during infiltration and UV polymerization of the Lowicryl mixture by supplemented uranyl acetate.

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8. REFERENCES 1.

Edelmann, L. Subcellular distribution of potassium in striated muscles, Scan. Electron Microsc., 875, 1984. 2. Edelmann, L. Freeze-dried embedded specimens for biological microanalysis, Scan. Electron Microsc., 1337, 1986. 3. Edelmann, L. Two opposing theories of the cell: Experimental testing by cryomethods and electron microscopy, in The Science of Biological Specimen Preparation, Müller, M., et al., eds., Scanning Electron Microscopy., Inc., AMF O'Hare, Chicago, IL, USA, 1986, 33. 4. Edelmann, L. Basic biological research with the striated muscle by using cryotechniques and electron microscopy, Physiol. Chem. Phys. Med. NMR, 33, 1, 2001. 5. Edelmann, L. Freeze-dried and resin-embedded biological material is well suited for ultrastructure research, J. Microsc., 207, 5, 2002. 6. Edelmann, L. and Ruf, A. Freeze-dried human leukocytes stabilized with uranyl acetate during low temperature embedding or with OsO4 vapor after embedding, Scan. Microsc. Suppl., 10, 295, 1996. 7. Linner, J.G. et al. A new technique for removal of amorphous phase tissue water without ice crystal damage: A preparative method for ultrastructural analysis and immunoelectron microscopy, J. Histochem. Cytochem., 34, 1123, 1986. 8. Sitte, H. et al. A new versatile system for freeze-substitution, freeze-drying and low temperature embedding of biological specimens, Scan. Microsc. Suppl., 8, 47, 1994. 9. Spehner, D. et al. Embedding in Spurr's resin is a good choice for immunolabelling after freeze drying as shown with chemically unfixed dendritic cells, J. Microsc., 207, 1, 2002. 10. Steinbrecht, R. and Müller, M. Freeze substitution and freeze drying, in Cryotechniques in Biological Electron Microscopy, Steinbrecht, R. and Zierold, K., eds., Springer, Berlin, Germany 1987, 149. 

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Part IV Freeze-Fracture and Metal Shadowing

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CONTENTS

GENERAL INTRODUCTION .................................................................................... 395 1.

PRINCIPLE OF THE METHOD .................................................................... 396 1.1. 1.2.

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 399 2.1. 2.2.

3.

Freeze-Drying and Metal Shadowing ....................................................... 403 Freeze-Fracture and Freeze-Etching......................................................... 405

ADVANTAGES/DISADVANTAGES.............................................................. 407 5.1. 5.2.

6.

Materials ................................................................................................... 400 Products .................................................................................................... 402

PROTOCOLS .................................................................................................... 403 4.1. 4.2.

5.

Freeze-Drying and Metal Shadowing ....................................................... 399 Freeze-Fracture and Freeze-Etching......................................................... 399

MATERIALS/PRODUCTS .............................................................................. 400 3.1. 3.2.

4.

Freeze-Drying and Metal Shadowing ....................................................... 396 Freeze-Fracture and Freeze-Etching......................................................... 398

Advantages ............................................................................................... 407 Disadvantages........................................................................................... 407

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 408 6.1.

6.2.

Freeze-Drying and Metal Shadowing ....................................................... 408 6.1.1. Visualization of fine surface details............................................ 408 6.1.2. Information on small molecules.................................................. 408 Freeze-Fracture and Freeze-Etching......................................................... 408 6.2.1. Freeze-fracture ............................................................................ 408 6.2.2. Freeze-etching............................................................................. 408

7.

OBSERVED RESULTS .................................................................................... 409

8.

REFERENCES .................................................................................................. 410



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GENERAL INTRODUCTION

Freeze-drying followed by unidirectional shadowing is a technique that enables one to directly observe the surface topography of biological objects. The contrast enhancement is limited to the surface exposed to the metal while the contrast contribution from non surface exposed areas is negligible. The appearance of a shadow is caused by the metal deposited under a fixed azimuthal and elevation angle. This provides images with a quasi 3D appearance.

Figure 16.1 Isolated membrane of an epithelial cell, observed after freeze-drying and shadowing. Bar = 200 nm

Freeze-fracture and freeze-etching are two related techniques. They allow electron microscopic investigation of cell surfaces and biological membranes in the frozen state. The fracturing of the frozen object produces cross-sectional views of the structure, but also views inside fractured membranes and surface views of organelles. Moor and Mühlethaler,1 in 1963, were the first to fully describe the yeast cell using the freeze-etching technique. Figure 16.2 Yeast spheroplast. Total view of a freeze-fractured cell after high-pressure freezing. Bar = 500 nm

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1. PRINCIPLE OF THE METHOD Shadowing or shadow casting means evaporating atoms of a heavy metal from a point source at an oblique angle to the specimen.2-6 Metal piles up on surfaces that face the source, but surfaces facing away from the source are shielded and receive no metal. This is the “shadow” that appears white under the electron beam. Given the angle of shadowing θ and the length of the shadow l, the height of the object h, which produces the shadow, can be determined simply from the formula: h = l * tan θ.

 Platinum (Pt) is mostly used in conjunction with carbon (Pt/C).

 Inverting the contrast will give contoured bright objects illuminated from the side by light and projecting a dark shadow.          

Figure 16.3 Isolated viral particles after shadowing. For θ = 45°, h = l = 27 nm. Bar = 100 nm   

1.1. Freeze-Drying and Metal Shadowing The goal is to freeze a hydrated suspension  of particles (virus, membrane, macromolecular complexes), then to freezedry the sample under vacuum and finally to shadow the surface of the specimen by metal evaporation. A sample suspension is applied to carboncoated grids and adsorbed. The grids are washed, blotted to remove excess water, quick frozen in liquid nitrogen and then transferred into a freeze-drying/metal shadowing unit (i.e., a freeze-fracture apparatus). In the apparatus, samples are freeze-dried for two hours at 80°C.

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Freeze-dried specimens are then shadowed with platinum–carbon (Pt/C) at a given elevation angle (commonly 45°), followed by evaporation of a layer of carbon at an elevation angle of 90° to prevent possible metal grain redistribution.

       

 

 

Figure 16.4 Freeze-dried ferritin molecules unidirectionally shadowed by Pt/C. θ = 45°. Bar = 50 nm               

 

Figure 16.5 Freeze-dried ferritin molecules rotationally shadowed by Pt/C. θ = 45°. Bar = 50 nm

  

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1.2. Freeze-Fracture and Freeze-Etching In the freeze-fracture technique, the specimen is frozen and broken into two pieces to expose fracture faces. The surface is immediately imprinted by making a shadow replica. In the freeze-etching variant, the specimen surface is allowed to freeze-dry slightly in order to remove the ice surface.7-9 Bulk biological samples are mounted between copper plates then frozen either by quick plunging in liquid propane or by high-pressure freezing. For freeze-fracture, the preparations are immediately shadowed at an elevation angle of 45° with 2 nm of platinum–carbon (Pt/C) and backed with a 10 to 20 nm-thick layer of carbon. For freeze-etching, after fracturing, the specimen temperature is raised to 100°C for about 2 min for etching. The specimen is then shadowed as above. After thawing, the replicas are cleaned of the remaining sample, rinsed in distilled water and mounted on copper grids.

 A method of specimen preparation for electron microscopy, in which a replica is made from a sample that has been rapidly frozen and then fractured along natural planes of weakness, mostly through the hydrophobic part of membranes9 to reveal its internal structure. 

Figure 16.6 Freeze-fractured microvilli from a midgut epithelial cell of an insect. Pt/C, θ = 45°. Bar = 100 nm

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The Shadow of Hydrated Biological Specimens

2. SUMMARY OF THE DIFFERENT STEPS 2.1. Freeze-Drying and Metal Shadowing 1. Cool the specimen stage of the freeze-fracture machine to 160°C. 2. Apply a 5 µL drop of suspension on a collodion carbon-coated grid. 3. Wash with a volatile buffer and/or distilled water. 4. Blot with filter paper. 5. Freeze in liquid nitrogen. 6. Introduce the specimen into the high vacuum chamber of the freeze-fracture machine. 7. Freeze-dry for two hours by raising the specimen stage to 80°C. 8. Evaporate Pt/C at a given elevation angle. 9. Evaporate carbon to stabilize the Pt/C layer at an angle of 90oC. 10. Warm up the specimen in vacuo to room temperature.

2.2. Freeze-Fracture and Freeze-Etching 1. Cool the specimen stage of the freeze-fracture machine to 160°C. 2. Mount the bulk biological sample between two copper plates. 3. Freeze the sandwich by quick plunging into liquid propane or better, if available, by highpressure freezing. 4. Introduce the specimen into the high vacuum chamber of the freeze-fracture machine.

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5. Fracture the specimen: The fracture can be done by using a knife or by breaking the sandwich into two pieces, depending on the equipment available. 6. Optional step of etching: Raise the temperature of the specimen stage to 100°C so as to allow sublimation of surface ice for 2 min. 7. Evaporate 2 nm of Pt/C at a given elevation angle (45°). 8. Evaporate at 90° a thick layer (20 nm) of carbon to stabilize the Pt/C replica. 9. Thaw the specimen and clean the replica by digesting the tissue. Wash the replica in distilled water. 10. Collect the replicas on uncoated grids.

3. MATERIALS/PRODUCTS 3.1. Materials  Equipment for rapid freezing.   Freeze-fracture/-etching unit.



© 2009 by Taylor & Francis Group, LLC

 See Part I, Cryo-Fixation Methods

Figure 16.7 JFD-9000 Freeze-Etching Equipment from JEOL, Tachikawa, Tokyo, Japan.

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

  Evaporating material



Figure 16.8 Cryofract 190 from ReichertJung, (now Leica Microsystems, Vienna, Austria). 

Figure 16.9 BAF 060 from BALTEC, Balzers, Liechtenstein, (now Leica Microsystems, Vienna, Austria). . 

Figure 16.10 Carbon tip (left), platinumcarbon tip (right) and ring-shaped filament, components of the electron beam evaporation gun10 of the Jeol JFD-9000.

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 Specimen holders  For unidirectional or rotary shadow  See Figure 16.11. casting (Jeol) with five magnetic slots for holding five nickel grids.  For freeze-fracture or freeze-etching  See Figure 16.12. (left: Cryofract, right: Jeol).

Figure 16.11 Specimen holder.  Left: The holder can receive four sandwiches made with two copper plates. The sandwiches are broken by opening the holder.  Right: The holder can receive one disk, whose surface has been scratched for better adherence of the object.   Dewar vessels  Filter paper  Nickel or copper grids, 300 to 400 mesh  Platinum loop  Porcelain spot plates  Tweezers  Wide bore pipette

3.2. Products    

2% (w/v) Ammonium acetate Carbon 2% (w/v) Collodion in amyl acetate 30% (w/v) Chromium oxide

 Distilled water  Household bleach (10 to 12% sodium hypochlorite)  Liquid nitrogen  Pt/C  Sample suspension for freeze-drying/ metal shadowing  70% (v/v) Sulphuric acid

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Figure 16.12 Specimen holders.

 The depth of the well (6 to 8 mm) should allow manipulation of replicas.

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4. PROTOCOLS 4.1. Freeze-Drying and Metal Shadowing 1. Cool down the specimen stage of the freeze-fracture apparatus to approximately 160°C. 2. Grids  Nickel or copper grids, 300 to 400 mesh, covered with a carbon film or a carbon collodion film.  Glow discharge the grids for 30 sec in low pressure air (2 × 10-1 Torr).

     To create positive charges.

3. Sample suspension  Spot on the grids 5 to 10 µL of the  It is helpful to examine the sample by sample suspension at approximately negative staining to determine the right 0.1 to 0.5 mg/mL for 1 min. concentration and time needed for contact of the sample with the grid. 4. Wash  3 to 5 times with distilled water or  This step is crucial to avoid salt with a volatile solvent like precipitation during freeze-drying. If ammonium acetate followed by buffering is necessary, a freshly made water. solution of 2% ammonium acetate is most often used. 5. Blot  Remove excess liquid with filter  Leave a very thin layer of water. paper. 6. Freeze  Quickly plunge the grid into liquid  Liquid ethane and liquid propane may nitrogen. also be used because they have better  Mount the grid on the specimen cryogenic properties than liquid nitrogen. holder maintained at liquid nitrogen temper-ature. 7. Dry  Introduce the specimen into the high vacuum chamber of the freezefracture/freeze-etching device.  Raise the temperature of the temperature controlled specimen stage to 80°C and maintain the specimen at this temperature for about two hours.

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 The freeze-drying process can be followed by monitoring the pressure of the chamber. During water sublimation, the pressure increases progressively to reach a plateau, which corresponds to the freezedrying state of the specimen. A simple alternative is to examine the grid through a binocular; the specimen is ready when ice is no longer seen.

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

 The grain size (and ultimate resolution) of the deposited metal depends on the Turn on the high voltage (HV) temperature of the specimen prior to switch and set the voltage to 3000 V. evaporation. Cooling down the specimen to 100°C or lower will reduce the grain size.5 Select the Pt/C source.



Tilt the specimen stage to 45°.



Raise gradually the emission current to 40 mA.



Monitor the thickness of the metal with the quartz crystal thickness monitor. When the evaporation is complete, return emission to 0 mA.



Return the specimen stage to its horizontal position.

8. Evaporate Pt/C 

 Rotary shadowing is recommended for studying small objects like macromolecules (see Figure 16.5). The specimen is mounted on a revolving holder, tilted under a chosen angle and rotated during Pt/C evaporation.  For small particles, DNA, filamentous proteins, use a low angle (5 to 30°).

 If the device for quartz crystal thickness measurement is not available or is not working properly, the alternative to obtain reproducible results is to calibrate the metal deposition by timing for a given current emission (i.e., 30 sec for 40 mA emission).

9. Evaporate C 

Select the C source.



Gradually raise the emission current to 40 mA.



Monitor the thickness of the C. When the evaporation is complete, return emission to 0 mA.

 See above.

10. Warm up the specimen in vacuo to  Good results are obtained for a Pt/C room temperature. thickness of 1 nm and a C thickness of 10 nm.

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4.2. Freeze-Fracture and Freeze-Etching 1. Cool down the specimen stage of the  freeze-fracture apparatus to approx-   imately 160°C.   Freezing is a crucial step for the success 2. Freeze  Mount the bulk biological specimen of the experiment. These methods are between two copper or aluminum presented and fully documented in Part I, Cryo-Fixation Methods, of this book (see plates. Chapters 26).  Freeze the sandwich by high-pressure  Instead of making a sandwich, one can freezing (see Chapters 5, 6). use one copper disk with a scratched or surface for better adherence of the object. A  Drop or plunge the object into liquid small droplet is placed on the disc and then propane. dropped into liquid propane.   Introduce the specimen into the high vacuum chamber of the freezefracture/freeze-etching device.  Mount the object on the precooled  Wait for a brief period until the specimen stage. temperature is stable. 3. Fracture  Freeze-fracture: Strike the sample mounted on one copper disk with the cold knife or break the sandwich into two pieces. Then immediately evaporate the metal (see Step 4). or  Freeze-etching: After the fracture process, the sample stage is warmed up to 100°C, while a cold trap (below 150°C) is held over the sample. The etching process lasts usually between one and two minutes.

 Freeze-fracture refers to experiments where ice removal is negligible; it is performed at low temperature (usually 150°C), and Pt/C evaporation is started before breaking the specimen.  The cold knife held above the specimen is often used as a cold trap. It traps the water, which sublimates out of the fracture surface and prevents its redeposition onto the fracture surface.

4. Evaporate  Turn on the HV switch and set the voltage to 3000 V.  Select the Pt/C source.  Most freeze-fracture methods use  Tilt the specimen stage to 45°.  Gradually raise the emission current to unidirectional shadowing at a nominal angle of 45°. However, fine details can be 40 mA. enhanced by using a lower angle of, e.g., 10 to 30°.

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 Rotary shadowing is also an alternative to reveal fine details in replicas. The specimen is mounted on a revolving holder, tilted under a chosen angle and rotated during Pt/C evaporation.  Monitor the thickness of the metal  A 2 nm-thick layer of Pt/C provides with the quartz crystal thickness good results. monitor. When the evaporation is complete, return emission to 0 mA.  Return the specimen stage to its horizontal position.  Select the C source.

 A 20 nm backing layer of carbon will strengthen the replica for the cleaning  Gradually raise the emission current to procedure. 40 mA.

 Monitor the thickness of the C. When the evaporation is complete, return emission to 0 mA. 5. Clean  The thin replica needs to be lifted off the  Remove the specimen from the freeze- sample before it can be viewed in the fracture device and leave it in distilled electron microscope. water for thawing.  Float the replicas on sulphuric acid  The digesting solution should remove (70%, 12 hours). the sample without harming the replica. Bleach, sulphuric acid, chromium oxide are usually used, but there is no universal recipe. This step can last from hours to days, depending on the tissue analyzed.  Transfer to a bath of distilled water for  Transfer from one liquid to the next with washing (five minutes). a platinum loop or wide-bore pipette.  Transfer to a bath of household bleach (2 to 12 hours).  Transfer to bath of distilled water (20 minutes).  Repeat the previous step until the bleach is replaced with distilled water.  Collect the replicas on uncoated 100 to 300 mesh copper grids.

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 The major problem is the breakage of the replica due to swelling and shrinkage of the specimen in the cleaning solutions. A device has been described for washing replicas11 or methods for strengthening the replica12 have been developed.

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5. ADVANTAGES/DISADVANTAGES 5.1. Advantages   Shadowing is interesting to examine:  Fine order surface details, such as  membrane proteins, which cannot be observed in any other way.3,5,6,13  Small objects of relatively low electron density because their contour may not be clearly defined otherwise.  Freeze-fracture and freeze-etching are  straightforward methods to obtain fine structural details within the cell. Specific details are revealed, based on the fracturing behavior of each individual tissue sample.  In freeze-fracture, the preferential fracture plane goes through the lipid bilayer.14 This is widely used for studying distribution and localization of integral membrane proteins.15-17 

5.2. Disadvantages  The grain size of Pt/C (2 nm) limits the  The alloy tungsten–tantalum gives finer grains than Pt/C (1 nm) but is more difficult resolution. to evaporate.13  Freeze-drying can induce shrinkage of  the material.6  Freeze-etching on model systems has  To reduce these artifacts, rapid freezing demonstrated that cryo-fixation can procedures and high-pressure freezing introduce artifacts as a result of slow methods have been proposed (see Chapters freezing like segregation of dispersed 26). material from the solvent or of intramembrane particles in biological membranes.18  Fracturing proceeds at its own will. It  The preferential fracture plane goes 14 is not possible to choose a particular through the lipid layer of the membranes. path for the fracture plane to follow.  Freeze-fracture apparatuses are expensive.

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6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Freeze-Drying and Metal Shadowing  Shadowing is of great interest to  examine:  Small objects of relatively low electron density because their contour  may not be clearly defined otherwise. 6.1.1. Visualization of fine surface details 

 Protruding membrane proteins.

 See Figure 16.1.

 One-sided information on tubular  crystals to determine the pitch of a   helical structure.   

6.1.2. Information on small molecules  Rotary shadowing can give informa-  See Figure 16.5. tion on the oligomeric state of small proteins.    DNA etc.   

6.2. Freeze-Fracture and Freeze-Etching  Freeze-fracture and freezeetching are  straightforward methods to obtain fine structural details within the cell. 6.2.1. Freeze-fracture



 Freeze-fracture provides a high mag-  nification view of the physical relationships between membrane components (see also Chapter 22).

6.2.2. Freeze-etching



 Freeze-etching is useful to study  relationships between cytoskeletal elements within the cell.

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7. OBSERVED RESULTS  Figure on the Chapter’s title page Isolated membrane of an epithelial cell,  Only one side of the membrane is observed after freeze-drying and shad- observed. Membrane proteins forming small aggregates are clearly seen (Pt/C, owing. θ= 45°, unidirectional shadowing).  Figure 16.1

 See General Introduction. Both sides of the membrane are Isolated membrane of an epithelial cell, observed, showing that the membrane has observed after freeze-drying and shad- an asymmetrical structure (Pt/C, θ = 45°, owing. unidirectional shadowing).   Figure 16.2

 See General Introduction.  Nucleus, vacuole-like structures and mitochondria are clearly observed (Pt/C, Yeast spheroplast. Full view of a freezeθ = 45°). fractured cell after high-pressure freezing.  Figure 16.3

 See Section 1.  The metal accumulates on the dark side while the shadow on the other side appears Isolated viral particles after freeze-drying white (Pt/C, θ = 45°). and shadowing.   Figure 16.4 Ferritin molecules freeze-dried and unidirectionally shadowed.   Figure 16.5 Ferritin molecules freeze-dried rotationally shadowed.

 See Section 1.1.  Pt/C, θ = 45°

 See Section 1.1.  The metal accumulates around the and spherical molecule and, in this case is structurally more informative than unidirectionally shadowing Pt/C, θ = 45°.

 See Section 1.2.  Figure 16.6 Freeze-fractured microvilli from a midgut  Pt/C, θ = 45° epithelial cell of an insect. Intramembrane particles (IMP) form a regular network within the microvilli membranes, with a constant 8 nm repeat unit. 

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8. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17. 18.

Moor, H. and Mühlethaler, K. Fine structure in frozen-etched yeast cells. J. Cell Biol., 17, 609, 1963. Kistler, J., Aebi, U. and Kellenberger, E. Freeze drying and shadowing a twodimensional periodic specimen, J. Ultrastruct. Res., 59, 76, 1977. Studer, D., Moor, H. and Gross. H. Single bacteriorhodopsin molecules revealed on both surfaces of freeze-dried and heavy metal-decorated purple membranes, J. Cell Biol., 90, 153, 1981. Fowler, W.E. and Aebi, U. Preparation of single molecules and supramolecular complexes for high-resolution metal shadowing, J. Ultrastruct. Res., 83, 319, 1983. Gross, H., et al. High resolution metal replication quantified by image processing. Ultramicroscopy, 16, 287, 1985. Gross, H. High resolution metal replication of freeze-dried specimens. In Cryotechniques in Biological Electron Micoscopy, Steinbrecht, R. A. and Zierold, K. eds., Springer-Verlag, Berlin, Heidelberg; Germany, 1987, 203. Steere, R.L. Electron microscopy of structural detail in frozen biological specimens. J. Biophys. Biochem. Cytol., 3, 45, 1957. Moor, H. et al. A new freezing-ultramicrotome. J. Biophys. Biochem. Cytol., 10, 1961. Branton, D. Fracture faces of frozen membranes. Proc. Nat. Acad. Sci. USA., 55, 1048 1966. Moor, H. Evaporation and electron guns. In Freeze-Etching Techniques and Applications, Benedetti, E.L. and Favard, P., eds., Société Française de Microscopie Electronique, Paris, France,1973, 27. Hohenberg, H. and Mannweiler, K. Semiautomatic washing device for simultaneous cleaning of surface replicas under identical conditions. Mikroskopie (Wien), 36, 145, 1980. Stolinski, C., Gabriel, G. and Martin, B. Reinforcement and protection with polystyrene of freeze fracture replicas during thawing and digestion, J. Microsc., 132, 149, 1983. Bachmann, L., et al. Decoration and shadowing of freeze-etched catalase crystals. Ultramicroscopy, 16, 305, 1985. Branton, D. The fracture process of freeze-etching. In Freeze-Etching Techniques and Applications, Benedetti, E.L. and Favard, P., eds., Société Française de Microscopie Electronique: Paris, France, 1973, 107. Hohenberg, et al. Plasma membrane antigens detected by replica techniques. In Science of Biological Specimen Preparation 1985, Müller, M., Becker R.P., Boyde, A. and Wolosewick, J.J. eds., SEM Inc. AMF O'Hare, IL, USA, 235. Fujimoto, K. SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distribution of integral membrane proteins and phospholipids in biomembranes: Practical procedure, interpretation and application. Histochem. Cell. Biol., 107, 87. 1997. Bron, P., et al. Oligomerization state of MIP proteins expressed in Xenopus oocytes as revealed by freeze-fracture electron microscopy analysis. J. Struct. Biol., 128, 287, 1999. Costello, M.J. and Gulik-Krzywicki, T. Correlated x-ray diffraction and freezefracture studies on membrane model systems: Perturbation induced by freezefracture preparative procedures, Biochim. Biophys. Acta, 455, 412, 1976. 

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CONTENTS

GENERAL INTRODUCTION .................................................................................... 415 1.

PRINCIPLES OF THE METHOD .................................................................. 418 1.1.

Preparation of the Sample, Preservation of the Internal Structure and Environment by Rapid Freezing ............................................................... 418

1.2. 1.3. 1.4. 1.5.

Transfer and Fracture under Vacuum and at Low Temperature: 170°C. 419 Shadowing and Replication ...................................................................... 419 Dissolving the Material and Washing the Replicas .................................. 420 Recovery of the Replicas and Observation............................................... 420

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 420

3.

MATERIALS/PRODUCTS .............................................................................. 421 3.1. 3.2.

Materials ................................................................................................... 421 Products .................................................................................................... 421

4.

PROTOCOL ...................................................................................................... 422

5.

ADVANTAGES/DISADVANTAGES.............................................................. 424 5.1. 5.2.

Advantages ............................................................................................... 424 Disadvantages........................................................................................... 424

6.

VITREOUS ORGANIC SOLVENT MADE SIMPLE ................................... 425

7.

OBSERVED RESULTS .................................................................................... 425

8.

REFERENCES .................................................................................................. 429

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GENERAL INTRODUCTION A number of difficulties in carrying out electron microscopic studies are due to the physico-chemical state of the sample. For “hard materials”, such as ceramics and metal alloys, the structure of the sample depends very little on their environment and many techniques can be used routinely to study their internal organization (ion milling, polishing, focused ion beam (FIB), etc.). The difficulties increase when the samples to be characterized have a structure that highly depends on their solvent content. This is, in fact, the case for most biological samples. Many chapters in this book examine how microscopists have overcome these problems in the field of biology. Biological objects can be observed in dilute conditions, in thin films, by cryo-TEM. There are a number of instances where dilution is undesirable because it may destroy the architecture of the material or it does not allow observation in the native state. Thin films are also inadequate for large objects such as large liposomes or tissues. Therefore, cryo-fracturing was developed by several groups9,10,22 to allow the investigation of large samples (tissue pieces or whole cells) or lipid phases,11 where the concentrations are the same as under physiological conditions (see also Chapter 16). These technical advances were achieved exclusively in aqueous medium. However, in organic solvents, microscopy has not yet reached this state-of-the-art. This sharp contrast is unfortunate because there is a growing demand from material scientists to examine objects in organic solvents. In the last decades, material science has exploited the concept of self-assembly to fabricate new materials.13,26 Self-assembly is the property that some molecules possess to associate spontaneously with themselves or with other molecules to form larger structures. In the field of chemistry, molecules have a limited size, typically at most 1 nm. But, the selfassembled objects they can form have dimensions as large as 10 to 1000 nm. The unique properties of self-assembled materials emerge from those large aggregates and not from the single molecules. Therefore, microscopic techniques with a resolution of a few nm are necessary to evaluate the shape and the structure of the aggregates. Among these materials, one can find liquid crystals and noncovalent polymers. This section will be devoted only to the case of organogelators because they illustrate most of the difficulties that can arise during the structural analysis of self-associated systems in organic solvents. Organogelators23,25 are a growing class of compounds that are able to gel organic solvents at low concentrations (typically a few percent per weight). This property is due to their ability to form self-assembled structures with a high aspect ratio (fibrils, platelets, ribbons, nanotubes, etc.). These structures form a 3-D network that is responsible for the visco-elastic properties of the materials. The self-assembly proceeds through noncovalent bonds, such as H-bonds, dipole or - interactions and Van der Waals forces. The weakness of these interactions is expressed at the macroscopic level by the thermoreversibility of the gels; when they are heated, a sharp transition to a sol phase is observed, and the gel forms again upon cooling. The rheological properties of organogels explain their use in many industrial applications, mainly as thickeners or rheomodifiers; they are used in coatings, in cosmetics or lubricants. The formation of a network with a high specific area enables their use as nucleating agents for semicrystalline polymers.19 Besides these widespread applications, recent work has shown that they can be used to template inorganic or organic compounds to form tubes or mesoporous materials.16,21,24 In the medical field, biocompatible gelators have been used as drug delivery systems3,14,15 or supports for tissue growth.8,12,20

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The interest in these compounds is also triggered by more fundamental questions. For instance, it is still hard to establish a clear relationship between the chemical structure of their gelators and the ability to form gels. The analysis of the shape and the dimensions of the self-assemblies are necessary to explore this relationship. Moreover, it is interesting to relate the morphology of the self-assembled fibrils to the rheological properties of the gels or to the conditions of their formation (cooling rate during gelation, concentration, etc.). Each of these questions requires an efficient tool to systematically measure the size of the self-assemblies. At this scale, only a few techniques are available to investigate the shape of the objects. Among them are small angle neutron or x-ray scattering techniques but these measurements often require beam time on large facilities. Electron microscopy is precious in this regard because it can provide a well-established method to study the structure of individual self-assemblies. The cryo-fracture technique of preparation and observation of organogels was chosen because of its specificity. We describe below some of the properties of organogels that determined this choice. 1. The structure of the self-assembled objects strongly depends on the solvent. The formation of fibrils and gels occurs in certain solvents only. In other organic solvents, the gelators may just form crystals or may remain soluble. Some of the behavior can be rationalized easily with basic chemical concepts. For instance, Hdonor or H-acceptor solvents can form H-bands with the gelators, competing with the intermolecular bonds of the self-assemblies, which results in a solubilization of the compounds. But very often it is hard to rationalize why certain solvents favor the formation of gels. The shape of the fibrils also varies with the solvent in which they are formed. 2. The structure of the self-assembly depends on its concentration. Self-assembly is governed by the following chemical equilibrium between single molecules, on the left and associated ones on the right:

Figure 17.1 As a consequence of the mass action law, the equilibrium is expected to shift toward the formation of single molecules upon dilution of the mixture. The phase diagram has been established for many organogelators. An example is given in the figure below. It can be easily observed that for a given temperature, there is a concentration C*, namely the gel concentration or minimal gel concentration, below which, the gel can no longer form.

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Figure 17.2 Phase diagram of gelators. For each temperature (here 25°C) the gel concentration C* is the concentration below which the gel does not form. The phase diagrams can be more complex, and for instance, the shape of the selfassemblies can vary with the concentration. The sol phase can be constituted of soluble self-assemblies, but their shape may differ from the ones in the gel. Both the properties described above result in severe limitations for the choice of the techniques used to prepare the sample — the solvent must be preserved in the samples and the concentration must be kept constant. For instance, when the observation is carried out on a solution that has been deposited on a carbon grid, blotted and shadowed, selfassembled objects can be observed, but their relevance to the objects in the real gel is always questionable. Either the concentration is kept constant during the preparation of the sample, and then, what is observed are the self-assemblies that are actually present in the sol, but not in the gel. It is more probable that the solvent evaporates during the preparation, but in this case, the final composition is not known. In this regard, it must be reminded that many organic solvents are more volatile then water. For instance, the vapor pressure at 25°C is 24 mmHg for water, 300 for ethyl acetate, 309 for cyclohexane, and 623 for chloroform. The evaporation of the solvent is a real concern when handling organogels. Other difficulties arise from the rheological properties themselves. The samples studied are gels. Their high viscosity forbids any spreading in thin films. Because of their high solvent content, they are soft materials and, hence, difficult to cut into ultrathin sections suitable for direct observation. Cryo-ultramicrotomy would be necessary, but it needs to be implemented for organic solvents first. These reasons have stimulated the implementation of the well-know technique of cryofracture to study the ultrastructure of organogels. The technique is based on rapid freezing of the sample to prevent crystallization of the solvent and to obtain it in a vitreous state. The benefits of the process that has been widely exploited in biology are also ideal to study the case of the organogelators because the solvent environment of the objects is preserved and the concentration is fixed. Rapid freezing is also expected to prevent any reorganization of the molecules within the objects, and thus allows observation of the self-assemblies at equilibrium and in their native state. Talmon has written that “freezefracture replica, regrettably a dying art, should be revived and used more for imaging high-viscosity systems.”5

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This chapter will present how to perform freeze-fracture experiments in organic solvents to study self-assembled or high-viscosity systems. We will present here some protocols that help in overcoming many of the obstacles associated with organic media. A number of papers illustrate the success of the method.1,2,4,6,7,17,18 Herein we will deliberately show some pictures resulting from negative experiments or careless protocols as well. These examples may help to identify problems in the case of poor results. However, the proposed protocols have been improved to make cryo-fracture a routine technique.

1. PRINCIPLES OF THE METHOD 1.1. Preparation of the Sample, Preservation of the Internal Structure and Environment by Rapid Freezing  The choice of the solvents in which the self-assembly will be performed is the first important step. The propensity of organic solvents to be vitrified is variable.

 Some solvents tend to crystallize upon rapid freezing, whereas others become amorphous. Unlike cryo-fracture in aqueous medium, no cryoprotectant (like glycerol in water, for instance) can be added.

 So far, no rule has been established  From our experience, we can empirically that predicts how easily a solvent can distinguish three kinds of solvents. (1) be vitrified. Good solvents that seldom crystallize. These solvents are dichloromethane,4 toluene,17,18 cyclohexane2,6,7 acetonitrile, pyridine,4 tetrachloroethane, mixtures of solvents. (2) Borderline solvents that can be vitrified, but often crystallize in the same sample. Obtaining clean pictures with the latter is still possible although difficult. This is the case of nonane,1 or tetralin. (3) Poor solvents that exclusively give rise to crystals. No other structures can be observed after fracture. This is the case of dodecane or heptane.  As discussed in the introduction,  For example, replacing dodecane by changing the solvent may change the nonane or cyclohexane structure of the self-assembly. However, it might sometimes be wise to switch to a solvent with close chemical properties, but with a different behavior in the freezing process.

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Cryo-Fracture of Self Assembled Systems in Organic Solvents

 The second step is the rapid freezing of the sample between two copper cups. The goal of this high rate is twofold: preserve the structure and obtain a vitreous solvent. The size of the sample is chosen so that it is as small as possible.

419

 The rate of freezing must be as fast as possible so the solvent will be amorphous (104 K/s for water). In the case of organogelators, care must be taken so that the gel does not undergo a phase transition or additional texturation due to a shear distortion while preparing the sandwiches.

1.2. Transfer and Fracture under Vacuum and at Low Temperature: 170°C  After transferring the samples into the  At low temperature (–170°C) and at high apparatus by taking care that the vacuum (between 10-8 to 10-7 mbar). holder is always in a liquid nitrogen bath, the fracturing can be performed.  The fracture will occur along the plane  It is well known that for lipid bilayers it that offers least resistance to the occurs mainly between the aliphatic chains. applied forces. Etching (optional step)  In some cases, etching is performed.

 First, an experiment is carried out without etching and the replicas are observed. If the structures appear embedded in the solvent, a second set of experiments has to be done with an etching step.

 In this case, after fracturing, the  The optimal time of etching cannot be temperature is raised to the known in advance, but has to be determined temperature of sublimation of the for each sample by several assays. solvent (for toluene, it is 90°C) and left for a few minutes. The temperature is then lowered to 170°C and the shadowing step is performed.

1.3. Shadowing and Replication  The replication process should faithfully reproduce the 3-D surface revealed by the fracturing. Replication is a two-step process.

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 First, a metal layer is evaporated at an oblique angle (45°, in general) to provide contrast and then reinforced by a supporting layer of carbon (C) at an angle of 90°.

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 Shadowing is often performed using an electron gun and two metals are most suitable for this purpose: Platinum/carbon (Pt/C) or tungsten/tantalum (W/Ta). The metal layer will cover the inner structures of the gel revealed by the fracturing process.

Pt/C is the most used as it is easier to evaporate, but Ta/W provides a finer grain allowing a better resolution. The thickness of the deposited metal layer is 2 nm and the C layer 20 nm, measured with a quartz balance.

1.4. Dissolving the Material and Washing the Replicas  At this point, it is important to choose a solvent in which the sample is highly soluble. This choice is determined by chemical considerations. For instance, a solvent with H-donor or H-acceptor groups can disassemble molecules associated through H-bands. Very often chloroform is the most suitable solvent. In some instances, heating the solvent has been shown to better dissolve the remaining sample. If heating is necessary, take care to perform all the steps under a fume hood.

1.5. Recovery of the Replicas and Observation  The floating replicas are picked up with naked 400 mesh copper grids. Finally the grid is air-dried and observed under standard transmission electron microscope (TEM) conditions.

 If the replicas break down into small pieces, higher mesh-size grids or grids covered with a supporting carbon film can alternatively be used.

2. SUMMARY OF THE DIFFERENT STEPS 1. Preparation of sandwiches. 2. Rapid freezing of sandwiches. 3. Transfer into the cryo-fracture apparatus. 4. Fracture under high vacuum and low temperature.

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5. Optional etching. 6. Evaporation of the metal (Pt/C or TaW). 7. Evaporation of the carbon layer. 8. Entry of air and increasing temperature. 9. Washing the replicas. 10. Catching the replicas on copper grids with or without a carbon support film. 11. Observation in the TEM.

3. MATERIALS/PRODUCTS 3.1. Materials  Cryo-fracture apparatus

 Balzers, Cressington, or homemade apparatus (see Chapters 5, 6).

 Binocular microscope  High-pressure freezing apparatus



 Electron microscope  Ultrasonic bath  Fast freezing device

 Plunge-freeze: Chapters 3, 4, 7).

3.2. Products  Solvents for washing and cleaning  Product for cleaning copper  400 mesh copper grids

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 Technical grade.

“Guillotine”

(see

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4. PROTOCOL 1. Carefully clean the cups supplied with the cryo-fracture apparatus (either dilute HCl or a common copper cleaning household product) and sonicate 10 minutes in acetone), dry.

Figure 17.3 Two different cups used 2. Deposit a drop or a portion of the substance under investigation on one cup and gently cover with a second one.

Figure 17.4 Closing the sandwich 3. Close the sandwich while taking care not to leave any substance on the sides. Figure 17.5 Not good

Figure 17.6 Good

4. Hold firmly the sandwich with a pair of tweezers. 5. Plunge the sandwich very quickly into the cryogen (either liquid ethane if the solvent is not miscible in it or liquid nitrogen.

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 The plunging can be done either with a freezing device ,manually or though a highpressure freezing machine (see Chapters 3, 4, 7).

Cryo-Fracture of Self Assembled Systems in Organic Solvents

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6. Position the sandwich tightly into the object holder of the cryo-fracture  The entire process is carried out in liquid apparatus maintained in liquid nitrogen. nitrogen.

Figure 17.7 “Escaig”-like object holder in the closed state at room temperature.

7. Introduce apparatus

into

the

cryo-fracturing

Figure 17.8 Our cryo-fracture apparatus was developed and built by Dr. J.-C. Homo.

 Wait until vacuum has reached the  The higher the vacuum the smaller the highest possible level (about 10-8 size of the metal grains deposited on the mbar). specimen. 8. Begin evaporation of metal and as soon as the quartz crystal thin film monitor detects a deposit, begin the fracture step. If an etching step is necessary, it will be performed before metal evaporation.

 By evaporating first and then fracturing, it is possible to avoid the readsorption of dirt on the surface that appears after fracturing (dirt may be residual solvent vapors in the apparatus or substances of other origin).

Figure 17.9 Holder in the open state with one fractured sandwich.

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9. After depositing about 2 nm Pt/C or Ta/W measured by the thickness detector, a 20 nm carbon layer is deposited at a 90° angle.

 The carbon layer improves the physical strength of the replica and, therefore, the latter can be more readily handled (see Chapter 16).

10. The holder is removed and heated up to room temperature under a dry nitrogen stream. 11. The cup is put into a recipient containing a solvent in which the  The choice of solubilizing solvent initial product is totally soluble. directly influences the final result. 12. The replicas are gently recovered  If necessary, grids with a smaller mesh onto 400-mesh naked copper grids. size or with a carbon supporting film can be used. This is mainly helpful if the replicas have broken into small pieces.

5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  Preservation of the native  environment (solvent is maintained).  Enables structural studies on “large”  Greater than 150 nm. objects or concentrated solutions.  Preservation of the three-dimensional organization.  Enables access to the internal and external structure of vesicular systems.

5.2. Disadvantages  Requires sophisticated equipment.



 Not all solvents are suitable for such  analysis (crystallization may be a problem).  Time consuming.   Low resolution.  Unsuitable for small objects.

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 About 2 nm.  Less than 2 nm if ordered, otherwise 10 nm.

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6. VITREOUS ORGANIC SOLVENT MADE SIMPLE The method works in most situations provided it is possible to find a solvent in which the substance being investigated is highly soluble. Chloroform has been found to be one of the most useful solvents in our experience.

7. OBSERVED RESULTS Figure on Chapter’s title page

F N

F B

HN

 Figure 17.10  Chemical structure of the molecule (1)

N

O

O C16H33O

 Nanotubes of self-assembled diamides 3 forming a gel in cyclohexane.

O N H

C16H33O OC16H33

N H

   OC16H33   OC16H33  OC16H33       Figure 17.11  Poor freezing: In gels of 1 in nonane, solvent crystals have formed. They appear as terraces corresponding to the molecular layers and present generally a faceted shape.          

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     Figure 17.12 Good preservation of structures in nonane.  Cryo-fracture of a gel formed by selfassembly of borondipyrromethenes in nonane. These molecules associate as small fibers 20 nm wide and up to 500 nm in length (arrows) and form a thermotropic liquid crystal. The gel also presents some interesting fluorescence properties.1

R1

   Figure 17.13 Chemical structure of the diamide gelators (compounds 2 to 5) visualized in figures 17.14 to 17.20.

O

O

O

O

Compound 2 3 4 5

R2

R2 O

O

NH

HN R3



R3

R1 R2 R3 C11H23 C5H10 C7H15 C10H21 C5H10 C7H13 C10H21 C10H21 C11H23 C20H41 C5H10 C7H15

     Figure 17.14  Poor freezing of a gel of the diamide gelator 2 in cyclohexane, solvent crystals appear as angular structures (arrows).    

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     Figure 17.15 Same sample as in Figure 17.14 at a higher magnification. Selfassembled fibers of the diamide 2 (arrows) are seen lying between the solvent crystals (star). 

     Figure 17.16 Poorly washed sample (gel of 3 in toluene, 2% (w/v)). The black structures (arrow) overlying the replica are the remains of the sample. Underneath, the real structures are visible as straight platelets (arrowheads).           Figure 17.17 Replica of a gel of diamide 4 in toluene 2% (w/v). The structures are bathing in the solvent and, therefore, difficult to visualize.       

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     Figure 17.18 Same sample as in Figure 17.17 but after freeze-etching at –90°C for three minutes. The structures are clearly visible as flat ribbons. 

 Figure 17.19 Replica of a gel of 5 in cyclohexane 2% (w/v) after freeze-drying. Excessive freeze-drying has entailed collapse of the structures, which appear as dense fibers (arrows).

 Figure 17.20 Same sample as above but this time well preserved.

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8. REFERENCES 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Camerel, F. et al. Highly luminescent gels and mesogens based on elaborated borondipyrromethenes, J. Am. Chem. Soc., 128, 4548, 2006. Camerel, F. et al. Tuning the thermotropic and lyotropic properties of liquidcrystalline terpyridine ligands, Eur. Chem. J., 12, 4261, 2006. Couffin-Hoarau, A.-C. et al. In situ-forming pharmaceutical organogels based on the self-assembly of L-alanine derivatives, Pharm. Res., 21, 454, 2004. Cuccia, L.A. et al. Encoded helical self-organization and self-assembly into helical fibers of an oligoheterocyclic pyridine-pyridazine molecular strand, Angew. Chem., Int. Ed. Engl., 39, 233, 2000. Danino, D. and Talmon, Y. Direct-imaging and freeze-fracture cryotransmission electron microscopy of molecular gels, in Molecular Gels. Materials with SelfAssembled Fibrillar Network, Weiss Richard, G. and Terech, P., eds., Springer, Dordrecht, The Netherlands, 2006, 253. Diaz, N. et al. Self-assembled nanotubes in organic solvents, Macromol. Symp., 241, 68, 2006. Diaz, N. et al. Self-assembled diamide nanotubes in organic solvents, Angew. Chem., Int. Ed. Engl., 44, 3260, 2005. Ellis-Behnke, R.G. et al. Nano neuro knitting: Peptide nanofiber scaffold for brain repair and axon regeneration with functional return of vision, Proc. Nat. Acad. Sci. USA, 103, 5054, 2006. Escaig, J. and Nicolas, G. Cryofractures of biological material performed at very low temperatures in ultravacuum, C. R. Acad. Sci. (Paris), 283, 1245, 1976. Gross, H., Bas, E., and Moor, H. Freeze-fracturing in ultrahigh vacuum at -196 degrees C, J. Cell Biol., 76, 712, 1978. Gulik-Krzywicki, T. Electron microscopy of cryofixed biological specimens, Biol. Cell, 80, 161, 1994. Holmes, T.C. et al. Extensive neurite outgrowth and active synapse formation on self-assembling peptide scaffolds, Proc. Nat. Acad. Sci. USA, 97, 6728, 2000. Lehn, J.M. Supramolecular chemistry, Science, 260, 1762, 1993. Murdan, S. Organogels in drug delivery, Expert Opin. Drug Deliv., 2, 489, 2005. Murdan, S., Gregoriadis, G., and Florence, A.T. Sorbitan monostearate/polysorbate 20 organogels containing niosomes: A delivery vehicle for antigens?, Eur. J. Pharm. Sci., 8, 177, 1999. Ono, Y. et al. Organic gels are useful as a template for the preparation of hollow fiber silica, Chem. Commun., 1477, 1998. Schmidt, R. et al. New bisamides gelators: Relationship between chemical structure and fiber morphology, Tetrahedron Lett., 44, 3171, 2003. Schmidt, R. et al. Organogelation properties of a series of oligoamides, Langmuir, 18, 5668, 2002. Shepard, T.A. et al. Self-organization and polyolefin nucleation efficacy of 1,3:2,4-di-p-methylbenzylidene sorbitol, J. Polym. Sci. Part B: Polym. Phys., 35, 2617, 1997. Silva, G.A. et al. Selective differentiation of neural progenitor cells by high-Epitope density nanofibers, Science, 303, 1352, 2004.

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Simon, F.X. et al. Formation of helical mesopores in organic polymer matrices, J. Am. Chem. Soc., 129, 3788, 2007. Steere, R.L. Electron microscopy of structural detail in frozen biological specimens, J. Biophys. Biochem. Cytol., 3, 45, 1957. Terech, P. and Weiss, R.G. Low molecular mass gelators of organic liquids and the properties of their gels, Chem. Rev., 97, 3133, 1997. van Bommel, K.J.C., Friggeri, A., and Shinkai, S. Organic templates for the generation of inorganic materials, Angew. Chem., Int. Ed. Engl., 42, 980, 2003. Weiss, R.G. and Terech, P. Molecular Gels. Materials with Self-Assembled Fibrillar Network. Springer, Dordrecht, The Netherlands, 2006. Whitesides, G.M., Mathias, J.P., and Seto, C.T. Molecular self-assembly and nanochemistry: A chemical strategy for the synthesis of nanostructures, Science, 254, 1312, 1991.



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Part V Analysis

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Progressive Lowering of Temperature PLT

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CONTENTS

GENERAL INTRODUCTION .................................................................................... 437 1.

PRINCIPLES OF PROGRESSIVE LOWERING OF TEMPERATURE ... 438 1.1. 1.2.

PLT Dogma .............................................................................................. 438 General Outline of the PLT Method ......................................................... 438

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 440

3.

MATERIALS/PRODUCTS/SOLUTIONS ...................................................... 442 3.1. 3.2. 3.3.

4.

PROTOCOLS .................................................................................................... 446 4.1.

4.2. 4.3.

4.4.

4.5. 4.6. 4.7.

5.

Materials ................................................................................................... 442 Products .................................................................................................... 443 Solutions ................................................................................................... 443

Chemical Fixation of the Sample of Interest ............................................ 446 4.1.1. Tissue ............................................................................................ 446 4.1.2. Cell suspensions ............................................................................ 447 4.1.3. Cell monolayers............................................................................. 447 4.1.4. Adherent cells grown on sapphire disks as monolayers ................ 448 4.1.5. Polarized cell monolayers ............................................................. 448 4.1.6. Precious samples ........................................................................... 449 Preparation of the Low-Temperature Embedding Apparatus ................... 450 Programming the Apparatus According to the Resin Chosen .................. 450 4.3.1. For Lowicryl K4M ........................................................................ 450 4.3.2. For Lowicryl HM20 ...................................................................... 451 4.3.3. For AFS2....................................................................................... 451 Dehydration .............................................................................................. 452 4.4.1. Postfixation ................................................................................... 452 4.4.2. Dehydration................................................................................... 452 Embedding................................................................................................ 453 Polymerization.......................................................................................... 454 Further Processing .................................................................................... 454 4.7.1. Room temperature sectioning........................................................ 454 4.7.2. Immunolabeling ............................................................................ 455 4.7.3. In situ hybridization....................................................................... 455

ADVANTAGES/DISADVANTAGES.............................................................. 458 5.1. 5.2.

Advantages ............................................................................................... 458 Disadvantages........................................................................................... 458

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436

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Handbook of Cryo-Preparation Methods for Electron Microscopy

WHY AND WHEN TO USE A SPECIFIC METHOD.................................. 459 6.1. 6.2. 6.3.

With Regards to the Tokuyasu Immunostaining Method......................... 459 Compared to Cryo-Fixation ..................................................................... 459 New Technique ........................................................................................ 459

7.

OBSERVED RESULTS.................................................................................... 460

8.

REFERENCES .................................................................................................. 464



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Progressive Lowering of Temperature PLT

437

GENERAL INTRODUCTION The progressive lowering of temperature (PLT) procedure was introduced in the 1950s when it was common to use acrylic resins. These methacrylate-based resins, however, were unstable under the electron beam, unreliable and difficult to section. Therefore, they disappeared giving way to epoxy resins until the 1980s when they became the resin of choice for immunocytochemistry or in situ hybridization. In fact, Kellenberger et al.14 reintroduced the acrylic resins, now commonly called Lowicryls, a combination of the name of the company Lowi Werke and the term “acryl.” Their formulation was also improved. The advantages of these new resins are multiple: they have low viscosity, therefore, they infiltrate rapidly into tissues; they are fluid at low temperatures, some of them are miscible with water; they are hydrophilic when polymerized so that they have no nonspecific attraction for immunoreagents; and, in addition, they have good electron beam stability. The Lowicryl resins were not only developed for the low temperature embedding procedure, but also to enable optimal ultrastructural preservation and to allow observation of unstained samples in scanning transmission electron microscopy (STEM). Indeed, they give low scattering ratios due to their low density as compared to the higher density of tissue.3 The contrast (ratio of elastically and inelastically scattered electrons known as the Z contrast) is provided by the tissue itself.5 However, the ability to view biological samples in STEM without contrasting agents is not applicable to transmission electron microscopy (TEM). The contrast required to view cellular structures embedded in Lowicryl K4M can be obtained by conventional methods or enhanced using the method described by Roth et al.24 A variety of acrylic resins have been developed and are known under the following trademarks:  LR White and LR Gold (London Resin, UK)  Lowicryl K4M, HM20, K11M and HM23 (Lowi Werke, Germany)  Bioacryl (Unicryl – British BioCell International, UK) In the meantime, colloidal gold was developed and the perspective of fine localization of macromolecules at the ultrastructural level12 required improved methods of specimen preparation. It was then clear that fixation, dehydration and resin embedding altered the specimen, therefore, the low temperature and freezing methodologies developed in the 1950s were considered as better alternatives to improve our understanding of ultrastructure and develop the science of immunocytochemistry. The PLT method, the freeze-substitution method (see Chapter 13) and the Tokuyasu method (see Chapter 19) are the three main procedures used today for immunocytochemistry. A new emerging technique based on cryo-fixation, rehydration and Tokuyasu cryo-sectioning (see Chapter 14) also appears promising.

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1. PRINCIPLES OF PROGRESSIVE LOWERING OF TEMPERATURE 1.1. PLT Dogma  In the PLT method, the temperature of the specimen is gradually decreased as water is removed from it by increasing the concentration of organic solvents so as to minimize the denaturing effects of solvents on proteins. This method originally developed by Fernandez-Moran6 was reintroduced by Carlemalm et al; in the 1980s.4

 Macromolecules in the specimen are immobilized in an “inert” manner.  Causes less conformational changes to the tertiary structure of protein molecules.  Reduces lipid extraction  The final temperature depends on the resin chosen.

 The samples are then impregnated with increasing concentrations of acrylic resin in solvent at the low temperature most suitable for the resin chosen. Finally, the sample is floating in a 100% acrylic resin solution.  The resin is then polymerized by UV  Inhibition of the polymerization step can irradiation, which activates a occur if osmium has been used as a postphotoinitiator mixed into it. fixative or if a colored pigment is present in the sample (e.g., intense color of the erythrocytes in blood vessels. Use perfusion fixation that will wash out the red blood cells).  The resin is maintained at a low temperature in a cooled ethanol bath  But there is no injection of nitrogen gas during polymerization, thus only a into the specimen chamber during minimal temperature increase can be polymerization. measured during polymerization.7

1.2. General Outline of the PLT Method  The sample is chemically fixed.

 The sample is dehydrated.

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 The sample should be fixed as soon as the cells are collected or the organ extracted from an animal.  Perfusion fixation is preferred.  Mild fixation is preferred because it does not interfere with antigenicity.  The overall structure is preserved.  Water is progressively replaced by the organic solvent in a series of steps.  The solvent environment should remain as “water like” or as polar as possible during the entire embedding procedure.

Progressive Lowering of Temperature PLT

 The temperature is lowered during the dehydration procedure.  Lowicryl embedding can be performed at temperatures between 70°C to 30°C depending on the type of resin.

 Other resins exist and have their advantages and disadvantages.  Polymerization is initiated by benzoin methylether and long wave UV irradiation (~ 360 nm, a wavelength that is not efficiently absorbed by proteins or nucleic acids) at low temperature.

© 2009 by Taylor & Francis Group, LLC

439 Based on this idea numerous attempts to develop water-soluble or water-compatible embedding media were made without success.  Ethanol is commonly used.  Acetone is unsuitable for the PLT technique as 70% acetone has a freezing point of 27°C and PLT requires the 70% step to be at 35°C.  The solvent concentration has to be carefully chosen such that the temperature is above the freezing point of the organic solvent concentration used in the previous step (see Graph 1 in the Lowicryl booklet supplied with the resin).  One must ensure that enough time is allotted to allow the interior of the sample to equilibrate with the dehydrating agent at each step.  The resin has to be soluble in the solvent used. K4M and HM20 are soluble in most dehydrating agents. However, ethylene glycol and dimetyl formamide are not miscible with HM20.  Until the polymerization temperature of the resin chosen is attained.  Lowicryls are the only low temperature embedding resins (below 30°C).  Different formulations of Lowicryls are available. We prefer K4M, which polymerizes down to 30°C or HM20 down to 45°C.  K4M was largely used because of its good properties in on-section colloidal gold labeling.23  HM20 was compared to K4M for immunodetection22 and was found to be easier to section and yield labeling comparable to K4M.  HM23 and K11M which polymerize at even lower temperatures are more dedicated to cryo-substitution.1  LR white (see Chapter 13).  LR gold (see Chapter 13).  Unicryl/Bioacryl.  In case of the use of Lowicryl.  First by indirect UV light.  For further hardening the block is exposed to direct UV light at room temperature for three days.

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2. SUMMARY OF THE DIFFERENT STEPS 1. Chemical fixation of the sample of interest 1.1. Tissue  Fixation by immersion  Sample size:  ~ 0.5 mm31 mm3  Samples not larger then 0.5 mm3 are recommended.  Fixation by perfusion  This is the method of choice.  Only possible on animals. 1.2. Cell suspensions  Add a two times concentrated fixative to the medium  Pellet the cells  Fix again 1.3. Adherent cell monolayers grown in culture flasks  Fix the cells  Scrap the monolayer with a rubber policeman  Centrifuge and discard the supernatant  Add fresh fixative over the pellet 1.4. Adherent cell monolayers grown on sapphire discs 1.5.

Polarized cell monolayers

1.6.

Precious samples

 Samples from human biopsies (e.g., bone marrow)  Samples from a Bio Safety Lab 3 when very few cells can be obtained.

   

A = Tissue fixation B = Cell suspension C = Cell monolayer D = Sapphire disc

Figure 18.1

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Progressive Lowering of Temperature PLT

2. Preparation of the low temperature  The Leica AFS25 is used in the authors’ laboratories and presented in this chapter embedding apparatus (see Figure 18.4). 3. Programming the apparatus depending on the resin chosen and on the apparatus  For Lowicryl K4M  For Lowicryl HM20  For the AFS2 4. Dehydration 5. Embedding depending on the sample and purpose of the work  Flat embedding  Mostly for tissue, useful to orientate the specimen.  Microtube embedding  For all purposes. Dehydration is done in Sarstedt tubes or in 1.5 mL microtubes. Embedding can be done in 0.5 mL Sarstedt microtubes, which can be directly fixed in the ultramicrotome after polymerization.  Leica molds

 Dehydration and embedding can be done in the same mold.

 Sapphire discs

 Dehydration and embedding are performed using the specific tools (Leica SD FS Unit).

 Embedding ization

after

protein

internal-  Step 1: A protein or antibody coupled to colloidal gold is internalized.  Step 2: Immunogold labeling on sections of the PLT embedded sample.

6. Polymerization 7. Further processing  Room temperature sectioning  Immunolabeling  In situ hybridization

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 See Chapter 23.  See Section 4.7.3.

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3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Apparatus for PLT

 Centrifuge  Chemical hood

 Homemade.27  Leica EM AFS or EM AFS2, Leica, Microsystems, Vienna, Austria.  RMC FS-7500, Boeckeler Instruments Inc., Tucson, Arizona, USA (no longer sold).  Used to pellet cells.  Used to prepare solutions.  Resins are very toxic and allergenic. Read the technical note before using them.

 Adapted gloves and chemical face  Caution: Methacrylates are aller-genic; mask please refer to the safety information provided with the product.  Agar Scientific or any other EM material  Gelatin capsules supplier. Size 00.  #16702738, perforated tubes used for  Leica plastic capsules freeze-substitution.  Glass bottles  Adapted to the chamber of the apparatus.  Some are furnished with the #16702737 Leica apparatus.  To scrape the cells off culture dishes.  Rubber policeman  Microcentrifuge tubes

 Sarstedt # 16702765.

 Styrofoam boxes

 To store ice.  for example, #16708826 or #16708864 Leica.  To recycle leftover solutions (should be performed by professionally qualified personnel). Polymerized resins are still toxic.  SD FS is a special tool for substitution and embedding of sapphire discs. Leica Microsystems, Vienna, Austria.  Sarstedt.  #16707150 Leica.  Sarstedt #16 440.

 Special waste containers

 Sapphire disc freeze-substitution unit  Sarstedt tubes for the AFS2  Microvettes CB300  Stove  Water bath

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 To keep the low melting point agarose and the instruments at 40°C.  To maintain the low melting point agarose fluid.

Progressive Lowering of Temperature PLT

443

3.2. Products  Cacodylic acid sodium salt trihydrate  Gelatin

 Uranyl acetate  Low melting point agarose  Gold colloids

 Silver enhancement  Methyl cellulose  Osmium tetroxide

 # 8.20670.0250, Merck.  To embed sparse cells, very small biopsies or very precious samples.  EMS #16560, Hatfield, Pennsylvania, USA or SPI Supplies, West Chester, Pennsylvania, USA or Merck 1.0470.  EMS: http://www.emsdiasum.com/microscopy/asp /a.aspx  Same use as gelatin (EMS #1670B).  Caution: Normal agarose may destroy the diamond knife.  Ultra-small colloidal gold and specific secondary antibodies: Aurion, The Netherlands, British BioCell International, U.K.  SE-EM, Aurion, The Netherlands.  25 centipoises.  10 x 0.25 g, # R 1016, Agar.

3.3. Solutions  Ethanol  30%, 50%, 75%, 100%

 #02860, Fluka.  The best method is to prepare ethanol solutions with an alcoholmeter.  Due to volume changes in mixtures of ethanol and water, dilutions are incorrect.

 Fixatives  2 to 4% (w/v) formaldehyde (FA) and  Cell culture: 2.5% FA + 0.1% GA. less then 0.5% (v/v) glutaraldehyde  Cell suspension: 5% FA + 0.2% GA. (GA) in 0.1 M cacodylate buffer;  Fixative preparation: pH 7.2  Dissolve 2.5 or 5 g paraformaldehyde in 45 mL H2O: 2 hours at 65°C.  Warm up to about 65C.  Stir vigorously for some time.  Add 20 to 80 L of NaOH 1 M, wait for 10 minutes.  If the paraformaldehyde has not dissolved into formaldehyde, repeat the NaOH step. Thereafter, add glutaraldehyde if desired and adjust to 50 mL with H2O.  Then add 50 mL double strength buffer and check the final pH.  Note: Can be stored frozen in aliquots.

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 Glutaraldehyde (GA)

 Uranyl acetate solutions (UA) 2%, 5% Aqueous uranyl acetate  0.5% Uranyl oxalate, pH 7.4

 Glutaraldyde 25% solution in water (EMS #16410).  Toxic by inhalation. Use under a fume hood.  In double-distilled water. Kept at 4°C in the dark.  Prepared as described in Chapter 19.

 Buffers  100 mM Phosphate buffer, pH 7.4 Preparation of 1 M stock solution:  Dissolve 14.19 g Na2HPO4 (MW 141.96) in 100 mL doubledistilled water  Dissolve 13.8 g NaH2PO4, H2O (MW 138.00) in 100mL double-distilled water  The pH should be 7.4, according to the phosphate sodium buffer chart

 Na2HPO4 disodium hydrogen phosphate, NaH2PO4 sodium dihydrogen phosphate.  Solution A.

 0.1 M Cacodylate buffer; pH 7.4 Preparation of 0.1 M Cacodylate buffer:  Dissolve 21.4 g in 1 L doubledistilled water  Add 1mM MgCl2 from a 1 M stock solution  Add 1mM CaCl2 from a 1 M stock solution  Add 2% sucrose.  Lowicryl resin:

 Cacodylic acid sodium salt trihydrate Na(CH3)2AsO2, 3H2O (MW 214.05).

 Ready to use K4M  Preparation of polar K4M:  Cross-linker A: 2.70 g or 3.6 mL  Initiator C: 0.10 g  Monomer B: 17.30 g or 25 mL  Procedure: Mix cross linker and monomer in a vial and take care not to introduce any air into the resin mixture. The initiator is then added and mixed the same way.

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 solution B.  Add 19 mL of solution A to 81 mL of solution B before use.  dilute 10-fold to make 100 mM.

 Check and adjust the pH to 7.2 to 7.4.  The osmolarity should be ~ 300 mosmoles.  Storage at 4°C or frozen in aliquots.  EMS or Polysciences Europe GmbH, Eppelheim, Germany.  K4M Monostep (EMS#14335).  Is hydrophilic.  Increase the amount to obtain harder blocks.  Benzoin methyl ether for UV polymerization.  Gives the polar property to the resin.  Bubble a small stream of dry nitrogen in the mixture through a Pasteur pipette.  Oxygen inhibits polymerization of any acryl resin.

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Progressive Lowering of Temperature PLT

 Ready to use HM20  Preparation of apolar HM20:  Cross-linker D: 2.98 g or 4 mL  Initiator C: 0.10 g  Monomer E: 17.02 g or 25 mL

 HM20 Monostep (EMS#14345).  Is hydrophobic.

 Benzoin methyl ether for UV polymerization.  Gives the apolar property to the resin.

 In Situ Hybridization solutions  SSC buffer 20X:  175.3 g sodium chloride (MW 58.44)  88.2 g sodium citrate (MW 294.10)  Fill up to 1 L sterile double-  Adjust the pH to 7.0 with sodium distilled water hydroxide 10 M.  Phosphate/NaCl buffer:  1 M Phosphate buffer  5 M Sodium chloride

 14.6 g Sodium chloride (MW 58.44) fill up to 50 mL with phosphate buffer.

 1 M Tris/HCl buffer (MW 121.16)

 C4H11NO3 .

 20 mM Tris/HCl–300 mM NaCl buffer  6 mL 5 M Sodium chloride  2 mL 1 M Tris  Fill up to 100 mL with sterile double-distilled water

 Adjust the pH to 7.6 with HCl.

 Blocking buffer  1% BSA in Phosphate/NaCl buffer  Washing buffer  20 mM Tris/HCl–300 mM NaCl buffer or 2X SSC  Hybridization buffer:  2X SSC buffer  30% deionized formamide  2X Denhardt’s solution  250 µg/mL yeast tRNA in a 100 µL final volume

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 The hybridization buffer can be stored at 20°C with or without the probe.  Roche 11 814 320 001.  Roche 10 109 495 001.

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4. PROTOCOLS 4.1. Chemical Fixation of the Sample of Interest  Choose the fixative so that:

 Extraction of cellular components is reduced.  No denaturation is observed.  No alteration of epitopes occurs because immunolabeling is the purpose of the PLT.  No alteration of DNA or RNA for in situ hybridization (ISH) occurs.  The volume and the shape of the sample are preserved.

 A mild fixation is preferred  Formaldehyde enters organs or cell usually 2 to 4% FA supplemented suspensions slowly. with less then 0.5% GA  Preserves antigenicity.  The overall morphology is not well preserved.  Take care: The reaction is reversible during the washing steps. The sample should be kept in a 1/10 dilution of the initial concentration if it is not immediately treated.19  Glutaraldehyde at a low concentration is added to preserve the ultrastructure.11  To preserve membrane structures, a  Osmium tetroxide cannot be used postfixation with neutral uranyl acetate because it interferes with antigen reactivity. is recommended.

4.1.1. Tissue  Fixation by immersion  2 to 4% FA + < 0.5% GA

 The sample size should not exceed 0.5 mm3.

 Fixation by perfusion  2 to 4% FA + < 0.5% GA

 Method of choice: Only possible on animals.  Depends on the organ and the purpose of the investigation. Some tissues (e.g., lung) must be “washed” with 9‰ NaCl at 37°C before perfusion to prevent cells from plugging capillaries. Very soft or poorly structured tissues, such as spleen or bone marrow, are difficult to recover after perfusion, although it is not impossible to do it.

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Progressive Lowering of Temperature PLT

4.1.2. Cell suspensions  Freshly prepared FA.

1. Add an equal volume of 5% FA supplemented with 0.2 % GA in 0.1 M cacodylate buffer. 2. Pellet the cells

30 min at 4°C 5 min 2500 rpm

3. Transfer the cells in a microtube and further fix them for half an hour with 2.5% FA supplemented with 0.1% GA.

30 min at 4°C

4. Wash the cells with 0.1 M cacodylate  Pellet the cells 1 min at 7000 rpm. buffer. 5. If not further processed immediately, the pellet should be resuspended in cacodylate buffer containing 0.25% FA. 4.1.3. Cell monolayers  Avoid the use of trypsin-EDTA, which complexes the divalent ions and modifies cell membranes.  Chemical detachment also leads to gross changes of the morphology.

1. The culture medium is removed.

2. Add enough 2.5% FA supplemented with 0.1% GA to cover the monolayer. 3. Scrap the policeman.

cells

with

4. Pellet the cells. 5. Resuspend the pellet in the same fixative and transfer it into a microtube.

30 min at 4°C a

rubber

5 min 2500 rpm 30 min at 4°C

6. The cells are then washed with 0.1 M cacodylate buffer. 7. If not further processed immediately, the pellet should be resuspended in cacodylate buffer containing 0.25% FA.

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Handbook of Cryo-Preparation Methods for Electron Microscopy

4.1.4. Adherent cells grown on sapphire disks as monolayers 1. The cells are fixed with 2.5% FA supplemented with 0.1 % of GA.

2. Wash the cells with 0.1 M cacodylate buffer, pH 7.4.

 Some authors (see section 4.4 in Chapter 30 min - 1 h 5) coat the sapphire discs with carbon at 4°C before growing cells.

3 × 10 min at 4°C

3. If not further processed immediately, the pellet should be resuspended in cacodylate buffer containing 0.25% FA. 4.1.5. Polarized cell monolayers 1. The cells grown in a Petri dish are fixed with 2.5% FA supplemented with 0.1% GA.

 Such as Caco cells. 1 h  The objective is often to embed a single at 4°C cell layer and to section it transversally to or less the culture.  Cheaper compared to sapphire discs.

2. The monolayer in the Petri dish is cut into small ribbons with a razor blade. 3. With a thin rubber policeman, a ribbon of cells is removed and placed on a slide. 4. A thin layer of low-melting point agarose (2% in 1 M cacodylate buffer)  Let it solidify on ice. is poured over the ribbon. 5. The sample is hereafter treated as a  The embedding can be done: cell pellet.  To further section the cell layer (see Figure 18.2, Steps 5, 6).  To further section the cell layer transversally (see Figure 18.2, Step 7).

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Progressive Lowering of Temperature PLT

 1 = Ribbons on the Petri dish.  2 = Lift off the ribbon with a rubber policeman and place it on a slide.  3 = Pour agarose on the sample and let it solidify.  4 = Cut several pieces of the sample.  5 = PLT and embedding in flat molds (AFS2).  6 = Embedding in flat molds (AFS).  7 = Dehydration and embedding (for further transverse sectioning) in a Leica tube (AFS).

Figure 18.2 The different steps to embed a monolayer of polarized cells.

 Caco cells have been agarose embedded as described above. After PLT and Lowicryl K4M flat embedding, sections were cut through the cell monolayer. Only one cell layer can be observed and the apical and basal sites are clearly visible under the electron microscope. Figure 18.3 Observed results of above steps 4.1.6. Precious samples  Some very precious samples, such as human biopsies, are easily damaged and/or lost during the whole procedure. Therefore, after fixation in 2.5% FA supplemented with 0.1% GA:

 The low melting point agarose is kept in a water bath at 40°C after dissolution by boiling.  Pipettes and microtubes are kept at 40°C in the water bath or in a stove.

 Small biopsies are enwrapped in low-  Let it solidify on ice. melting point agarose.  Most of the agarose surrounding the sample is removed with a razor blade.  Isolated cells are pelleted in low-  An easy way to pellet the cells is to use melting point agarose. Microvette (Sarstedt).  Solidified cell pellets are cut into small cubes.

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4.2. Preparation of the Low-Temperature Embedding Apparatus  Fill with liquid nitrogen

 See Figure 18.4

Figure 18.4 Leica AFS.

4.3. Programming the Apparatus According to the Resin Chosen 4.3.1. For Lowicryl K4M  Settings for the AFS

Settings are made according to Carlemalm et al.4 and Whitehouse et al.27  The different steps are important: Do not forget any of them!  When in 30% ethanol, the temperature should be 0°C or it is better to set a slope from 0°C to 2°C or 4°C.  When in 50% ethanol, the temperature should progressively decrease from 0°C to 20°C.  It should also progressively decrease from 20°C to the next temperature: 25°C (K4M) or 45°C (HM20). This is possible with the AFS2 where two successive slopes can be selected. While not possible in the AFS1, a short step (T2) is inserted before the next slope.  The two steps in 100% ethanol are also important. Figure 18.5 Control unit of the AFS.

 T indicates the temperature chosen and S the slope or rate in decrease from one temperature to another. The temperatures are set in decimals: half an hour is 0.5 hour, 0.1 hour is 6 min.

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 The temperature can vary from one apparatus to another by ± 5°C. Check the temperature the first time you use it.  An easy test is to put some K4M Lowicryl in the apparatus at 30°C. If the viscosity of the Lowicryl has increased, the temperature is too low.

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Progressive Lowering of Temperature PLT

    

The program should be set at:  S2: 5 C; 1h  T3: 25 C; 48h  Another way is to put some ethanol 30% in the AFS chamber at 0°C. The ethanol should stay fluid. If this is not the case, your AFS is too cold. Set the program as above.

T1 : 0 C; 0.5h S1 : 20 C; 1h T2 : 20 C; 0.2h S2 : 10 C; 1h T3 : 30 C; 48h

4.3.2. For Lowicryl HM20      

Settings T1 : 0 C; 0.5h S1 : 20 C; 1h T2 : 20 C; 0.2h S2 : 25 C; 1h T3 : 45 C; 48h

 Same comments as above.

4.3.3. For AFS2  Settings An example of settings is shown in Table  When equipped with the freeze substitution processor (FSP), the AFS2 18.1. becomes a real robot: dehydration, embedding and UV polymerization are done automatically.

STEP

T°

T° End

Slope

Start

°C

°C/h

Time

Reagent

Content

Transfer

Agitation off

UV

01

0

0

0

0.05

Ethanol

30%

stay

02

0

15

30

0.30

Ethanol

50%

mix

on

03

15

30

30

0.30

Ethanol

75%

mix

on

04

30

35

10

0.30

Ethanol

100%

Exch/fill

on

05

35

35

0

0.30

Ethanol

100%

Exch/fill

on

06

35

35

0

1:00

HM20

30%

mix

on

07

35

35

0

1:00

HM20

60%

mix

on

08

35

35

0

1:00

HM20

100%

Exch/fill

on

09

35

35

0

15:00

HM20

100%

Exch/fill

on

10

35

35

0

30:00

HM20

100%

stay

off

X

11

35

20

13.8

4:00

HM20

100%

stay

off

X

Table 18.1 Settings of the Different Steps

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Pause

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4.4. Dehydration 4.4.1. Postfixation  0.5% Neutral uranyl acetate

 30 minutes on ice (not more!).  Neutral uranyl acetate is prepared in a Veronal-based Mickaelis buffer.  Note: Because Veronal is very toxic, a special authorization to manipulate this product is required. Therefore, we will not give the recipe in this chapter.  The uranyl oxalate prepared for the Tokuyasu method should be preferred (see Chapter 19).  

4.4.2. Dehydration Dehydration is done using ethanol at  The solvent must never freeze. The different concentrations. temperature of the chamber should always be well above the freezing point of the solvent to be used. Method:  For embedding in Lowicryl K4M:  30% Ethanol: 30 min, 0°C  50% Ethanol: 1h, 0°C to 20°C  75% Ethanol: 1h, 20°C to 30°C  100% Ethanol: 1h, at 30°C  100% Ethanol: 1h, at 30°C      

 Cool the pipette, discard the medium and add precooled 30% ethanol. At each step the next ethanol solution is precooled in the AFS chamber.

For embedding in Lowicryl HM20: 30% Ethanol: 30 min, 0°C 50% Ethanol: 1h, 0°C to 20°C 75% Ethanol: 1h, 20°C to 45°C 100% Ethanol: 1h, at 45°C 100% Ethanol: 1h, at 45°C

E Vol % 30 50 70 95 100 100

Temperature °C K4M

HM20

K11M

HM23

T min

0 20 35 35 35 35

0 20 50 50 50 50

0 20 50 60 60 60

0 20 50 60 80 80

30 60 60 60 60 60

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 Same comments as above.

 Use of Lowicryls (follow the supplier’s instructions).  E = Ethanol  T = Time Table 18.2 Typical dehydration scheme for PLT. (Carlemalm, personal communication, see Reference 10.)

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4.5. Embedding Embedding depending on the sample and the purpose of the work. 1.     

Procedure EthanolLowicryl: (1:1) (v/v) EthanolLowicryl: (1:2) (v/v) Pure Lowicryl Pure Lowicryl Pure Lowicryl

  For one hour  For one hour  For one hour  Overnight  For one hour before inserting in molds and embedding.

2. Flat embedding  After dehydration, the sample is placed in the adequate holder. Embedding is done as described before.

  Mostly for tissue.  The block can be orientated.  Caution: Flat embedding cannot be done in parallel with Leica mold embedding.  In the AFS2, flat embedding single-use molds are furnished.

3. Eppendorf embedding

 Convenient technique in the AFS but only with the tightly sealing Sarstedt microtubes.  Works well for cryo-substitution and Epon embedding.  In the AFS2, Eppendorf embedding is replaced by “Sarstedt” tube embedding.

4. Leica capsule embedding

 Convenient because dehydration and embedding can be done in the same capsule.  In the AFS2 “Leica” capsules are replaced by very convenient single-use molds.   Special tools exist; just follow the supplier’s manual.   It is possible to follow internalization of a protein coupled to colloidal gold and to perform an immunostaining on sections.15  

5. Cell culture on sapphire discs

6. Embedding after immunostaining

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 Procedure

  In this experiment, the heat shock protein Hsp-70 coupled to 10 nm colloidal gold particles was traced in human dendritic cells. The cells were then embedded in K4M Lowicryl using the PLT method. They were further immunolabeled with anti-MHC class II or anti-CD63 antibodies that were revealed by secondary antibodies coupled to Ultra-small gold. Ultra-small gold particles were amplified by silver enhancement.

Figure 18.6 A: Hsp-70 internalization and silver enhancement. The gold particles are localized in the so-called MHC class II compartments. B: CD63 (open arrow) co-localizes with Hsp-70 as all the gold particles were silver enhanced.

4.6. Polymerization  Polymerization of the resin is triggered by an activator sensitive to UV light, which is incorporated in the resin mixture. The reaction is very  Polymerization 24 to 48 hours under UV exothermic and controlled by the low light at the embedding temperature. temperature.

4.7. Further Processing 4.7.1. Room temperature sectioning  Trouble shooting.

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 See Chapter 2.

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4.7.2. Immunolabeling  See Chapter 23. 4.7.3. In situ hybridization 1. Principle The in situ hybridization (ISH) technique is based on the pairing reaction between the nucleic acid sequences which are under study in a biological material and the complementary labeled probes of a hybridization solution. Hybridization at the LM level was first described by John et al.13 and Pardue and Gall.20 It was developed afterwards for EM cryo-sections or resin embedded specimens by Morel et al.18

 Use labeled probes (oligonucleotides, cDNA or cRNA). Optimal conditions of in situ hybridization have been described.17,21  It is also possible to simultaneously detect a nucleic acid sequence and an antigen.16

 Three major types of probes are  They have to be labeled before use. The commonly used: label depends on the probe chosen.17  cDNA  Stable probe to be denatured before use.  Several different oligonucleotides can be  oligonucleotide(s) used simultaneously.  Single-stranded probe. The hybrids  cRNA obtained with these probes are more stable but these probes are more fragile.  Three types of antigenic markers  The revelation process depends on the exist: marker chosen.  Digoxigenin  Fluorescein  Biotin

 Three methods are available:  ISH pre-embedding

 Plant molecule that does not exist in animal specimens (less background). Do not use it for plant material.  In principle, could be observed at the LM level.  This is the smallest molecule available.  Endogenous biotin (vitamin H) often creates a background.  Each has its advantages and drawbacks.

 Hybridization is done on fixed material before embedding. Sensitive method, but time consuming.  ISH on resin-embedded sections  Easiest method, but is the least sensitive. After PLT embedding, the sensitivity is enhanced.  ISH on cryo-sections according to  Most sensitive method, but a cryoTokuyasu. ultramicrotome is required.

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 A general procedure involves the different steps:  Labeling the probe  Labeled oligonucleotide probes can be purchased commercially.  Tissue preparation and sectioning  As described in this chapter.  Pretreatment  To render the target accessible.  Hybridization  To form the complexes between nucleic acid and probe.  Washing  Washing out of nonspecific hybridization label.  Detection  Immunocytological reaction using colloidal gold particles.  Observation  Under an electron microscope 2. Protocol ISH is carried out on ultrathin Lowicryl  Easiest method. K4M or HM20 sections (100 nm thickness) using nucleic acid probes.  Labeling the probe  Oligonucleotide  100 pmoles/mL  Labeling a short oligonucleotide probe of hybridization (30 mers with a GC at the 3´end) is carried buffer out by the addition at the 3´ end of labeled nucleotides using an enzyme: terminal transferase (TdT).  The labeled nucleotides are carriers of antigenic molecules.17  Mixture solution  cDNA should be denatured.  Add the labeled probe (i.e.,  Oligonucleotides and cRNA probes do oligonucleotide probe) in the not have to be denatured. hybridization buffer and vortex  But cRNA should be hydrolyzed to  Centrifuge produce sequences of around 300 nuc Chill rapidly on ice leotides for the best results.  Hybridization  Place the grid on the mixture solution  Incubation  Washing  2X SSC  1X SSC

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 30 µL/grid  The volume can be reduced to a few µL. 3 h  Humid chamber. at RT 3 ×10 min  Necessary. at RT 2 × 5 min  Optional.

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 Detection  Stabilizing the structures  4% FA in 2X SSC

 Indirect immunocytological reaction.  Immunodetection is carried out on drops 5 min of 40 to 50 µL.

 Washing  2X SSC

5 min

 Blocking nonspecific sites in blocking buffer

1030 min  Necessary

 Incubate the first 60 min  See “Labeling the probe.” above. antibody antiprobe  40 µL/grid  Raised in species X. label diluted 1:50 in  Humid chamber. phosphate/NaCl buffer  Washing  Phosphate/NaCl 2 × 5 min buffer  20 mM Tris/300 mM 2 × 5 min NaCl buffer  Incubate colloidal gold immunoglobulin conjugate diluted in 20 mM Tris/300 mM NaCl buffer     

Washing TrisHCl/NaCl buffer 2X SSC Complex fixation 2.5% GA in 2X SSC

60 min  Antispecies secondary antibodies: IgG or  20 µL/grid Fab fragments (5mU/mL).  Detection of the complex.  Humid chamber.

2 × 5 min 5 min 5 min

 Staining  2% Aqueous uranyl acetate  Distilled water

 Observation

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25 min  Uranyl oxalate can be successfully used (see Chapter 19). in darkness 5 × 4 min  Finish washing in a jet of double distilled water from a wash bottle.  See Section 7, Figure 18.8.8

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5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  Fast and easy method to localize  Can localize proteins or protein comproteins in situ. plexes inside a cell or an organ.  Can localize heterologous proteins, e.g., expressed with a recombinant virus.  Can only localize the protein or overexpressed protein, biochemical experiments have to be performed to see if the protein is functional.  Fast and easy method to localize a  It can only localize; other experiments gene. have to be performed to see if the gene is active or not.  If DNA is destroyed by DNase treatment, only RNA can be detected. In this case, the messenger RNA detected reflects gene activity.  Lowicryl resin mixtures have the  K4M tolerates 5% water. advantage of keeping some hydration in the specimen.

 Lowicryl resin K4M and HM20 can be  75% K4M and 25% HM20, for example. mixed together to combine their advantages.

5.2. Disadvantages  If the gene of the protein to be  Intracellular proteins involved in localized is poorly expressed, labeling trafficking, such as those localized in rafts becomes difficult or even impossible. or scarce proteins.  Protein or nucleic acid localization can be tedious. In such situations, the Tokuyasu method is preferred because it is more sensitive.

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6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. With Regards to the Tokuyasu Immunostaining Method  Low-cost apparatus  No cryo-sections

 Can be homemade.  An ordinary ultramicrotome can do the job.  No experience in cryo-sectioning is needed.

 Block of resin

 Can be sectioned years after it was prepared.  Easy storage, no need of a special Dewar flask and liquid nitrogen.

 Good ultrastructural preservation

 In our hands with the ready-to-use resins, polymerization is reproducible and the overall morphology comparable to sections of Epon-embedded specimens.

 Antigenic sites preserved

 Perfect immunolabeling if the antigen is abundant.  PLT is the easiest method to start with the ISH technique.

 In situ hybridization

6.2. Compared to Cryo-Fixation  HPF followed by cryo-substitution

 Expensive apparatus.  Time consuming procedure.  Very little material to be sectioned.  Specific “how to do” is needed.  Slam-freezing followed by cryo-sub-  Less material available because the entire sample is not well preserved. stitution  Time consuming.

6.3. New Technique  High-pressure freezing, freezesubstitution fixation, rehydration, Tokuyasu cryo-sectioning, and immunolabeling

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 See Chapter 14.  Takes advantage of cryo-fixation and the classical Tokuyasu method.  To be considered in case of problematic samples. 

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7. OBSERVED RESULTS  Figure on the Chapter’s title page 1. Description

 Immunolocalization of a membrane protein in a prokaryote.

2. Method

 PLT.

3. Sample

 Mycoplasma.

4. Fixation

 4% FA + 1% GA, 30 minutes at 4°C.

5. Embedding

 Lowicryl K4M, 30°C.

6. Visualization for immunocytochemistry (ICC)

 Goat antirabbit coupled to 10 nm colloidal gold.

7. Comments

 The colloidal gold particles are found only on the plasma membrane.

 Figure 18.7 1. Description

 Cellular and subcellular distribution of AQP8 in the gastrointestinal tract.

2. Method

 PLT.

3. Sample

 Rat hepatocytes.

4. Fixation

 4% FA + 0.01% GA, 2 hours at 4°C.

5. Embedding

 Lowicryl K4M, 35°C.

6. Visualization for ICC

 Goat antirabbit coupled to 10nm colloidal gold.

7. Magnification

 40,000 ×; insets, 130,000 ×.

8. Comments

 APQ8 in the gastrointestinal tract of the rat may be involved in the (1) absorption of water in small and large intestine, (2) secretion of canalicular bile and (3) secretion of pancreatic juice. (Bc) bile canaliculus, (cyt) cytoplasm. (From Calamita, et al.2, with permission from Elsevier.)

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 Figure 18.8 1. Description

2. 3. 4. 5. 6.

Method Sample Fixation Embedding Visualization for ICC

7. Magnification 8. Comments

 Co-localization by immunolabeling of nucleocapsid-like particles in cells infected with a vaccinia virus recombinant encoding the measles virus nucleoprotein and phosphoprotein. (From Spehner, et al. 26, with permission from Elsevier.)  PLT.  Hamster BHK21cells.  2.5% FA + 0.1% GA, one hour at 4°C.  Lowicryl K4M at 25°C.  Specific antibody: Mouse antimeasles nucleoprotein; secondary antibody: Goat antimouse coupled to 5 nm colloidal gold.  Specific antibody: Rabbit antimeasles phosphoprotein; secondary antibody: Goat antirabbit coupled to 10 nm colloidal gold.  ~ 120,000 ×  Some vaccinia virus particles are visible in the top right of the picture. The electron dense mass corresponds to the accumulation of nucleocapsid-like structures. These structures are labeled with antiNP (5 nm) and antiP (10 nm) antibodies.

 Figure 18.9 1. Description

 In situ hybridization.9 Simultaneous detection of the three receptor subtype mRNAs in distal (A) and proximal tubules (B).

2. Method

 PLT.

3. Tissue

 Kidney cortex.

4. Fixation

 4% FA for 2 hours at 4°C.

5. Embedding

 Lowicryl K4M at 35°C.

6. Visualization for immunocytochemistry

 Goat antimouse coupled to 10 nm colloidal gold particles.

7. Scale bar

 0.5 µm.

8. Comments

 The 10 nm gold particles were localized in the cytoplasmic matrix near the nucleus (N), but not in mitochondria (m). (From Grandclement and Morel8, with permission from Portland Press.)

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8. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Acetarin, J.D., Carlemalm, E., and Villiger, W. Developments of new Lowicryl resins for embedding biological specimens at even lower temperatures, J. Microsc., 143, 81, 1986. Calamita, G. et al. Expression and immunolocalization of the aquaporin-8 water channel in rat gastrointestinal tract, Eur. J. Cell Biol., 80, 711, 2001. Carlemalm, E., Colliex, C., and Kellenberger, E. Contrast formation in electron microscopy of biological material, in Advances in Electronics and Electron Physics,, 1985, 269. Carlemalm, E., Garavito, R.M., and Villiger, W. Resin development for electron microscopy and an analysis of embedding at low temperature, J. Microsc., 126, 123, 1982. Carlemalm, E. and Kellenberger, E. The reproducible observation of unstained embedded cellular material in thin sections: Visualisation of an integral membrane protein by a new mode of imaging for STEM, Embo J., 1, 63, 1982. Fernandez-Moran, H. The fine structure of vertebrate and invertebrate photoreceptors as revealed by low temperature microscopy, in The Structure of the Eye, Smelser, G.K., ed., Academic Press, New York, NY, USA 1961, 521. Glauert, A.M. and Young, R.D. The control of temperature during polymerization of Lowicryl K4M: There is a low-temperature embedding method, J. Microsc., 154, 101, 1989. Grandclement, B. and Morel, G. Ultrastructural characterization of atrial natriuretic peptide receptors (ANP-R) mRNA expression in rat kidney cortex, Biol. Cell, 90, 213, 1998. Grandclement, B., Ronsin, B., and Morel, G. The three subtypes of atrial natriuretic peptide (ANP) receptors are expressed in the rat adrenal gland, Biol. Cell, 89, 29, 1997. Griffiths, G. Fine Structure Immuno-Cytochemistry. Springer-Verlag, Berlin, Heidelberg, New York, 1993. Hayat, M.A. Glutaraldehyde: Role in electron microscopy, Micron Microsc. Act, 1, 115, 1986. Hobot, J.A. Lowicryls and low temperature embedding for colloidal gold methods, in Colloidal Gold: Principles, Methods and Applications, Hayat, M.A., ed., Academic Press, San Diego, CA, USA, 1989, 75. John, H.A., Birnstiel, M.L., and Jones, K.W. RNA-DNA hybrids at the cytological level, Nature, 223, 582, 1969. Kellenberger, E. et al. Low Denaturation Embedding for Electron Microscopy of Thin Sections. Chemische Werke Lowi G.m.b.H., West Germany, 1980. Lipsker, D. et al. Heat shock proteins 70 and 60 share common receptors which are expressed on human monocyte-derived but not epidermal dendritic cells, Eur. J. Immunol., 32, 322, 2002. Morel, G. In situ hybridization on ultrathin frozen section, in Hybridization Techniques for Electron Microscopy, Morel, G., ed., CRC Press, Boca Raton, FL, USA, 1993, 163. Morel, G., Cavalier, A., and Williams, L. In Situ Hybridization in Electron Microscopy, ed. Morel, G., CRC Press, Boca Raton, FL, USA, 2001.

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18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

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Morel, G., Dubois, P., and Gossard, F. Ultrastructural detection of messenger RNA coding for growth hormone in the rat anterior pituitary by in situ hybridization, C. R. Acad. Sci. III, 302, 479, 1986. Newman, J.R. and Hobot, J.A. Resin Microscopy and On-Section Immunocytochemistry. Springer-Verlag, Berlin, Germany, 1993. Pardue, M.L. and Gall, J.G. Molecular hybridization of radioactive DNA to the DNA of cytological preparations, Proc. Nat. Acad. Sci. USA, 64, 600, 1969. Puvion-Dutilleul, F. and Puvion, E. Non-isotopic electron microscope in situ hybridization for studying the functional sub-compartmentalization of the cell nucleus, Histochem. Cell Biol., 106, 59, 1996. Robertson, D. et al. An appraisal of low-temperature embedding by progressive lowering of temperature into Lowicryl HM20 for immunocytochemical studies, J. Microsc., 168, 85, 1992. Roth, J. et al. Enhancement of structural preservation and immunocytochemical staining in low temperature embedded pancreatic tissue, J. Histochem. Cytochem., 29, 663, 1981. Roth, J., Taatjes, D.J., and Tokuyasu, K.T. Contrasting of Lowicryl K4M thin sections, Histochemistry, 95, 123, 1990. Sitte, H. et al. A new versatile system for freeze-substitution, freeze-drying and low temperature embedding of biological specimens, Scan. Microsc. Suppl., 8, 41, 1994. Spehner, D., Drillien, R., and Howley, P.M. The assembly of the measles virus nucleoprotein into nucleocapsid-like particles is modulated by the phosphoprotein, Virology, 232, 260, 1997. Whitehouse, R.L. et al. Immunolabelling of bacteriophage lambda receptor protein (LamB) on thin sections of E. coli embedded in Lowicryl, Biol. Cell, 51, 389, 1984.

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469

CONTENTS

GENERAL INTRODUCTION .................................................................................... 471 1.

PRINCIPLES OF CRYO-SECTIONING ACCORDING TO TOKUYASU472

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 474

3.

MATERIALS/PRODUCTS/SOLUTIONS ...................................................... 475 3.1. 3.2. 3.3.

4.

PROTOCOLS .................................................................................................... 480 4.1. 4.2. 4.3. 4.4. 4.5. 4.6.

5.

Materials ................................................................................................... 475 Products .................................................................................................... 476 Solutions ................................................................................................... 477

Preparation of Animal Cells and Tissue ................................................... 480 Preparation of Microorganisms ................................................................ 481 Preparation of Plant Tissue....................................................................... 483 Cryo-Sectioning........................................................................................ 484 Staining and Methyl Cellulose Embedding .............................................. 489 Cleaning Diamond Cryo-Knives .............................................................. 489

ADVANTAGES/DISADVANTAGES.............................................................. 490 5.1. 5.2.

Advantages ............................................................................................... 490 Disadvantages........................................................................................... 491

6.

WHY AND WHEN TO USE CRYO-SECTIONING ACCORDING TO TOKUYASU ....................................................................................................... 491

7.

OBSERVED RESULTS .................................................................................... 492

8.

REFERENCES .................................................................................................. 496



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471

GENERAL INTRODUCTION The desire to visualise biological ultrastructure in its aqueous surrounding is more than 50 years old. One of the great pioneers of cryo-electron microscopy, Humberto Fernández-Morán,1 described the first cryo-ultramicrotome in 1952.2 In the succeeding two decades, Wilhelm Bernhard,3 Christensen,4 Dollop and Sitte,5 and Sevéus6 and Barnard constructed the first functional cryo-microtomes, but it was not until the early 1970s that the technique had its breakthrough, initiated and propagated by Kiyoteru Tokuyasu.7-9 He made cryo-ultramicrotomy of chemically fixed and cryo-protected samples a standard method for sensitive immunogold labelling.10

Since then, the technique was further improved and brought to perfection by the group of Jan Willem Slot and Hans Geuze.11-13 This process of perfection would not have been possible without the new design and development of high-precision, reliable and easy to use cryo-ultramicrotomes.14 In general, today, Tokuyasu cryo-sectioning is as easily done as resin sectioning with the same quality. Even serial sectioning and sectioning parallel to the substrate are no longer impossible tasks.12,15 Today, the Tokuyasu cryo-sectioning method has become a standard method in combination with highly efficient immunogold labelling.12

Unfortunately, not all specimens are equally well suited for this method. Efficient and fast chemical fixation is impeded in samples containing air or large vacuoles (leaves), cell walls (plants, fungi) or cuticles, hydrophobic surfaces (insects, nematodes); and cryosectioning is more difficult with samples that exhibit differing internal stiffness, e.g., due to extracellular cell walls or cuticle-like barriers in plant, arthropod, and nematode tissues. Therefore, in plant as well as in nematode and arthropod research, there are only a few labs with a limited number of examples of successful application of the Tokuyasu cryosectioning method.16-18 Especially in these cases, a novel technique, combining cryofixation and ultrathin cryo-section labelling according to Tokuyasu offers a number of potential advantages19 (see Chapter 14).

Despite the drawbacks for a few biological specimens, cryo-sectioning according to Tokuyasu and described in this chapter, is the method of choice for most immunolabelling applications.

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1. PRINCIPLES OF CRYO-SECTIONING ACCORDING TO TOKUYASU  The biological material is chemically  Chemical fixatives cannot penetrate fixed with low concentrations of deeply into dense tissue. aldehydes.  Mammalian tissue is best preserved after a perfusion fixation. If perfusion fixation is not possible, the samples are cut in small 1 mm3 pieces in the presence of fixatives.  Tissue culture cells are fixed in situ and then scraped from the culture wells with a rubber policemen. They can also be used for flat-embedding according to a special protocol.15   The suspended cells are collected by  Caution: For scraped tissue culture centrifugation in 1.5 mL microtubes cells, e.g., human foreskin fibroblasts and washed several times in buffer. (HFF), 1% gelatine or 1% bovine serum albumin (BSA) should be added to the washing buffer to avoid sticking of the cell layers to the tube walls.15  Cells are pelleted into 10 to 12% gelatine, which is solidified on ice, and then small cubes, not more than 1 mm3, are cut.

 Tissue blocks are also infiltrated with 12% gelatine to fill empty spaces. Large differences in matrix densities have a negative influence on sectioning.

 The cubes of tissue or cells containing gelatine are infiltrated with 2.1 to 2.3 M sucrose or a mixture of polyvinyl pyrrolidone (PVP) and sucrose on a slowly rotating (4 rpm) spinning wheel at 4°C. Impregnation is done for 2 to 24 hours until the blocks do not float to the surface anymore.

 Sucrose or sucrose/PVP impregnation is important to avoid ice crystal damage during freezing and to make sure that the ice has good sectioning properties7,9 (see also Chapter 11).

 If needed, the blocks can be cut smaller and mounted on pins that can be fit into the holder of the microtome. Remove the excess of sucrose solution with a filter paper. Then the sample is frozen either in liquid nitrogen or in the sectioning chamber of the cryoultramicrotome.

 The smaller the block the less trimming is needed.  Due to the high sucrose concentration, the sample will vitrify independently of the way freezing is carried out.

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 Semithin and ultrathin sections are cut with either glass or diamonds knives at temperatures between 80°C to 140°C.

 The optimum temperature has to be found experimentally. It is mainly dependent on the desired thickness of the sections, but also on the texture of the sample.

 During trimming and sectioning an ioniser is directed toward the knife edge. The intensity is adjusted so that the sections glide from the knife edge without touching the surface, but do not fly away.

 The sectioning process causes electrostatic charging, which interferes with the gliding properties of the sections on the knife surface. Sticking to the surface can cause severe compressions (see also Chapter 11)!

 The sections are retrieved from the knife surface with a loop containing a drop of pick-up solution and transferred to room temperature on a Formvar- or Pioloform-carbon-coated electron microscopy grid or on coverslips or silane-coated glass slides for light microscopy applications.

 The exact timing of gently touching the cryo-sections with the surface of the freezing drop is important for the structural integrity of the cryo-section. It has to be found experimentally.

 At this step immunolabelling can be  See Chapter 21, 23. performed.  Then the sections are stained with  Methyl cellulose prevents drying uranyl acetate and embedded in artefacts and irregular shrinkage of the methyl cellulose. different cell organelles.  After drying, the sections are ready for analysis by electron microscopy.  For light microscopy, coverslips with  See Chapter 21. sections are mounted on a slide in a drop of mounting medium containing an antifading reagent.

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2. SUMMARY OF THE DIFFERENT STEPS 1. Chemical prefixation  Alternative: cryo-fixation (see Chapters 5, 6, 14) 2. Gelatine impregnation (optional) 3. Cryo-protection 4. Mounting/freezing (a,b) 5. Trimming (c: Top view on specimen holder (stub) with frozen sample)

6. Semithin cryo-sectioning for light microscopy (LM) (d) 7. Ultra-thin cryo-sectioning for EM (d) 8. Section retrieval (e)

9. Transfer to grid (EM) or coverslip (LM) (f,g)

10.

Immunolabelling (h)

11. Staining and methyl cellulose embedding (ultrathin cryo-sections) (i) or  Moviol embedding (semithin cryosections) Figure 19.1 (a-i) Outline of the cryosectioning and immunolabelling procedure of thin, thawed cryo-sections according to Tokuyasu.

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3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Cryo-ultramicrotome  Knife maker  Glass knives

 Diamond cryotrim

 Diamond cryo-knives

 Forceps  Loop 



  

 Leica EM UCT/EM FCS or EM UC6/EM FC6, Leica Microsystems, Vienna Austria.  Leica Knifemaker, Leica Microsystems, Vienna Austria. Please check the quality of the knife maker carefully before buying!  Made from glass strips: Gumhag shi 6.4 x 25/30, Glass Ultra Micro Co., Stockholm, Sweden; Glass strips (several sizes), Leica Microsystems, Vienna, Austria.  For breaking glass knives (see Reference 20, pp 145152).  Diamond trimming blades with a trimming angle of 20° and 45° (cryotrim 20, cryotrim 45; Diatome AG, Bienne, Switzerland).  There are special cryo-knives available with different cutting angles, 25°, 35°, 45°, e.g., Diatome AG, Bienne, Switzerland and Element Six, Cuijk, The Netherlands.  For example, Dumont #2a, #3, #4, #4N, #7, Dumont & Fils, Switzerland.

 Homemade loops from remanium wire or perfect loop, EMS, Fort Washington, Pennsylvania, USA. Rubber policemen/cell scraper or cell  Costar #3010, Corning B.V.; Corning New York, USA, Schiphol-Rijk, The lifter Netherlands.  Costar #3008, Corning B. V.  Swing-out centrifuge, e.g., Rotima 35, Table centrifuge Andreas Hettich GmbH&Co. KG., Tuttlingen, Germany.  A centrifuge with six different swing-out elements allows for more combinatorial freedom.  Centrifuge 5415C, Gerätebau Eppendorf Microfuge GmbH, Enelsdorf, Germany.  0.5 and 1.5 mL Eppendorf-NethelerMicrotubes Hinz GmbH, Hamburg, Germany or Sarstedt AG & Co, Nümbrecht, Germany.  Double-edged razor blades are best Razor blades suited for trimming the blocks.

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 Filter paper    

 Coffee filter paper or Whatman 50, Whatman International Ltd, Maidstone, England. H2O  Double distilled or MilliQ water. Coverslips  For polylysine coating, see Chapter 21. Glass slides  For immunolabelling, they may be silane-coated. Pioloform- or Formvar-coated and  Copper, nickel or gold grids, 50 to carbon-covered grids. 400 mesh: e.g., Stork Veco B. V., Eerbeek, The Netherlands; or Gilder Grids, Grantham, England.  For coating, see Reference 21.

3.2. Products  Gelatine  BSA, bovine serum Fraction V  Uranyl acetate (UO2(CH3COO)2 × 2H2O)  





 From the supermarket, e.g., Dr. Oetker. albumin,  A-7906, Sigma-Aldrich: Fluka, Buchs, Switzerland.  Stain for electron microscopy. EMS, Fort Washington, Pennsylvania, USA or SPI Supplies, West Chester, Pennsylvania, USA. Methyl cellulose  25 centipoises, Sigma M-6385. Fluorescent secondary antibodies  Against the species in which the primary antibody was produced.  Provided by several companies, e.g., Jackson Immunoresearch Europe Ltd, Suffolk, U. K. Gold-labelled secondary antibodies  Against the species in which the primary antibody was produced.  Provided by several companies, e.g., Dianova: Hamburg, Germany; Aurion, Wageningen, The Netherlands; British BioCell International (BBI) Ltd, Cardiff, U.K. For 1 nm gold markers, see Chapter 23.  Binds preferentially to the Fc region of Gold-labelled protein A rabbit, pig, human and mouse (subclasses IgG2a and IgG2b) antibodies (for details see Reference 20 pp 320323).  Can easily be homemade22,23 or purchased from Dr. G. Posthuma, Department of Cell Biology, Utrecht Medical Centre, Utrecht, The Netherlands.  Provided by several companies, e.g., Dianova, Aurion, and BBI, see above..

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 Paraformaldehyde  Disodium hydrogenphosphate  Na2HPO4.  Polyvinyl pyrrolidone (PVP, MW  Sigma PVP10-100G. 10,000)  Sodium carbonate  Na2CO3.  Sucrose  C12H22O11.  Sodium chloride  NaCl.  Potassium chloride  KCl.  Glycine  PIPES  1,4-Piperazine-bis(ethanesulfonic acid).  HEPES  2-[4-(2-Hydroxyethyl)-1-piperazine] ethanesulfonic acid.  Magnesium chloride  MgCl2.  Magnesium sulphate  MgSO4.  Potassium chloride  KCl.  EGTA  Ethyleneglycol- bis(β-aminoethyl)-N,N, N’,N’-tetracetic acid.  MES  2-(N-Morpholino)-ethanesulfonic acid.  Oxalic acid  Potassium dihydrogen phosphate  KH2PO4.  Glutaraldehyde (aqueous, e.g., 8%)  For example, Sigma #G-5882.  8% solution, Polysciences, # 00216A.  Keep at 4oC.  Sodium metaperiodate  NaIO4; Merck #106597.  Periodic acid  H5IO6; Merck #10052.  Ethanol  Triton X-100  Nonionic detergent, e.g., Fluka; Buchs, Switzerland.  Citric acid monohydrate  Merck 231211.

3.3. Solutions        

PBS buffer (pH 7.4) 137 mM sodium chloride NaCl 2.7 mM potassium chloride KCl 8.1 mM disodium hydrogenphosphate Na2HPO4 1.5 mM sodium dihydrogen phosphate NaH2PO4 PB buffer (200 mM, pH 7.4) 162 mM disodium hydrogenphosphate Na2H PO4 38 mM sodium dihydrogen phosphate NH2 PO4

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 Phosphate buffered saline, a standard buffer.

 Phosphate buffer, a standard buffer.  Make two stock solutions of 200 mM:  Buffer A: 27.6 g NaH2PO4 x 1H2O in 1 L H2O  Buffer B: 35.6 g Na2HPO4, 2H2O in 1 L H2 O

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 Add 19 mL buffer A to 81 mL buffer B to get 200 mM phosphate buffer; pH 7.4. Check pH and if necessary add more of the appropriate buffer to get pH 7.4.     

PHEM buffer (pH 6.9) 60 mM PIPES 25 mM HEPES 10 mM EGTA 2 mM MgCl2

 Citrate buffer  200 mM citric acid monohydrate

 Cytoskeleton buffer.24

 For low pH 1.55.0.  Used as a primary fixative for difficult to fix organisms, e.g., Dictyostelium discoideum.

 Buffers for plants  Often relatively low salt buffers.  Phosphate buffer 100 mM potassium phosphate buffer; pH 7.025  MTSB (microtubule stabilising  Cytoskeleton buffer. buffer)26  15 g PIPES (50 mM)  1.9 g EGTA (5 mM)  0.6 g MgSO4 (5 mM)  2,5 g KOH (solid) in 1 L H2O  pH 6.97.0  MS medium (pH 5.8)  Plant culture medium.27 Including micro and macro elements  Duchefa Biochemie B.V., Haarlem, The (0.5 ×). Netherlands.  Dissolve 2151.04 mg medium in 1 L H2O.  Fixatives  2 to 8% (w/v) formaldehyde

 For a 16% stock solution, suspend 16 g paraformaldehyde in 45 mL H2O, warm to about 60C (au bain Marie) and stir  2% (w/v) formaldehyde and 0.20.02% vigorously for some time. Then add 20 to (v/v) glutaraldehyde 80 l 1 M NaOH and wait for about  In PB, PHEM, or 200 mM 10 minutes. If the paraformaldehyde has not HEPES; pH 7.2 or dissolved into formaldehyde, repeat the  In MTSB; pH 6.97.0 or PB; NaOH step. Cool down on ice, filter and pH 7.0 adjust volume with H2O to 50 mL. If desired, glutaraldehyde is added to the working dilution of formaldehyde in buffer (HEPES pH 7.2, PB, PHEM, MTSB), check the final pH.  Note: Fixatives can be stored frozen in aliquots.

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 2.5 parts 8% formaldehyde in H2O  1.5 parts of a saturated solution of picric acid in H2O  5.0 parts citrate buffer  Add H2O to make 10 parts  Adjust pH to the pH of the growth medium.

 This fixative can be useful for difficult to fix samples like yeast (see Figure 19.13) or Dictyostelium discoideum.28  Note: The cytoplasm often gets a grainy appearance in the presence of picric acid.

 1 to 12% (w/v) gelatine in PBS

 Suspend in PBS, warm to about 60°C, stir until the gelatine powder has dissolved. Cool to 37oC, add sodium azide, aliquot (ca 2 mL) and store at 4oC.

 2.3 M sucrose in PBS or PHEM  Weigh 78.73 g sucrose in a 100 mL buffer volumetric flask. Add PBS or PHEM buffer pH 7.4 while stirring and wait until the sucrose has dissolved. Remove the stir bar and top up with the buffer to the 100 mL mark. The mixture is stored at 4oC or frozen in 10 to 20 mL aliquots.  Polyvinyl pyrrolidone (PVP)sucrose9  20% PVP  1.84 M sucrose in phosphate carbonate buffer

 Infiltration time is longer compared to pure sucrose.  To make 100 mL, mix 20 g PVP and 4 mL of 1.1 M disodium carbonate (Na2CO3), in 0.1 M disodium hydrogen phosphate (Na2HPO4) and 80 mL of 2.3 M sucrose in 0.1 M Na2HPO4.

 Pick-up solution7,12  2.3 M sucrose or  Reduces mechanical damage induced by  1% (w/v) methyl cellulose, 1.15 M spreading of sections during thawing on the sucrose in PHEM buffer surface of the 2.3 M sucrose drop.  Alternatively, one can use a mixture of one part sucrose in PBS and one part PVPsucrose to reduce spreading upon thawing.  Staining solution for transmission electron microscopy (TEM)  To avoid vesiculation of cellular  Neutral uranyl acetate (uranyl oxalate) membranes.8 2% (w/v) uranyl acetate in 0.15 M  Stir a 0.3 M oxalic acid solution in H2O oxalic acid; pH 7.4 vigorously and add slowly the same volume of a 4% (w/v) aqueous uranyl acetate solution. Adjust pH to 7.4 with 10% ammonium hydroxide.8 w  2% ( /v) aqueous uranyl acetate  Make 4% to add to the methyl cellulose for embedding.

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 Staining solution (LM)  Toluidine Blue O

 See Chapter 21.

 Methyl cellulose  Dissolve methyl cellulose in prewarmed  2% (w/v) methyl cellulose in H2O H2O and stir in the cold for several hours. (25 centipoises)10 Centrifuge the solution for 60 min at 100,000 g. Store in the refrigerator.  Embedding solution (TEM)  Methyl cellulose + uranyl acetate  9 parts methyl cellulose  More uranyl acetate can be added to  1 part 4% (w/v) aqueous uranyl increase contrast. acetate29  Embedding solution (LM)  Moviol

 See Chapter 21.

4. PROTOCOLS 4.1. Preparation of Animal Cells and Tissue 1. Chemical fixation of culture cells  Cells are grown in culture dishes or culture flasks to about 70% confluency.  Preferentially, growth medium is refreshed one day before fixation  Cells in culture are fixed for 1 to 2 hours at room temperature by adding equal volumes of double-strength fixatives to the growth medium.  Then the cells are scraped in the presence of 1% gelatine in buffer15 to reduce damage and loss of cells due to adherence to plastic or glass surfaces or to the walls of microtubes.  Suspension cells are directly fixed with the same volume of doublestrength fixative.  The cells are collected in a centrifuge tube and pelleted at about 800 to1200 g.  Then, they are washed three times with buffer containing 1% gelatine.

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 Routinely, we use two fixatives: One with formaldehyde only and one that contains formaldehyde and glutaraldehyde.  Fixative is made in phosphate, PHEM or HEPES buffer. PHEM and HEPES buffers have higher buffering capacity and generally result in better structural preservation.20  For adherent cells, the medium may be replaced by the fixative solution and the cells are fixed for 1 to 2 hours at room temperature.

 1000 rpm in a microcentrifuge.

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2. Chemical fixation of tissue  If possible, the tissue is fixed by  Perfusion fixation is preferred because perfusion fixation. tissue is very sensitive to the postmortem  Otherwise, the tissue is sampled, alterations and damage by excision. submersed into the fixative and cut into small pieces of about 1 mm3.  The tissue pieces are fixed for 1 to 2 hours at room temperature.  Thereafter, they are washed three times with buffer. 3. Gelatine impregnation  The cells are pelleted into gelatine in PBS or PHEM buffer and the gelatine is solidified on ice.  Small blocks of not more than 1 mm3 are cut from the gelatine-embedded cells.  The pieces of tissue are also impregnated with 12% gelatine for 30 minutes at 37oC on a rotating wheel. Before gelling, the excess gelatine is drained off with a piece of filter paper or tissue.

 The gelatine density should match the sample density; we usually use 10 to 12%.  The pellet should be loose. In dense pellets, the cells are closely packed and individual cells are difficult to photograph. The plasma membrane labelling may also be reduced.  Tissue blocks are infiltrated with gelatine to fill spaces, e.g., capillaries, with protein. Large differences in matrix densities can also impair the sectioning process.

4.2. Preparation of Microorganisms 1. Chemical fixation of bacteria  Bacteria are grown in growth medium or on agar plates to the desired density.  An equal volume of double-strength fixative is added to the growth medium. Cells grown on agar plates are collected with a spatula or a bacteria loop and suspended in singlestrength fixative.  After about 15 to 30 minutes the cells are pelleted at 800 to 1200 g and resuspended in fresh single strength fixative.  Fixation is continued for 1 to 2 hours overall.  The fixed cells can be stored in 1% formaldehyde at 4oC.

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 Note that the fixative solution should mimic the culture conditions as closely as possible, therefore, fix with the pH of the growth medium at the growing temperature. For acid or basic pH, appropriate buffers have to be chosen.  Adding equal volumes of doublestrength fixatives to the growth medium is the preferred initial fixation.

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2. Chemical fixation of yeast cells and other fungi  Yeast cells (fungi) are grown in culture medium or on agar plates to the desired density.  An equal volume of double-strength fixative is added to the growth medium (see above) and stirred well. Cells grown on agar plates are collected with a spatula or a bacteria loop and suspended in single-strength fixative.  After about 15 to 30 minutes the cells are pelleted at about 2500 × g and resuspended in fresh single-strength fixative at pH 7.2.

 For yeast cells it seems to be even more important that the fixative solution mimics the culture conditions. If disturbed by a change of environment or even by centrifugation, yeast cells can close their membrane pores and the fixation process will be delayed. As a result, yeast cells are not well fixed and can lose a large amount of cellular content.  4000 rpm in a Hettich swing-out centrifuge.  After initial fixation, the pH should be raised to 7.2 because at low pH aldehydes do not fix very well.20

 Fixation is continued for 1 to 2 hours overall.  After the cells are washed in buffer, then fixed cells can be stored at 4°C or even shipped in 1% formaldehyde.  Now the cells are washed three times in buffer, e.g., PHEM, and then incubated in 1% periodic acid in buffer for onr hour at ambient temperature. Alternatively, they are treated with 1% (w/v) sodium metaperiodate for one hour at 4°C.

3. Gelatine impregnation  The cells are pelleted in 12% gelatine in PBS or PHEM buffer and the gelatine is solidified on ice.  Small cubes or prisms of not more than 1 mm3 are cut from the gelatineembedded cells.

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 The treatment with periodate30 (De Maziere, A.M., personal communication) reduces detaching of the cell wall during sectioning. Frequently, the cell wall nicely lines the plasma membrane in contrast to standard fixation methods where the cell wall detaches and lies on the section like twisted bicycle tires.  Under these conditions, the oxidation process is very limited and the glucane chains in the cell wall can still be labelled with specific antibodies31 (see Figure 19.13).

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4.3. Preparation of Plant Tissue 1. Chemical fixation of tissue without enclosed air  Seedlings grown on agar are transferred to single strength fixative  Seedlings treated with reagents, e.g., dissolved in half-strength (0.5 ×) MS medium27 (pH 5.8) are fixed by adding the same amount of double strength fixative in PB, or MTSB.  After a few minutes, the seedlings are transferred to fresh single-strength fixative at ~ pH 7.0.

 For example, meristem tissue like root tips.  Samples are fixed either with 4% or 8% formaldehyde or with a mixture of formaldehyde and glutaraldehyde, in a relatively low ionic strength buffer (100 mM PB, MTSB).  Highly vacuolated cells (with high internal pressure) may collapse.  In order to enhance wetting of samples and to facilitate fixative diffusion into cells/tissues covered by hydrophobic surfaces (e.g., ovules, anthers/pollen), the fixation buffer can be supplemented with low concentrations of detergent (e.g., 0.01% Triton X-100). Caution: Detergent treatment increases extraction!  In case of detaching of the cell wall during sectioning and section transfer, sodium periodate or periodic acid treatment may help (see Section 4.2.2).

2. Chemical fixation of tissue containing air  The tissue of interest is transferred to  Samples are fixed either with 4% or 8% single strength fixative and is cut in formaldehyde or with a mixture of smaller pieces (1 to 2 × 1 to 2 mm). formaldehyde and glutaraldehyde, in a relatively low ionic strength buffer (PB, MTSB).  Degassing during fixation (pressure  Removes air and improves fixative should not be lower than 200 to 300 diffusion. mbar to prevent boiling of buffer). 3. Gelatine impregnation  Tissue pieces are embedded in 12%  Optional, may improve sectioning properties (see 3. in Section 4.1). gelatine.  May be done in several steps, e.g., 2%, 4%, 8%, and 12% gelatine, to reduce deleterious osmotic effects, such as vacuole collapse and shrinkage of highly vacuolated plant tissue.

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4.4. Cryo-Sectioning 1. Sucrose impregnation  The small blocks of tissue or gelatine embedded cells are put into a microtube containing 2.3 M sucrose. The tube is mounted on a rotating wheel and the tubes are rotated head over.  Impregnation is done for 4 to 20 hours in a cold room at 4oC.

2. Mounting the sample  The gelatine or tissue blocks are cut smaller if needed (in cubes or prisms) and mounted on the stubs of the cryoultramicrotome.  Drain off the sucrose solution from the surface of the block, but retain a conical-shaped portion around the bottom of the block at the stub. Then the block is frozen on the stub either by immersing it in liquid nitrogen or in the cold atmosphere of the ultramicrotome.

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 After successful impregnation, the blocks float in the solution or sink to the bottom. Under no circumstances should they swim.  Infiltration in several steps, e.g., 0.7 M, 1.4 M, 2.3 M, may reduce possible osmotic effects, such as vacuole collapse and shrinkage of highly vacuolated plant tissue.  Alternatively, PVP-sucrose may be used, if gelatine embedding was omitted. PVP does not permeate cells and may reduce differences in matrix densities, which can impair the sectioning process.9  The smaller the block, the shorter the time of trimming.  Please remember that the thinner the section has to be cut, the smaller the length of the edge: 600 μm edge for 200 nm section; 400 μm for 100 nm; 300 μm for 80 nm and 200 μm for 60 nm, 150 μm for 40 nm and 100 μm for 30 nm (H. Gnägi, Diatome AG, personal communication).  The conical-shaped drop stabilises the block during sectioning.  Excess sucrose can cause cracking and the block may break off.  Before mounting, the surface of the stub is scratched, then the stubs are cleaned with acetone in a sonicator to improve attachment of the sample.  In the case of PVP-sucrose, samples have to be processed very rapidly to prevent evaporation.  In contrast to cryo-fixation, here slower freezing is preferred to prevent fast changes in material shrinkage during the freezing process. Unequal shrinkage of the metal and the sample can lead to breaks at the stub — block interface and loss of the sample. The high sucrose content guarantees vitrification even at low freezing rates. Figure 19.2 Mounting and freezing.

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3. Trimming  The temperature of the cryoultramicrotome is set to about 80oC to 100oC.  The tip of the ioniser is placed close to  To avoid sticking of the sections to the the blade and set to full power. surface of the trimming tool.  The blocks are trimmed to the desired size either with a glass knife or the diamond cryotrim.  Trimming can be done at high speed with a feed of a few hundred nanometres.

 With the glass knife, the angle of the pyramid will be 90o; with the cryotrim 20; the angle will be 70°; and with cryotrim 45, it will be 45°. The smaller the angle the more stable the resulting pyramid, but the faster the sections grow in size.

Figure 19.3 Trimming with the cryotrim diamond knife. Top view on the frozen gelatine block mounted in the pin/stub. 4. Semithin sections  The temperature of the cryoultramicrotome is set to about 100°C, for PVP-sucrose to about 80°C.  The tip of the ioniser is placed close to the blade and set to full power.  The knife, glass knife or cryo-diamond knife slowly approaches the face of the pyramid.

 The sections are cut with a nominal feed of 200 to 400 nm at a speed of about 1 mm/sec.  With an eyelash mounted on a wooden stick, some sections are brought together.

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 Semithin sections are very useful for correlative light and electron microscopy (see Chapter 21), but also for screening new antibodies and orientation in the tissue.  The reflection of the knife edge mirrored in the clean surface of the pyramid shows whether the knife is parallel to the pyramid and indicates the distance from it.  During sectioning, the strength of ioniser is adapted by the power, but also by changing the distance to the knife edge so that the sections float above the knife surface but do not fly away. When the sections stick to the surface, it is advisable to use a fresh edge of the knife.

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 A drop of pick-up solution is mounted in a loop and held into the cryochamber close to the sections. At the moment a drop of 2.3 M sucrose freezes, indicated by a thin trail of fog, the sections are touched and will stick to the drop. With a mixture of sucrose/methylcellulose the sections are picked up before the drop has completely frozen and turned white.

 Caution: The ioniser has to be switched off during section retrieval.  Electrical contact between the ioniser and the microtome can damage the circuits of the cryo-ultramicrotome!  The ioniser also changes the freezing behaviour of the pick-up solution and impairs section pickup.  When the drop is to close to the sections, they may jump to the drop by electrostatic interactions.

Figure 19.4 Semithin cryo-sectioning and retrieval or pickup of the sections. The sections are arranged on the knife with an eyelash.  The loop is brought to room temperature and thawed. Depending on the pick-up solution the sections spread more or less evenly on the surface of the melting drop.  Then the sections are placed on a coverslip or glass slide.

Figure 19.5 Transfer of thawed cryosections to ambient temperature and mounting on a coverslip, which is placed upside down on a drop of buffer for subsequent immunolabelling. Alternatively, coverslips can be handled as described in Chapter 21.

 The sections can be treated with common histological stains, e.g., Toluidine blue; or for immunofluorescence studies and correlative  Observe preferably with bright-field or microscopy, (see Chapter 21). phase contrast illumination.

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Figure 19.6 Immunolabelling for LM in a moist chamber on Parafilm (see Chapter 21, 23). 5. Ultrathin sections  The temperature of the cryoultramicrotome is set between 120°C and 140°C, for PVP-sucrose to about 110 to 115°C.  The tip of the ioniser is placed close to the blade and set to full power.  The knife, glass knife or cryo-diamond knife should slowly approach the face of the pyramid.

 The reflection of the knife edge mirrored in the clean surface of the pyramid shows whether the knife is parallel to the pyramid and indicates the distance from it.

 The sections are cut with a nominal  The Diatome diamond knives come with feed of 50 to 100 nm at a speed of an indication of the preferential cutting about 1 mm/sec. speed. The optimum cutting speed, however, is dependent on the material, the temperature, the thickness of the section, the quality of the knife and has to be determined empirically.  A drop of pick-up solution is mounted  Caution: The ioniser has to be switched in a loop and the sections are picked off during section retrieval!  Caution: The methyl cellulose/sucrose up as described above. solution freezes much faster than sucrose alone.  The loop is brought to room  In 2.3 M sucrose, the sections spread temperature and thawed. Depending considerably, a phenomenon that can lead on the pickup solution, the sections to severe changes of the ultrastructure. In spread more or less evenly on the the methyl cellulose/sucrose solution,12 the ultrastructure is of much superior quality. surface of the melting drop.  The methyl cellulose/sucrose solution should be used with perfect sections only, compressions and folding of the section will remain.

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 The moment of pickup is important, too early may lead to folding of the sections and too late may mechanically destroy the sections or they may not attach at all. Figure 19.7 Picking up/retrieval of ultrathin cryo-sections, after removing them from the knife edge with the help of an eyelash.  Then the sections are placed on a Formvar- or Pioloform-carbon-coated electron microscopy grid. Now the sections are ready for immunogold labelling (see Chapter 23) and/or embedding into methyl cellulose for inspection by electron microscopy.

 Copper grids can be used for morphological analysis or for immunolabelling procedures, which do not exceed one day. Copper grids can also be used for long-term storage of sections.32  For overnight incubation and silverenhancement (see Chapter 23), inert material, such as nickel or gold, is preferred.  For plastic coating and carbon covering of grids, see Reference 21.

Figure 19.8 Transfer of thawed cryosections to a Formvar/Pioloform and carbon-coated grid and incubation of the grid (upside down) on a buffer drop for subsequent immunolabelling.

Figure 19.9 Immunolabelling in a moist chamber on Parafilm. Transfer of grids can be done with forceps or a loop.

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4.5. Staining and Methyl Cellulose Embedding  After thawing or immunolabelling, respectively, the sections are carefully washed on several drops of H2O.  Then they are stained for 10 minutes on uranyl oxalate at pH 7.4.

 Here the full embedding procedure is given. Depending on the application and the intensity of the contrast in a given application, some of the steps can be omitted: staining with aqueous uranyl acetate only or directly embedding in  The sections are quickly washed over uranyl acetate containing methyl cellulose. two drops of H2O. Other combinations are also valid.  Stained for two minutes on aqueous  The main purpose of the neutral uranyl uranyl acetate. oxalate is to protect the morphology of delicate membrane structures.8  Incubated for five minutes on a drop of  The methyl cellulose embedding is done methyl cellulose or methyl cellulose on ice because the solution is less viscous with uranyl acetate. in the cold.  The grids are removed individually from the drops with a loop.  The excess methyl cellulose is drained away with a wet piece of tissue paper or filter paper, such that a very thin film remains.  Then the grids are dried for about 30 minutes before they are carefully removed from the loop with forceps. Figure 19.10. Uptake of grids in loop (left) and removal of excess methyl cellulose from the grid, placed in a loop, using a piece of filter paper (right) (the distance between grid rim and loop is larger then in reality).  After silver-enhancement the grids should be stored in dry air, under nitrogen gas or under vacuum because the silver layer is not stable in a humid atmosphere (see Chapter 23).

4.6. Cleaning Diamond Cryo-Knives  Remove knife from the cryo-chamber before heating up.  Flush it with running tap water until the water does not freeze anymore on the knife.

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 Caution: A diamond knife is very delicate! In order not to damage your diamond knife, please refer to the manufacture’s instructions prior to cleaning. This is the method used routinely in our lab by experienced personnel; however, we do not take any liability!

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 The tip of the polystyrene rod is cut to an angle of about 90° using a razor blade.  The sectioned polystyrene tip is dipped in 50% ethanol/water.  Gently run the rod tip across the cutting edge of the diamond knife without applying lateral pressure.

 The rod is available from Diatome and supplied with new diamond knives.  Caution: The razor blade must be cleaned with white spirits before use. The remaining oil layer destroys the coating of the diamond knife (H. Gnägi, Diatome, personal communication).  Caution: Do not touch the glue (if present) between knife and knife holder.

Fig. 19.11 Moving the polystyrene rod tip along the knife edge.  When sections or debris have dried  Caution: Refer to the user manual of onto the knife edge, place the knife in manufacturer. H2O overnight before cleaning.

5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  Highly efficient immunolabelling  The sample is only partly dehydrated and the proteins remain in their natural aqueous environment.  The topology of the thawed sections enables better access to the antibodies.  Excellent definition of intracellular membrane systems.

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 Especially suited for low-copy number antigens.  20 to 30% water remains in the sample.  Suited for antigens sensitive to solvents (ethanol) and resin components.  Partial penetration of antibodies and ultra-small gold markers into the section is possible (see Chapter 23).  See Reference 12 and 20.

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5.2. Disadvantages  The biological material is chemically fixed by aldehydes.  An undefined number of soluble proteins may be lost.  Suboptimum preservation of the cellular ultrastructure.

 Aldehyde fixation is selective.

 Usually only a weak aldehyde fixation is used.  Alterations induced by chemical fixation, mainly in membrane architecture33-35 (see Part I, Cryo-Fixation Methods; and Chapters 11, 13).  The contrast in the cytoplasm is  Stronger fixation leads to less loss of dependent on the fixation. proteins. Therefore, there is very little difference in contrast, especially in the smaller cytoplasmic structures, e.g., ribosomes and cytoskeletal components cannot be differentiated. As an additional note, stronger fixation also prevents antibodies from penetrating the cryosections, which may result in reduced labelling efficiency.36

6. WHY AND WHEN TO USE CRYO-SECTIONING ACCORDING TO TOKUYASU  Cryo-sectioning according to Tokuyasu is the most sensitive preparation method for on-section immunolabelling.

 The proteins remain in their aqueous environment.  Suited for antigens sensitive to ethanol or methacrylates.  Especially suited for low-copy number antigens.  The thawed sections have a rough surface topology exposing more antigens than resin sections.  Antibodies can access antigens in the sections of weakly fixed material.37  The method is very fast.  If necessary, within one day a sample can be prepared, sectioned, immunolabelled and analysed.  It is best suited for locating proteins in  It excels in membrane contrast. correlation to cellular compartments.

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7. OBSERVED RESULTS  Figure on the Chapter’s title page

 A light and electron microscopy image of rat liver labelled for catalase (correlative microscopy). Rat liver was perfusion-fixed with 2% formaldehyde and 0.2% glutaraldehyde in phosphate buffer. Cryo-sections of about 300 and 80 nm thickness were cut in a cryoultramicrotome and labelled with a rabbit anticatalase antibody. For fluorescence light microscopy (upper left corner), the semithin sections were detected with a secondary FITC-conjugated antibody (bright dots) and stained with DAPI to visualise the DNA (grey contours). For electron microscopy labelling (lower right corner), the primary antibody was detected with a goat antirabbit antibody coupled to ultra-small gold particles (Aurion). The particles were silver-enhanced with RGENT SE-EM (see Chapter 23) ca. 40 min at 23°C, see below.

 Figure 19.12

 Electron micrograph of a cryo-section of rat liver labelled for catalase, see above. Bar = 500 nm

 Figure 19.13

 Yeast cells were fixed in 2% FA, 15% saturated picric in 100 mM citrate buffer pH 4.0 at 30°C for about 15 min. Fixation was continued in 2% FA and 0.02% GA in 200 mM HEPES, pH 7.4 for 1 h, then the cells were incubated in 1% NaIO4 in water for 1 h at 4°C. Finally, 100 nm-thick cryosections were labelled for 1-6 β-glucan and detected with 10 nm gold particles.31,38 This result confirms that the oxidation by iodate did not dramatically change the chemical structure of the glucan chains. Bar = 200 nm

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494  Figure 19.14

 Figure 19.15

 Figure 19.16

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 Arabidopsis root tip cell in mitosis. Seedling root tips were fixed with 4% (30 min) and 8% FA (90 min) in MTSB, pH 7.0. Root tips were infiltrated with PVPsucrose, mounted on a pin, frozen in LN2, and sections of about 100 nm were cut. A monoclonal mouse antimyc antibody was used to label the myc-tagged version of the cytokinesis specific syntaxin KNOLLE. The primary antibody was detected with a secondary antibody conjugated to ™ Nanogold . Nanogold was silver-enhanced with HQ Silver™ for 8.5 min (see Chapter 23). KNOLLE is necessary for fusion of Golgi-derived vesicles with the newly formed cell plate (CP) membranes between the two daughter cells. KNOLLE can be also detected on the trans-Golgi network (TGN).  Arabidopsis seedling root tips were processed as described above. The GFPtagged Sialyltransferase, which localizes to trans-Golgi cisternae and TGN was labelled with a rabbit anti-GFP antibody and detected with a secondary antibody coupled to ultra-small gold. The ultra-small gold colloids were silver-enhanced with RGENT SE-EM for 45 min (see Chapter 23).  Arabidopsis seedling root tips were processed as described above. Clathrin coats on TGN vesicles were detected with rabbit anticlathrin antibodies and secondary antibodies conjugated to Nanogold. Nanogold was silver-enhanced with HQ Silver for 8.5 min (see Chapter 23). The gap between the trans-Golgi stack and TGN vesicles is a stretching artefact caused by the use of sucrose alone for section retrieval. MC-sucrose mixture is less harmful to the thawing cryo-section.  G = Golgi  M = mitochondrion  TGN = trans-Golgi network Bars = 250 nm

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8. REFERENCES 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Fernández-Morán, H. Low-temperature preparation techniques for electron microscopy of biological specimens based on rapid freezing with liquid Helium II, Ann. N. Y. Acad. Sci., 85, 689, 1960. Fernández-Morán, H. Application of the ultrathin freezing-sectioning technique to the study of cell structures with the electron microscope, Arkiv Fysik, 4, 471, 1952. Bernhard, W. Ultramicrotomie à basse température, Ann. Biol., 4, 5, 1965. Christensen, A.K. Frozen thin sections of fresh tissue for electron microscopy with a description of pancreas and liver, J. Cell Biol., 51, 1971. Dollhopf, F. and Sitte, H. Die Shandon-Reichert-Kühleinrichtung FC-150 zum Herstellen von Ultradünn- und Feinschnitten bei extrem niedrigen Temperaturen. I. Gerätetechnik, Mikroskopie, 25, 15, 1969. Sevéus, L. Preparation of biological material for x-ray microanalysis of diffusible elements. I. Rapid freezing of biological tissue in nitrogen slush and preparation of ultrathin frozen sections in the absence of trough liquid., J. Microsc., 112, 269, 1978. Tokuyasu, K.T. A technique for ultracryotomy of cell suspensions and tissues, J. Cell Biol., 57, 551, 1973. Tokuyasu, K.T. A study of positive staining of ultrathin frozen sections, J. Ultrastruct. Res., 63, 287, 1978. Tokuyasu, K.T. Use of poly(vinylpyrrolidone) and poly(vinyl alcohol) for cryo ultramicrotomy, Histochem. J., 21, 163, 1989. Tokuyasu, K.T. Immunocytochemistry on ultrathin frozen sections, Histochem. J., 12, 381, 1980. Geuze, H.J. et al. Use of colloidal gold particles in double-labelling immunoelectron microscopy of ultrathin frozen tissue sections, J. Cell Biol., 89, 653, 1981. Liou, W., Geuze, H.J., and Slot, J.W. Improving structural integrity of cryosections for immunogold labelling, Histochem. Cell Biol., 106, 41, 1996. Slot, J.W. and Geuze, H.J. A new method of preparing gold probes for multiplelabelling cytochemistry, Eur. J. Cell Biol., 38, 87, 1985. Sitte, H. Process of ultrathin sectioning, in Science of Biological Specimen Preparation 1983, Revel, J.P., Barnard, T., and Haggis, G.H., eds., SEM Inc., AMF O'Hare, IL, USA, 1984, 97. Oorschot, V. et al. A novel flat-embedding method to prepare ultrathin cryosections from cultured cells in their in situ orientation, J. Histochem. Cytochem., 50, 1067, 2002. González-Melendi, P. et al. New in situ approaches to study the induction of pollen embryogenesis in Capsicum annuum L, Eur. J. Cell Biol., 69, 373, 1996. Dettmer, J. et al. Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis, Plant Cell, 18, 715, 2006. Pimpl, P. et al. In situ localization and in vitro induction of plant COPI-coated vesicles, Plant Cell, 12, 2219, 2000. Van Donselaar, E. et al. Immunogold labelling of cryo-sections from high-pressure frozen cells, Traffic, 8, 471, 2007. Griffiths, G. Fine Structure Immunocytochemistry. Springer-Verlag, Berlin, Heidelberg, Germany, 1993.

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21. 22. 23. 24. 25. 26. 27. 28. 29.

30.

31. 32. 33. 34. 35. 36. 37. 38.

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Hayat, M.A. Principles and Techniques of Electron Microscopy. Biological Applications. 4th ed., Cambridge University Press, Cambridge, UK,2000. Faulk, W.P. and Taylor, G.M. An immunocolloid method for the electron microscope, Immunochemistry, 8, 1081, 1971. Slot, J.W. and Geuze, H.J. Sizing of protein A-colloidal gold probes for immunoelectron microscopy, J. Cell Biol., 90, 533, 1981. Schliwa, M., van Blerkom, J., and Porter, K.R. Stabilization of the cytoplasmic ground substance in detergent-opened cells and a structural and biochemical analysis of its composition, Proc. Nat. Acad. Sci. USA, 78, 4329, 1981. Hinz, G. et al. Vacuolar storage proteins and the putative vacuolar sorting receptor BP-80 exit the Golgi apparatus of developing pea cotyledons in different transport vesicles, Plant Cell, 11, 1509, 1999. Goodbody, K.C. and Lloyd, C.W. Immunofluorescence techniques for analysis of the cytoskeleton, in Plant Cell Biology. A Practical Approach, Harris, N. and Oparka, K.J., eds., IRL Press, Oxford, UK, 1994, 221. Murashige, T. and Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures, Physiologia Plantarum, 15, 473, 1962. Humbel, B.M. and Biegelmann, E. A preparation protocol for postembedding immunoelectron microscopy of Dictyostelium discoideum cells with monoclonal antibodies, Scan. Microsc., 6, 817, 1992. Griffiths, G. Selective contrast for electron microscopy using thawed frozen sections and immunocytochemistry, in Science of Biological Specimen Preparation, 1983, Revel, J.P., Barnard, T., and Haggis, G.H., eds., SEM Inc., AMF O'Hare, IL, USA, 1984, 153 Kärgel, E. et al. Candida maltosa NADPH-cytochrome P450 reductase: Cloning of a full-length cDNA, heterologous expression in Saccharomyces cerevisiae and function of the N-terminal region for membrane anchoring and proliferation of the endoplasmic reticulum, Yeast, 12, 333, 1996. Müller, W.H. et al. Immuno-electron microscopy in yeast cell research, Recent Res. Devel. Mol. Microbiol., 1, 119, 2002. Griffith, J.M. and Posthuma, G. A reliable and convenient method to store ultrathin thawed cryosections prior to immunolabelling, J. Histochem. Cytochem., 50, 57, 2002. Van Harreveld, A., Crowell, J., and Malhotra, S.K. A study of extracellular space in central nervous tissue by freeze-substitution, J. Cell Biol., 25, 117, 1965. Murk, J.L.A.N. et al. Influence of aldehyde fixation on the morphology of endosomes and lysosomes: Quatitative analysis and electron tomography, J. Microsc., 212, 81, 2003. Szczesny, P.J., Walther, P., and Müller, M. Light damage in rod outer segments: The effects of fixation on ultrastructural alterations, Curr. Eye Res., 15, 807, 1996. Stierhof, Y.-D., Schwarz, H., and Frank, H. Transverse sectioning of plasticembedded immunolabeled cryosections: Morphology and permeability to protein Acolloidal gold complexes, J. Ultrastruct. Mol. Struct. Res., 97, 187, 1986. Stierhof, Y.-D. et al. Yield of immunolabel compared to resin sections and thawed cryosections, in Colloidal Gold: Principles, Methods, and Applications, Hayat, M.A., ed., Academic Press, Inc., San Diego, CA, USA, 1991, 87. Humbel, B.M. et al. In situ localization of β-glucans in the cell wall of Schizosaccharomyces pombe, Yeast, 18, 433, 2001.

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CONTENTS

GENERAL INTRODUCTION .................................................................................... 503 1.

PRINCIPLES OF THE METHODS ................................................................ 504 1.1. 1.2.

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 506 2.1. 2.2.

3.

Materials ................................................................................................... 509 Products .................................................................................................... 511 Solutions ................................................................................................... 512

PROTOCOLS .................................................................................................... 512 4.1. 4.2. 4.3.

4.4.

4.5.

4.6.

4.7.

5.

Chemical Procedure.................................................................................. 506 Cryo-Procedures ....................................................................................... 507

MATERIALS/PRODUCTS/SOLUTIONS ...................................................... 509 3.1. 3.2. 3.3.

4.

Energy Filtered Transmission Electron Microscopy (EFTEM)................ 504 Secondary Ion Mass Spectroscopy (SIMS) .............................................. 505

Sampling................................................................................................... 512 Chemical Procedure.................................................................................. 513 Cryo-Fixation ........................................................................................... 514 4.3.1. Slamming .................................................................................... 514 4.3.2. High-pressure freezing................................................................ 515 Dehydration of Cryo-Fixed Samples ........................................................ 516 4.4.1. Freeze-substitution...................................................................... 516 4.4.2. Freeze-drying .............................................................................. 516 Resin Embedding...................................................................................... 517 4.5.1. Low viscosity epoxy resin mixture ............................................. 517 4.5.2. Lowicryl HM® ............................................................................ 518 Sectioning ................................................................................................. 519 4.6.1. Sectioning on fluid...................................................................... 519 4.6.2. Dry sectioning............................................................................. 520 Elemental Analysis ................................................................................... 520 4.7.1. EFTEM ....................................................................................... 520 4.7.2. SIMS ........................................................................................... 521

ADVANTAGES/DISADVANTAGES.............................................................. 522 5.1.

Advantages ............................................................................................... 522 5.1.1. Chemical procedure .................................................................... 522 5.1.2. Cryo-fixation............................................................................... 522 5.1.3. Dehydration ................................................................................ 523

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5.2.

6.

5.1.4. Resin embedding ........................................................................ 523 5.1.5. Sectioning................................................................................... 524 5.1.6. Elemental analysis ...................................................................... 524 Disadvantages .......................................................................................... 525 5.2.1. Chemical procedure.................................................................... 525 5.2.2. Cryo-fixation .............................................................................. 525 5.2.3. Dehydration ................................................................................ 526 5.2.4. Resin embedding ........................................................................ 526 5.2.5. Sectioning................................................................................... 527 5.2.6. Analysis ...................................................................................... 527

WHY AND WHEN TO USE A SPECIFIC METHOD.................................. 528 6.1. 6.2.

6.3.

6.4.

6.5.

6.6.

Chemical Procedure ................................................................................. 528 Cryo-Fixation ........................................................................................... 528 6.2.1. Cryo-fixation by slamming......................................................... 528 6.2.2. Cryo-fixation by high-pressure freezing..................................... 528 Dehydration.............................................................................................. 529 6.3.1. Dehydration by freeze-substitution............................................. 529 6.3.2. Dehydration by freeze-drying..................................................... 529 Resin Embedding ..................................................................................... 529 6.4.1. Embedding in Spurr’s resin ........................................................ 529 6.4.2. Embedding in Lowicryl HM23................................................... 530 Sectioning................................................................................................. 530 6.5.1. Sectioning on fluid ..................................................................... 530 6.5.2. Dry-sectioning ............................................................................ 530 Analysis.................................................................................................... 531 6.6.1. EFTEM....................................................................................... 531 6.6.2. SIMS........................................................................................... 531

7.

OBSERVED RESULTS.................................................................................... 532

8.

REFERENCES .................................................................................................. 536



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GENERAL INTRODUCTION         Nowadays some analytical techniques, such EFTEM = Energy Filtered Transas EFTEM3,4 or SIMS imaging8,9 allow mission Electron Microscopy mapping of most of the chemical elements SIMS = Secondary Ion Mass Specpresent in samples. trometry Identification, localisation and quantification of intracellular chemical elements are important questions in many areas of biological research, especially in pharmaco-toxicology to understand mechanisms by which drugs interfere with living processes.

During such analysis, samples are subjected to drastic conditions (vacuum, bombardment energy, etc.) that prevent observation of living cells. Therefore, all samples should be prepared prior to any analysis. First, biological processes should be immobilized so that the samples stay as close as possible to the living state. The samples are then dehydrated and generally embedded in a plastic resin. When preserving samples, the preparation methods should first and foremost minimize artefacts, particularly delocalisation of the molecule of interest at the level of resolution of the imaging technique, which is in a nanometric range for analytical methods using electron microscopy.

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 Hydrated samples are not compatible with the high vacuum conditions of the analysis chamber.

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1. PRINCIPLES OF THE METHODS 1.1. Energy Filtered Transmission Electron Microscopy (EFTEM)  For more details see Reference 7.  In a transmission electron microscope (TEM) when incident electrons from the electron beam go into a sample, they interact with the constituent atoms. These interactions depend on the atomic structure of the sample. Some of these electrons are scattered. When inelastic collisions with the electron shell of the sample’s atoms occur, the primary electrons undergo a deviation of their trajectories and a loss of energy, which is specific for every element present in the sample. This phenomenon is the basic principle of electron energy-loss spectroscopy (EELS). E = Energy of incident electrons E = Energy lost during inelastic scattering Figure 20.1 Electron scattering by a single Chemical information can also be atom. determined by other analytical techniques, which are secondary events of inelastic  Such as auger or x-ray emissions. collisions of electron beam and sample atoms.  Characterization of energy losses of scattered electrons is obtained by using different kinds of dispersive energy filters placed either in or below the column of the microscope. S = Sample P = Alpha filter in the column M = Electrostatic mirror E = Energy of incident electrons E = Energy lost during inelastic scattering Figure 20.2 Example of an in column filter (Alpha filter) in an energy filtered transmission electron microscope (EFTEM).

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1.2. Secondary Ion Mass Spectroscopy (SIMS)  When a solid sample is bombarded by primary ions of a few keV in energy, molecular fragmentation occurs and particles are emitted from the surface. The portion of sputtered material that leaves the sample surface as ions (secondary ions) is accelerated into a mass spectrometer where it is separated by its mass-to-charge ratio, and detected. Secondary ion emission supplies information about either the elemental and isotopic composition of the surface uppermost atomic layers by dynamic SIMS5 or the molecular composition by static SIMS (TOF-SIMS).2 SIMS is the most sensitive surface analysis technique and can be applied to any type of material that can be maintained under vacuum. PI = Primary ion beam S = Sample SI = Sputtered secondary ions Figure 20.3. Primary ion bombardment produces a collision cascade and sputter elements from the sample surface.  Elemental or isotopic distribution imaging can be obtained by rastering the primary ion probe upon the surface.17 D = Detectors ES = Electrostatic sector M = Magnet MS = Mass spectrometer PI = Primary ions IS = Primary ions source S = Sample SI = Secondary ions 1, 2, 3, 4, 5 = Images of the chemical element distribution observed from the data measured on detectors d1, d2, d3, d4 and d5. Figure 20.4 SIMS-instrument (NanoSIMS) scheme.

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2. SUMMARY OF THE DIFFERENT STEPS Because living samples cannot be observed directly by EFTEM or SIMS, preparation steps are essential and specific strategies must be adapted to each problem and each kind of sample.

2.1. Chemical Procedure

1. Chemical fixation

 Numerous procedures are described in all classical laboratory manuals. For more details, see Reference 10.  Glutaraldehyde or a mixture of glutaraldehyde / formaldehyde.

2. Dehydration by solvent

 Water is usually removed by passing the specimen through solutions of ascending concentrations of organic solvents (ethanol, acetone): 50 to 100%.

3. Resin infiltration

 In epoxy resin by passing the specimen through solutions of ascending concentrations of resin mixture without accelerator (50%, 75% and pure resin twice) followed by resin mixture with accelerator.

4. Polymerization by heat at 65°C

 At 65°C in resin mixture with accelerator 12 to 24 hours depending on the resin.

5. Sectioning at room temperature and A = Embedded specimen collecting sections by floating B = Diamond knife C = Sections D = Water or glycerol boat E = Section on a glass slide for light microscopy F = Section on a grid for EFTEM analysis G = Section on stainless steel or silicon holder for SIMS analysis Figure 20.5 Sectioning by floating on fluid. 6. EFTEM and/or SIMS analysis

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 Sections 40 to 50 nm thick for EFTEM, adjacent 200 nm thick sections for SIMS.

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2.2. Cryo-Procedures 1. Cryo-fixation Cryo-fixation of specimens can be carried out in a number of ways, but we have chosen to use either slamming or highpressure freezing:  Slamming (see Chapter 2).

 Quick freezing is the best way for rapid immobilisation of any biological material. Depending on the experimental conditions, the freezing process can lead to formation of different ice states going from vitreous to hexagonal ice. Hexagonal crystals alter drastically cellular ultrastructure by solute segregation and could redistribute erratically some intracellular elements. A = Injector rod with pneumatic damping system B = Specimen holder: metal plate, foam rubber and double-sided adhesive tape C = Moist sample fixed downward onto the adhesive tape D = LN2-cooled, gold-coated copper mirror Figure 20.6 Scheme of specimen slamming onto cooled metal mirror.

 High-pressure freezing (see Chapter 6).

A = Flat specimen holder (perforated steel envelope) B = Flat specimen carrier (gold-coated copper) C = Direct liquid nitrogen jets D = Thin tube transmitting pressure P (about 2000 bar) to specimen holder

Figure 20.7 Schematic drawing of the high-pressure principle according to Studer.

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2. Dehydration of cryo-fixed samples  Freeze-substitution12,13 (see Chapter 13).

 Dry acetone with or without osmic acid at 90°C for about three days; ice crystal formation can occur at 90°C without apparent damage to the sample at the nanometre scale.

 Freeze-drying6 (see Chapter 15).

 Freeze-drying process starts around 100°C.

3. Embedding in a resin

 Fluid epoxy resin or Lowicryl® HM23.

4. Polymerization at 65°C or by UV  The aim of embedding is to fill the light at 70°C "water space" in the biological specimen by fluid resin that, after polymerization, will harden the sample. The resin acts also as a homogenous matrix for the analytical beam. 5. Sectioning as in Section 4.6.1. or dry sectioning

A = Embedded specimen B = Diamond knife C = Sections E = Ionizer (to neutralize electrostatic charges on the dry section by an ion emission) F = Section on a glass slide for light microscopy G = Section on a grid for EFTEM analysis H = Section on stainless steel or silicon holder for SIMS analysis Figure 20.8 Dry sectioning. 6. EFTEM and/or SIMS analysis

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3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Refridegerator  Magnetic stirrer  Oven 65°C  Horizontal self pressurized LN2 container with a roller base  A little LN2 container with handle  Slam-freezing device

 High-pressure freezing device

 Freeze-substitution device

 Freeze-drying device  Ultramicrotome  Diamond knifes

 Diamond knife with a boat  knife angle: 35°

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 Storage of fixatives and embedding products used in chemical procedure.  To mix chemicals and resin mixtures.  To dry flasks, moulds and for heat polymerization of epoxy resins.  50 litres: intermediate LN2 storage inside laboratory.  4 to 5 litres to gently fill the freeze devices.  Commercially available. For example:  EM MM80E, or EM CPC, Leica Microsystems, Vienna, Austria.  RMC MF-7000, Boeckeler Instruments Inc. Tucson, Arizona,USA.  Commercially available. For example:  HPM 010, BAL-TEC product. Now available from Boeckeler.   A new modified apparatus HPM 100 is available from Leica  EM PACT or more recently  EM PACT 2 + RTS device, Leica  HPF Compact 01, Engineering Office M. Wohlwend GmbH, Sennwald, Switzerland.  Commercially available. For example:  EM AFS or more recently  EM AFS2, Leica  RMC FS-7500  Commercially available. For example:  EM CFD, Leica (upon request)  MED/020 GBF, Leica.  Commercially available. For example:  EM UC6, Leica  Power Tome X and XL, RMC.  Diamond knives are available from various manufacturers (e.g., Diatome AG, Bienne, Switzerland or Element Six, Cuijk, The Netherlands).  Sectioning by floating on fluid.  Biological tissues, heterogeneous (in term of hardness) blocks (i.g., non decalcified spongy bone).

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 knife angle: 45°

 Recommended for uniformly hard samples.

 Cryoimmuno diamond knife  knife angle: 35°

 Dry sectioning thick sections up to 200 nm.

 Ionizer

 To neutralize electrostatic charges on the dry section to facilitate manipulations.  Commercially available. For example, Static Line II by Diatome Ltd.

 Grid storage box with 50 to 100 alphanumeric labelled compartments  Uncoated hexagonal nickel or gold  600 mesh to support sections in EFTEM specimen grids analysis.  250 mesh to support sections in SIMS analysis.  Carbon-coated, square specimen grids with alphabetic identification  Small items:  Sapphire disks  1.4 mm diameter, 50 µm thick. Commercially available: Leica Product.  Thermanox® film  Nalge NUNC Products, Rochester, NY, USA.  Scalpels, scissors, fine forceps, biopsy  To dissect and prepare samples. needles  Glass vials, flasks and beakers  25 mL and 60 mL for storage and mixing the different resins kept in a dry oven (60°C).  Magnet bars  Used to mix resin solutions.  Disposable plastic Pasteur pipettes  3 and 1.5 mL.  Watch-glass  Used to transfer samples under resin if necessary.  Embedding flat moulds  Embedding gelatine capsules  In any microscopy product catalogue, e.g., Pelco International, Clovis, California, USA.  Supports with holes  Used to maintain gelatine capsules vertically during resin polymerization.  Small section pickup loop  Used to pickup sections obtained by flotation.  Gloves: latex and nitrile  Important: Some components for embedding resins can be very toxic.

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3.2. Products  Cacodylic acid sodium salt trihydrate  50% Glutaraldehyde  Formaldehyde in powder  2% OsO4 in water  Acetone GR dry  Calcium chloride in powder  Spurr’s epoxy resin mixture reported by Spurr:18  ERL® 4206 or VCD (vinyl cyclohexene dioxyde): diepoxide

  

 

 Toxic by inhalation and if swallowed.  Toxic by inhalation. Use under a fume hood.  Toxic by inhalation. Use under a fume hood.  Extremely toxic for eyes, skin, lung, kidneys at very low concentrations. Use under a fume hood.  Maximum 0.01% water.  Available from various manufacturers (e.g., Fluka Chemie GmbH, Switzerland).

as  Viscosity of the complete mixture: about 60 cP at 25°C, polymerization at 65°C.  Harmful if swallowed, eye and skin irritant, may cause allergic skin reaction, causes cancer in laboratory animals.  Production of ERL®4206 is discontinued and is actually replaced by ERL®4221. ® DER 736 (Diglycidyl ether of  Eye and skin irritant. polypropyleneglycol): softener NSA (Nonenyl succinic anhydride):  Harmful if swallowed, eye and skin hardener irritant. DMAE (Dimethylaminoethanol):  Eye, skin and mucous membrane accelerator irritant, may injure the central nervous system.  Use and prepare Spurr’s mixture under a fume hood. Lowicryl® HM23 hydrophobic, non-  See Reference 1. polar resin  Low viscosity at –80°C. Monomer G: a mixture of Ethyl  Methacrylates may cause eczema and methacrylate and n-Butyl metha- allergic reactions: wear nitrile and latex crylate gloves.

 Cross-linker: 1,3 Butanediol dimethacrylate  Photo-initiator J  LN2: Cryogen

© 2009 by Taylor & Francis Group, LLC

 BDA: Benzyl dimethyl acetal.  For polymerization at –80°C or –70°C by UV radiation (360 nm).  May cause severe burns; wear protective clothes, glasses and gloves for handling LN2. Always use LN2 in wellventilated areas.

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3.3. Solutions  Phosphate buffer 0.15 M according to Sörensen (see Reference 10)  0.2 M Cacodylate buffer  1% Glutaraldehyde in 0.15 M phosphate buffer  1.6% Formaldehyde, 3% glutaraldehyde, 0.05% CaCl2 in 0.1 M cacodylate buffer  1% OsO4 in buffer or small crystals  Increasing concentrations of ethanol from 50% to 100%  Spurr’s mixture, manufacturer’s standard medium:  VCD: 10.0 g  DER 736: 6.0 g  NSA: 26.0 g  DMAE: 0.4 g  Lowicryl mixture (HM23) for infiltration and UV polymerization below 70°C:  Cross-linker: 1.10 g  Monomer G: 18.90 g  Initiator J: 0.15 g  Lowicryl now also exists as premixed ready to use solutions.

 Di Na/mono K, pH 7.4  pH 7.4  For chemical fixation of cultured cells.  For chemical fixation of samples.  When post fixation is required.  Chemical procedure.

 Polymerization at 65°C for 8 hours minimum.  UV polymerization at –70°C: at least two days, if necessary complete by UV polymerization for one day to three days at 50°C.

4. PROTOCOLS 4.1. Sampling Because in vivo and in vitro observations by EFTEM and SIMS are so far impossible, the first step in any analysis consists in collecting samples. This step is crucial and should be performed while minimizing cellular stress  Cellular stress induces drastic changes that could modify molecular distributions. in free ion distribution. Biological materials come from:  Cell or microorganism cultures that are  Cells cultured on organic support homogeneous and simple models and that films (Thermanox™ type) or on mineral come in adherent or suspended form. disks minimize stressing steps.  Biopsies (plant or animal).

© 2009 by Taylor & Francis Group, LLC

 Reflect more tissue heterogeneity.  Useful for pharmacological or trace element studies.

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4.2. Chemical Procedure 1. Fixation 1.1. Cultured cells on support  1% Glutaraldehyde in 0.15 M phosphate  First replace culture medium by warm buffer, pH 7.4 fixative mixture (35°C) for 5 min, then change for cold (4°C) fixative mixture for 20 min.  Wash buffer 3 × 10 min at 4°C  Postfixation: 30 min 1% OsO4 in buffer at RT  Wash buffer 3 × 10 min at RT 1.2. Biopsy samples  1.6% Formaldehyde, 3% glutaraldehyde in 0.1 M Cacodylate buffer, 0.05 % CaCl2, pH 7.4  Wash buffer 0.1 M Cacodylate buffer, 0.2 M Sucrose, 0.03 M CaCl2, pH 7.4  Postfixation: 1% OsO4 in 0.1 M Cacodylate buffer  Wash buffer

4-5 h at 4°C

3 × 10 min at 4°C 45 min at RT 3 × 10 min at RT

2. Dehydration  Ascending concentrations of organic  Usually ethanol or acetone. solvents:  30%, 50%, 70%, 95% 510 min/each at 4°C  100% twice 10 min  For cultured cells dehydration time RT can be reduced. 3. Resin infiltration  Ethanol + Spurr’s resin: 1/1  Ethanol + Spurr’s resin : 1/3  Spurr’s 100% 4. Polymerization

© 2009 by Taylor & Francis Group, LLC

60 min at RT 60 min at RT 112 h (2 baths)  For cultured cells infiltration time can at RT be reduced. 10 h at 65°C

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5. Sectioning  EFTEM: Usually section thickness varies between 40 to 50 nm.  SIMS: Typical sections are 100 to 500 nm-thick.

 For more details see Section 4.6.  The thinner the sections, the easier the analysis.  Below 100 nm, the sections are too thin to resist ion bombardment for a long period of analysis, while over 500 nm the electrostatic charge of the sample could perturb the analysis.

4.3. Cryo-Fixation 4.3.1. Slamming Cryo-fixation using EM MM80 E, Leica.

 For more details, see Chapter 2. 

 For cultured cells on Thermanox™ type film or sapphire disks or thin biopsies.  Parameter settings:  Force: 11  Speed: 11  Thickness: 4  Depending on specific studies, cell stimulation can be carried out at this step.  Attach the fresh sample onto the  See Figure 20.9 One to three samples specimen holder and impact it onto the can be attached onto the holder and cryoprecooled metal block. fixed at the same time. Just before fixation excess culture medium should be gently removed with a piece of filter paper.  After freezing, the specimens are stored in LN2 or directly transferred in LN2 to the freeze drying device. A = Cultured cells:  Scheme of process.  On Thermanox® type film (3 × 3 mm)  On sapphire discs (diameter 1.4 mm, 50 µm thick) or small biopsies (1mm3) coated with 1 µl culture medium, 20% foetal calf serum: (omit if stimulation will be done later). B = Stimulation if necessary (if studies of dynamic processes) C = Gently remove excess medium with filter paper D = Sample fixed downward onto the adhesive tape E = Slamming onto the LN2 cooled metal mirror Figure 20.9 Scheme of the slamming process.

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4.3.2. High-pressure freezing High-pressure freezing using the EM PACT apparatus from Leica with specimen carriers for:  1. Cultured cells on sapphire disks 2. Thin and regular samples

 For more details, see Chapter 6.  Note: In this part, terms describing small tools come from the EM PACT’s Leica catalogue.   1.4 mm diameter, 50 µm thick for sapphire.  For example, one-day-old rat calvarium or plant leaf cut with a biopsy needle (diameter 1 mm).

A = View from the top of an empty carrier B = Sectional drawing of a carrier with a sample (cells cultured on a sapphire disk) in culture medium. Figure 20.10 Scheme of a flat specimen carrier for sapphire disks.  Adherent cells are cultured on the  To avoid handling the disk before sapphire disk. If necessary, the cavity fixation, cell cultures are set up directly of the specimen carrier is filled with in the specimen carrier. appropriate culture medium.  Samples are punched with the biopsy  If cell stimulation is needed proceed at needle and pushed directly in the this step. cavity of the specimen carrier prefilled with 0.6 µL of culture medium.  Once the flat specimen carrier is filled with sample and medium, it is sandwiched in the flat specimen holder using a torque wrench.

 The outer ring of the specimen carrier should be dry; if necessary, gently remove medium with filter paper.  Applying a force of 20 N/cm2.  Total time for sampling and filling the carrier should be minimized and be below 30 seconds.

 The specimen holder is mounted on the rod and transferred in the EM PACT and freezing is automatically carried out.

 Two persons are needed for this operation, one to prepare the specimen carrier (filling, specimen stimulation, positioning, etc.) and the other to fix it on the rod and freeze it.

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 The HPF device operates up to  It takes about 15 ms to freeze down from 2000 bars with a cooling rate of about 30°C to 190°C with a pressure near 7000 to 10000°C/s. 2000 bars.  After freezing, the specimen carriers are separated from the holder by unscrewing under LN2 with the torque wrench and left in LN2 or directly transferred in LN2 to the freezesubstitution device.

 After use some of the flat specimen carriers are deformed. Samples from damaged carriers should be rejected to ensure the quality of freezing.

4.4. Dehydration of Cryo-Fixed Samples 4.4.1. Freeze-substitution



 Frozen samples are transferred in  For handling of the AFS, see Chapter 18. liquid nitrogen into a freeze-  AFS2 now in Leica catalogue. substitution device (AFS, Leica).  Ice is removed by substitution using  Commercial dry acetone (maximum pure dry acetone (90°C, three days). 0.01% water). Avoid molecular sieves that can carry impurities altering the analyses.

4.4.2. Freeze-drying  Freeze-drying in the cryo-sorption  For details, see Chapter 15. freeze-drying (CFD) apparatus accor-   ding to Sitte.16 A = Chamber filled with cold LN2 gas  B = Object table heating (regulation of temperature against LN2 temperature) C = Samples D = Cold trap wings E = Translucent glass F = Connection for pumping with valve V1 = Valve for communication of molecular sieve with chamber A V2 = Valve connection with cold LN2 gas (breaking vacuum) MS = Molecular sieve (zeolite)

Figure 20.11 Schematic cross-section of freeze-drying chamber.  

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 Before freeze-drying, the molecular sieve should be regenerated by heating.  Fill the FD device with 30 L LN2.  Precool the chamber to 150°C.  Transfer cryo-fixed samples in the precooled object table (190°C).  Place the cold trap wings over the samples.  Primary vacuum is obtained in the chamber of the CFD by a membrane pump.  Secondary vacuum is obtained by cryosorption pumping (zeolite).  Freeze-drying process is achieved by increasing temperature slowly up to 0°C.  Dehydration is then stopped by breaking vacuum with LN2 gas entry.

 At last 12 hours at 90°C, valves F and V1 open, V2 closed and glass E in position.  Allow 40 L for filling the machine.  From freeze-slamming or high-pressure freezing experiment.

 18 mbars.  2 × 10-3 mbars.  To better preserve molecular structure and biological functions, freeze-drying can be stopped earlier (60°C or 10°C).

4.5. Resin Embedding 4.5.1. Low viscosity epoxy resin mixture We use the Spurr formulation18 (see Section 3.2.) with a low viscosity, which facilitates infiltration of densely packed tissues.  Freeze-dried samples  When the dehydration process is stopped (at 10°C or 0°C) (see Section 4.4.2) chamber A (see Figure 20.11) can be opened.  Pure Spurr’s resin is dropped on samples and infiltration is carried out with a weak vacuum (about 500 mbar) while the temperature is increased to 20°C (1°C/hour).  Resin infiltration is then completed at room temperature in small dry flasks outside the CFD apparatus.  Place infiltrated samples in flat moulds or in gelatine capsules.  Process polymerization in an oven.

© 2009 by Taylor & Francis Group, LLC

 The viscosity of the resin mixture is lower than 60 cP.

 Freeze-drying process atmospheric pressure.

stopped

 Infiltration time depends on sample.  Embedding medium is renewed.  Previously dried in an oven at 65°C.  At 65°C for 10 hours.

at

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 Freeze-substituted samples  After freeze-substitution, resin infiltration is carried out in ascending  50, 75, 100% ERL. concentrations of ERL in acetone for about 24 hours from –40°C to 10°C.  Infiltration is completed in Spurr’s  Infiltration time depends on sample. mixture for one or two days at 20°C.  Process polymerization in an oven.  At 65°C for 10 hours.

4.5.2. Lowicryl HM® Lowicryl HM resins are nonpolar and hydrophobic. They are methacrylate-based  Lowicryl HM20 (can be used at 45°C). and remain fluid at low temperature.1  Lowicryl HM23 (can be used at 80°C).  Polymerization by UV light takes place at  Polymerization with Lowicryl HM20 can low temperatures. be carried out at 45°C.  Polymerization with Lowicryl HM23 can We use a Lowicryl HM23 mixture: be carried out at 80°C.  Freeze-dried samples  When freeze-drying is achieved (see Section 4.4.2), infiltration of resin is carried out at 60°C by adding a few drops of resin mixture onto the samples in the LN2 cold gas-vented CFD chamber.   Polymerise at 60°C, 48 hours.  UV polymerization.  If necessary, complete the polymerization for 24 to 72 hours at 50°C.  Freeze-substituted samples  When freeze-substitution is achieved (see Section 4.4.1), infiltration of resin is performed:  50% resin in acetone at 90°C, three hours  100% resin twice 30 hours at 80°C.  Polymerize at 70°C, 48 hours.  Using UV.  If necessary, complete the polymer-  If resin blocks are too soft or if a liquid ization for 24 to 72 hours at 50°C. film remains at the surface.

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4.6. Sectioning Sections are cut with an ultramicrotome at  low speed (1 mm/sec) or manually using a  diamond knife (angle: 35° or 45°).   4.6.1. Sectioning on fluid Usually, sections are collected on a water  boat. Glycerol may be used to minimize element losses (see Section 2.1. and Figure 20.5 for general procedure).  For EFTEM  EFTEM analysis requires ultrathin slices (thinner than 100 nm) to avoid multiple electron scattering through the section that alters the transmitted signal.  Sections are collected on fluid and recovered on hexagonal 400 to 600 mesh nickel or gold grids  Sections floating on water are picked up quickly. Only one or two sections are transferred on one grid without any coating film.  If sections are collected on anhydrous glycerol, work in a dry room.  Grids are stored in a grid box until observation.  For SIMS  Section thickness can vary from 100 nm to 500 nm and sections are made using a histological diamond knife (angle: 45°).

  Ultrathin sections: 40 to 50 nm thick.

 Dry carefully the grids on a filter paper to remove fluid before storing.  Only one or two sections can be cut and picked up in a short time (about 10 seconds).  To reduce glycerol hydration.  Hygrometry < 50%. 

 Less than 100 nm sections are too thin to resist ion bombardment for a long period of analysis, while in the case of over 500 nm sections electrostatic charges of the sample could perturb the analysis.  See Figure 20.5 (G).  Sections are floated on water and put  Standard stainless steel or silicon holders on a warm holder with a small section for SIMS analysis have a 10 mm diameter. pickup loop.  If TEM imaging is performed on the same section for ultrastructural  Section thickness can be reduced to 80 to identification, then sections can be 100 nm. directly put on 250 mesh carboncoated grids.  Sections are dried for one hour on a hot plate and then stored in a vacuum  To keep the sections dry. oven at 50°C or directly in a storage chamber of the SIMS apparatus for impending observations.

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4.6.2. Dry sectioning  Dry sectioning is recommended for examination of highly diffusible elements and it is performed manually at room temperature using a 45° immuno cryo-diamond knife.  Dry sections for EFTEM are thicker than sections obtained on fluid (up to 60 nm).  Static Line II (ionizer) should be operated during the entire sectioning procedure.  Sections are grabbed with fine tweezers and put on prewarmed holders and stored at 50°C.

 See Section 2.2. and Figure 20.8.

 Ion emission neutralizes electrostatic charges on dry sections and avoids folding until sections are put on the holder. 

4.7. Elemental Analysis 4.7.1. EFTEM  After locking a grid with sample sections in the column of the electron microscope, element specific signals are identified by energy loss of the transmitted electrons that interact with the sample.  Data can be acquired on:  Spectrum mode  EELS: (electron energy loss spectroscopy). The energy loss of the imaged electrons on one spot (10 nm diameter) is scanned in a large window (100 eV) and the electron intensity is recorded and processed.  PEELS: (parallel electron energy loss spectroscopy). Beyond the spectrometer, the electron beam is dispersed according to the energy of the electrons. The spectrum is visible on the image plane as a line with varying brightness and can be imaged via the camera.

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 Element specific signals (ionization edge) are superimposed on a background that has to be removed from data to obtain the net distribution of each element of interest.

 Acquisition is relatively time consuming for just one spectrum (about two minutes) and damages the sample locally. 

 Intensity profile of the image is recorded and processed to identify elements present locally in the sample.  The time required for acquisition of the spectrum is much faster (<1 sec).

Cryo-Preparation Procedures for Elemental Imaging by SIMS and EFTEM

 Image mode  ESI: (electron spectroscopic imaging) A short series of images is acquired around a specific energy edge of the element of interest.

 Image EELS: (combination of spectrum and images) A series of images (up to 100) obtained at different energy-loss values (typically with an energy increment of 1 eV) are processed to either spectra or element distribution images.

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 Image signal contains specific element information buried in unspecific background. Therefore, the signal of each pixel has to be processed to extract the useful information and to map the distribution of the specific element.  Acquisition of images and processing for each element takes typically less than one minute.

 A complete spectrum is associated to each pixel. Therefore, after processing, the chemical distribution of various elements can be easily mapped.

4.7.2. SIMS  Sample sections are placed and  A good vacuum is essential for efficient maintained under vacuum for several transmission of secondary ion beams. hours in the SIMS apparatus.  Implantation of the sample surface  In dynamic SIMS, implantation is with primary ion beam. required so that secondary ion current reaches a steady state.  Setting the microprobe and detectors for analysis.  The probe is rastered over the area of interest for analysis. Emitted secondary ion beam goes then through the mass spectrometer where it is analysed.  Data processing.

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 In dynamic SIMS each secondary ion species is detected within specifically tuned detectors.  Typical acquisition time varies from 5 to 30 minutes.   Mapping of selected chemical species and correlation with light or TEM images for ultrastructural characterisation.

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5. ADVANTAGES/DISADVANTAGES 5.1. Advantages 5.1.1. Chemical procedure  A well-established method various types of samples.  Quick and easy to do.

with

 Quality of fixation is reproducible.

 A lot of references.  Specimen preparation takes about two days.  All specimens in an experiment are identical in quality.

5.1.2. Cryo-fixation  All biological processes and biochemical reactions are quickly stopped  (in a few milliseconds). 1.   

Cryo-fixation by slamming No specific sampling is required. Can be used with every kind of biopsy. Method is quick and easy to perform.

 Time between sampling and freezing  Typically less than 10 seconds with can be very short. cultured cells.  Quality of fixation is quite satisfactory.  Usually, according to the resolution of the analytical method, a thickness up to 10 µm is usable for EFTEM studies and up to 20 µm for SIMS analyses (see. References 15 and 16). 2. Cryo-fixation by high-pressure freezing  HP-freezing is very efficient  See Reference 19. (temperature and pressure are well controlled).  With specimen carriers (see Figure 20.10), high quality freezing can be obtained on thicker samples than with slamming.  Up to 200 µm.  The copper tube holder is very suitable for suspensions of cells, bacteria, virus, etc.  New developments in the HPF device  The new EMPACT2 + Rapid Transfer simplify the sampling procedures System (RTS) in the Leica apparatus reduce (shorter and easier to do). the interval to about three seconds between stimulation and the freezing of the material.

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5.1.3. Dehydration Cryo-dehydration procedures tend to  minimize or to suppress element losses and/or delocalization during ice removal from cryofixed samples. 1. Dehydration by freeze-substitution  Procedure is very easy.  Apparatuses are commercially available and widely used.  Requires minimum handling of  Especially with the new AFS2 (Leica) samples during freeze-substitution. using the FSP (Freeze-Substitution Processor).  Some commercial devices (such as the EM AFS) include extraction of toxic fumes directly from the cryosubstitution chamber.  Safer to use. 2. Dehydration by freeze-drying  The best way to minimize loss or  See Reference 6. redistribution of elements (better than cryo-substitution).  No handling of samples during cryosorption.

5.1.4. Resin embedding Embedded specimens can be stored for unlimited periods of time. Sections can be handled and observed at room temperature. 1. Embedding in Spurr’s resin  Good resistance to analytical beams (electrons or ions).  When samples are freeze-dried prior to embedding in pure resin, there is very little loss or delocalization of labile ions or molecules.  Easy sectioning of Spurr’s embedded samples by floating on fluid. 2. Embedding in Lowicryl HM 23 resin  Mixture fluid down to –80°C  Polymerization at –80°C  Under UV light.  Enhanced preservation of protein and membrane structures.

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5.1.5. Sectioning 1. Sectioning on fluid The cutting process generates section compression and subsequent charging phenomena. Floating sections on a fluid allows surface tensions to stretch the sections and restore them to initial dimensions.  Easy to perform  Ultrathin and regular sections (40 to 50 nm thick) can be obtained.  Glycerol (anhydrous solvent) can reduce element loss during sectioning (but it should remain dry).

 Ultrathin and regular sections are required for ETEM analysis.  Work in dry atmosphere (hygrometry lower than 50%) with fresh pure glycerol.

2. Dry sectioning  Avoids element losses during  When very soluble elements are sectioning. analysed.  Dry sectioning is easier with Lowicryl  Ionizer should be used. HM23 resin than with Spurr’s resin.

5.1.6. Elemental analysis 1. EFTEM  Particularly well adapted for light  Atomic number (Z): 3 to 30 element analysis.  However, some elements with Z > 30 can be analysed.  Most significant and detectable elements in biological samples are N, O, Mg, P, S, Ca and Fe, etc.  Some exogenous elements can be  Applications in trace element and detected (Al, Ni, In, Pt, etc.). pharmacological studies (see Reference 11).  High local resolution of element distri-  In biological samples, elements can be bution localized with a resolution of about 5 nm.  High sensitivity

 With respect to x-ray microanalysis.  Adapted for elements concentrated in very small volumes.  Easy correlation between ultrastruc-  An ultrastructural image is acquired in tural information and chemical the same area as the analytical data. mapping  Possible correlation with other  Particularly with methods using the same analytical methods type of fixed sample.

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2. SIMS  High sensitivity in elemental analysis.  Most significant and detectable elements in biological samples are CN, O, P, S, Br, I, Pt, Ca, Fe, etc.

 Slightly less in molecular analysis with static SIMS (TOF-SIMS).  CN, P, S are useful for structural characterisation.  I, Br, Pt can be used in molecules as tracers for pharmacological studies.  Trace element analysis.  Particularly for elements with high electron affinity (Pt, Se, Ni, Ag, etc.).  The only technique that can  Isotope analysis makes it possible to differentiate between isotopes of the study intracellular and/or intercellular same chemical element. metabolic pathways by use of stable isotope-labelled tracer molecules.  Subcellular resolution.  Particularly in dynamic SIMS.

 Possible methods.

correlation

with

other  Particularly useful, for example, with TEM to correlate chemical mapping with subcellular structures.

5.2. Disadvantages 5.2.1. Chemical procedure  Fixation occurs progressively from  Loss of membrane functions precede outer cell to inner cell. inner fixation of cells; free water and ion displacements occur across the cell membrane.  Fixation and dehydration solutions  Small molecules and free ions are facilitate diffusion and/or precipi- subjected to diffusion. tation of cellular material.  Severe extraction constituents.

cell  For instance, solvents used for dehydration and resin infiltration extract lipids.  Deformation of surface membranes and organelle volume.14 of

some

5.2.2. Cryo-fixation  Cryo-fixation is restricted to small  samples.  Cryoprotectant agents that improve the quality of freezing cannot be used for analytical purposes.  Parameters to control high-quality freezing are not easily determined and conditions have to be defined and adapted for each new sample.

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1. Cryo-fixation by slamming  For analytical purposes, slamming is  HPF is better for this kind of sample. less adapted for fixing biological suspensions than for biopsies and adherent cell cultures. 2. Cryo-fixation by high-pressure freezing  Specific care is required concerning sampling.  Sampling of some tissues is very difficult.  Some tissues cannot be cryofixed by HPF.  Due to the tiny size of the holder, sample handling is not easy and takes at least 10 to 15 seconds to place the sample and fill properly the carrier prior to freezing.

 For example, muscular fibre or plant root.  Lung tissue, for instance.  Inner diameter of carriers for EM PACT is only 1.5 mm.

5.2.3. Dehydration 1. Dehydration by freeze-substitution  Samples are in contact with organic  Risk of loss or at least redistribution of solvents for a long period of time up to soluble elements. the embedding step. 2. Dehydration by freeze-drying  Very time-consuming process.

 More than 8 days are needed for dehydration because a slow rate of increase in the temperature is required (typically 0.4°C/hour).  The cryosorption chamber is not really  Osmium is useful for ultrastructural adapted for treating samples with observations by EFTEM. osmium vapours.  Some samples are difficult to  Particularly for plant tissues. dehydrate properly. 5.2.4. Resin embedding 1. Embedding in Spurr’s resin  Only fluid at room temperature.

 Viscous below 0°C.

 Polymerised by heat.

 65°C.

2. Embedding in Lowicryl HM23 resin  Poor resistance to analytical beams  For analytical purposes, sections cannot (electrons or ions). be protected by support film.

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5.2.5. Sectioning 1. Sectioning on fluid  Soluble elements from sample can be removed or displaced during section floating.  Glycerol modifies the resin.  Glycerol is a hydrophilic absorbent for atmospheric water.

 Sections have to be quickly picked up (10 seconds maximum) to minimize element delocalization.  Temporary softening of the resin  Sectioning should be carried out in a room where hygrometry is lower than 50%.

 Potential contamination of the sample sections by glycerol. 2. Dry sectioning  Dry sectioning of epoxy resin blocks  Almost impossible to obtain 50 to 70 is tricky. nm-thick dry sections for EFTEM.  Typical dry sections are thicker than 100 nm.  Laying down dry sections flat on a  Thickness variation alters EFTEM TEM grid is very difficult. analytical data. It is easier to put dry sections flat on a prewarmed SIMS holder. 5.2.6. Analysis 1. EFTEM  Complicated method for mapping a limited number of elements in each experiment.  Analysis is restricted to a limited area.  No molecular information.  Data are related to the global concentration of an element.  The evaporation process can occur under the electron beam for some elements.  Although the carbon signal is intense, its biological usefulness is limited.  K mapping is very difficult to obtain.

Analysis is limited to a region of a cell.  Free and bound elements cannot be distinguished.  Na, K, Cl.  Carbon is extensively present in embedding medium.  K signal is masked by the strong carbon signal.

2. SIMS  Only fixed samples can be analyzed.  In dynamic SIMS, organic molecules can only be visualized through specific exogenous atoms or isotopes labelling.  The data provide the global con-  Free and bound elements cannot be centration of an element. distinguished.  Identification of subcellular structures is difficult and usually requires correlation of TEM imaging.

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6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Chemical Procedure  Why:  A lot of well-established procedures for various kinds of samples. 

 For more than 50 years, a number of articles and books have been written concerning problems in processing specimens from both animal and plant domains.   When:  For example, to analyse components  For structural TEM, EFTEM and strongly bound to cellular structures and/or SIMS analysis when other methods that cannot be easily removed by solvents are not required or failed. (insoluble crystals, clusters such as ferritin,  etc.).  

6.2. Cryo-Fixation 6.2.1. Cryo-fixation by slamming   Why:  Quick and easy procedure that can be used with biopsies of almost any type of biological tissue.  When:  Using cultured cells.  Nature of tissue does not allow HPF fixation.  Structure of tissue is not compatible with an easy sampling for HPF system.  Fixation of relatively large specimens is required.  Studying dynamic processes.

 Particularly on Thermanox® type film.  For instance, lung tissue cannot be fixed by the HPF method.  Muscle or lung tissue is difficult to sample for an HPF carrier.  Up to 25 mm2.

6.2.2. Cryo-fixation by high-pressure freezing  Why:   HP freezing is the only method to properly freeze biological samples up to 200 µm in depth.  When:  Organ biopsies, cultured cells on  Tools have been created specifically: sapphire disks or biological biopsy needles, carriers. suspensions.  

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6.3. Dehydration 6.3.1. Dehydration by freeze-substitution   Why:  Easy procedure that can be used with cryofixed biopsies of almost any type of biological tissue.  When:   Analysis is restricted to components strongly bound to cellular structures and/or that cannot be easily removed by solvents.  Dehydration will take too much time  For example, freeze-drying of plant by freeze-drying. tissues can require more than three weeks.   6.3.2. Dehydration by freeze-drying  Why:   To avoid use of any solvent prior to analysis.  When:   Anytime molecular redistribution can  potentially occur with use of solvent.  To minimize chemical localisation artefacts.  

6.4. Resin Embedding 6.4.1. Embedding in Spurr’s resin  Why:  Low viscosity (about 60 cP) at room  Infiltration of sample is possible without using any solvent. temperature.  Ultrathin sectioning is very easy by floating sections on water.  Particularly useful for EFTEM analysis.  Resin withstands electron or ion bombardment.

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  When:  Samples of even thin thicknesses are  EFTEM analysis requires ultrathin sections: 40 to 50 nm-thick. required.   A good resistance of section to  electron bombardment is required for  For example, samples are subjected to severe irradiation during EELS analysis. specific analysis.   6.4.2. Embedding in Lowicryl HM23  Why:  Embedding is temperatures.

achieved

at

low  Down to 80°C. 

 When:   Antigenic properties of samples have to be preserved for ultrastructural characterisation by immunolabelling to complete analytical studies.  Dry sectioning is needed.  It is easier to perform dry-sectioning on Lowicryl than on Spurr’s resin.  

6.5. Sectioning 6.5.1. Sectioning on fluid  Why:  Thin and regular sections are easily  40 to 50 nm-thick for EFTEM analysis. collected on water.  When:  Standard use.

  However, sections have to be collected quickly to avoid potential molecular redistribution. 

6.5.2. Dry-sectioning  Why:   To avoid contact of samples with any solvent.  When:   Analysing the distribution of labile  components.  

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6.6. Analysis 6.6.1. EFTEM   Why:  Elemental analysis at the subcellular level.  When:   High spatial resolution in elemental  About 2 to 5 nm. analysis is needed.  To map clusters of a few hundred atoms.  6.6.2. SIMS  Why:  It is the only technique that can differentiate between isotopes of the same chemical element.  To analyse trace elements or drug targeting.

   It is possible, for instance, to differentiate in the same mapping 12C and 13 C or 14N and 15N to analyse the cellular distribution of a 15N or 13C labelled molecule.  Highly sensitive analytical technique.     When:   Mapping distribution of specific,  exogenous atoms or isotope labelled  Lateral resolution of about 100 nm. molecules at a cellular level.              

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7. OBSERVED RESULTS  Figure on the Chapter’s title page

Left: Cryo-fixed cultured osteoblasts. Top left: Energy filtered image by EFTEM. Bottom left: Calcium mapping in the same region by ESI. Experimental EELS spectrum for calcium recorded from the same area is superimposed. Right: SIMS imaging of cultured osteoblasts. Top right: Phosphorus mapping. Bottom right: Sulphur mapping.

 Figure 20.12, A-D: EFTEM data (see colour insert following page )  Calcium distribution in quiescent 1. Description cultured osteoblasts (left) and in osteoblasts perfused with 5 µM ionomycin (right).  EFTEM analysis. 2. Method  Cell culture. 3. Tissue  Cryo-fixation by slamming. 4. Fixation  Spurr’s resin. 5. Embedding  EELS and ESI with an energy filtered 6. Visualisation transmission electron microscope.  0.5 µm. 7. Bar 8. Comments  Calcium mapping is in grey spotted (in red, see the colour insert), superimposed on an energy filtered image obtained at 250 eV (inverted contrast without staining).  White arrows: mitochondria with dense or clear matrix, E.R.: endoplasmic reticulum.  A and C: Data for unstimulated cells. Dense mitochondria exhibit a weak calcium signal. In experimental EELS spectrum (purple in C, see colour insert) recorded in circle in A, Ca-L2,3 signal (344 eV) is below the limit of detection.  B and D: Data for cells stimulated by 5 µM ionomycin. Dense mitochondria exhibit a strong calcium signal and endoplasmic reticulum an identifiable weak calcium signal. In experimental EELS spectrum (green in D, see colour insert) recorded in circle in B, Ca-L2,3 signal is clearly visible.  C-K predominant carbon signal in C and D.  For more information, see Reference 4.

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      

       

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 Figure 20.12, E- H: SIMS images (see colour insert following page) 1. Description 2. 3. 4. 5. 6.

Method Tissue Fixation Embedding Visualisation

7. Bar 8. Comments

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 Ultrastructural distribution of an iodinated drug within B16 melanoma cells from a mouse lung colony.  SIMS analysis.  Small sample of lung.  Cryo-fixation by slamming.  Spurr’s resin.  SIMS images (carried out with a CAMECA NanoSIMS 50 microprobe).  Field of view: 25µm.  Iodobenzamide (BZA) was injected intravenously (2.6 µmoles) in tumour bearing mice six hours before sacrifice.  E: Elemental distribution of CN- ions (m = 26). Melanosomes containing the melanin grains rich in carbon and nitrogen appear as bright spots.  F: Elemental distribution of S- ions (m = 32). Part of the melanin biopolymer (pheomelanin) contains sulphur.  G: Elemental distribution of I- ions (m = 127). Iodine is representative of the drug distribution and appears to be specifically located within melanosomes.  H: Merge (CN-: blue, S-: green, I-: red). Co-localisation of the observed ions leads to colour variations. It appears that melanosomes containing mainly eumelanin (arrow “a”) without the sulphur element bind BZA (magenta colour corresponding to merge blue (CN-) and red (I-) while melanosomes richer in pheomelanin (arrow “b”) do not bind BZA and appear in cyan colour (merging of CNin blue and S- in green). Arrow “c” corresponds to white melanosomes (merging of blue, green and red) containing a balanced mixture of pheomelanin and eumelanin (for colour indications, see colour insert). For more information, see Reference 9.

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8. REFERENCES

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

Acetarin, J.D. et al. Developments of new Lowicryl resins for embedding biological specimens at even lower temperatures, J. Microsc., 143, 81, 1986. Benninghoven, A. Surface investigation of solids by the statical method of secondary ion mass spectroscopy (SIMS), Surf. Sci., 35, 427, 1973. Bordat, C. et al. Calcium distribution in high-pressure frozen bone cells by electron energy loss spectroscopy and electron spectroscopic imaging, Histochem. Cell Biol., 109, 167, 1998. Bordat, C. et al. Direct visualization of intracellular calcium in rat osteoblasts by energy-filtering transmission electron microscopy, Histochem. Cell Biol., 121:31, 2004. Castaing R. and Slodzian G., Microanalyse par émission ionique secondaire, J. Microsc., 1, 395, 1962. Edelmann, L. Freeze-dried and resin-embedded biological material is well suited for ultrastructure research, J. Microsc., 207, 1, 5, 2002. Egerton, R.F. Electron Energy Loss Spectroscopy in the Electron Microscope. 2nd ed., Plenum Press, New York,USA, 1989. Guerquin-Kern J.L. et al. Progress in analytical imaging of the cell by dynamic secondary ion mass spectroscopy (SIMS microscopy), BBA, 1724, 228, 2005. Guerquin-Kern, J.L. et al. Ultra-structural cell distribution of the melanoma marker iodobenzamide: Improved potentiality of SIMS imaging in life sciences, Biomed. Eng. Online, 3:10, 2004. Hayat, M.A. Principles and Techniques of Electron Microscopy. Biological Applications. 4th ed., Cambridge University Press, Cambridge, UK, 2000. Jeanguillaume, C. Electron energy loss spectroscopy and biology, Scan. Microsc., 2, 437,1987 Kellenberger, E. The potential of cryo-fixation and freeze substitution: Observations and theoretical considerations, J. Microsc., 161, 2, 183, 1991. Nicolas, G. Advantages of fast-freeze fixation followed by freeze-substitution for the preservation of cell integrity, J. Electr. Microsc. Tech., 18 (4), 395, 1991. Murk, J.L. et al. Influence of aldehyde fixation on the morphology of endosomes and lysosomes: Quantitative analysis and electron tomography. J. Microsc., 212, 1, 81, 2003. Reipert, S. et al. Cryo-fixation of epithelial cells grown on sapphire coverslips by impact freezing, J. Microsc., 209, 2, 76, 2003. Sitte, H. et al. Cryo-fixation without pre-treatment at ambient pressure, in Cryotechniques in Biological Electron Microscopy, R.A. Steinbrecht and K. Zierold, eds., Springer-Verlag, Berlin, Germany, 87, 1987. Slodzian, G. et al. Scanning secondary ion analytical microscopy with parallel detection, Biol. Cell, 74, 43,1992. Spurr, A.R. A low viscosity epoxy-resin embedding medium for electron microscopy, J. Ultrastruct. Res., 26, 31, 1969. Studer, D. et al. A new approach for cryo-fixation by high-pressure freezing, J. Microsc., 203, 3, 285, 2001.

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CONTENTS

GENERAL INTRODUCTION .................................................................................... 541 1.

PRINCIPLES OF THE METHOD .................................................................. 543 1.1. 1.2.

Preparation................................................................................................ 543 Imaging..................................................................................................... 543

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 544

3.

MATERIALS/PRODUCTS/SOLUTIONS ...................................................... 545 3.1. 3.2. 3.3.

4.

PROTOCOLS .................................................................................................... 549 4.1.

4.2. 4.3. 4.4. 5.

Protocols for Materials Needed for On-Section Immunolabelling ........... 549 4.1.1. Poly-L-lysine coating of coverslips ............................................ 549 4.1.2. Mounting solution for fluorescence microscopy......................... 549 Sample Preparation................................................................................... 550 Toluidine Blue O Staining ........................................................................ 551 Protocol for On-Section Immunolabelling ............................................... 551

ADVANTAGES/DISADVANTAGES.............................................................. 556 5.1. 5.2.

6.

Materials ................................................................................................... 545 Products .................................................................................................... 546 Solutions ................................................................................................... 547

Advantages ............................................................................................... 556 Disadvantages........................................................................................... 556

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 556 6.1. 6.2. 6.3. 6.4.

Specimen Preparation ............................................................................... 556 Marker Systems ........................................................................................ 557 Light Microscopy ..................................................................................... 558 Staining for Electron Microscopy............................................................. 559

7.

OBSERVED RESULTS .................................................................................... 560

8.

REFERENCES .................................................................................................. 564



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GENERAL INTRODUCTION

On the search of the holy grail to image cellular ultrastructure in its native state, electron microscopists have established a wealth of preparation techniques, which result in excellent preservation of cellular components.1-11 Cryo-preparation techniques form this groundwork as dealt with in depth throughout this book. In light microscopy, recent years have resulted in major developments in: 1. Imaging: Confocal microscopy12-15 and deconvolution algorithms.16 2. Detection: A large variety of fluorochromes with a wide range of excitation and emission spectra as well as improved photostability. 3. Live imaging: Green fluorescent protein (GFP), their variants17 and alternatives (ReAsh, Lumio Technology).18 These improvements have almost exclusively been in the area of fluorescence microscopy. Dark-field imaging techniques, such as epifluorescence microscopy have improved contrast, which results in a high signal-tonoise ratio. Nevertheless, in light microscopy, little attention has been paid to preparation methods that remain crude and harsh, live imaging excluded. In fluorescence microscopy the deterioration of cellular ultrastructure is invisible and, therefore, neglected. It should, however, be kept in mind that in the dark-field mode, particles smaller than the resolution of a light microscope can be detected, in the same way the eye can see microscopical particles of dust in a beam of sunlight. We suggest combining the advantages of the developments in (fluorescence) light microscopy with the knowledge of high resolution preparation techniques in electron microscopy. The basic concept is to inspect serial (ultra) thin sections of the very same sample block, stained and immunolabelled, in both light and electron microscopes. Histological stains are used for orientation in the tissue; the labelling used under the light microscope identifies the structure of interest, which is further analysed at high resolution in the electron microscope The high-resolution objectives of light microscopes have inevitably a very low depth-offield, much smaller than the thickness of even one cell layer. This results in out-of-focus information, hence, blurring the image and reducing the final resolution. The principle of confocal (fluorescence) light microscopy or the application of deconvolution algorithms partially overcomes this drawback by generating three-dimensional stacks of in-focus optical sections.

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However, we would like to encourage the use of very thin (50 to 500 nm) sections for histological analysis by conventional bright-field, phase contrast or interference contrast light microscopy and localisation of cellular components by wide-field epifluorescence microscopy.19-21 Physical sectioning produces much higher z-resolution data than can be obtained with confocal light microscopy of thick specimens. The section is thinner than the depth-of-field of the high-resolution objectives and, therefore, all structures imaged are perfectly in focus. In addition, the high-quality preparation methods used in electron microscopy guarantee the preservation of high-resolution structural information. A further advantage is the fact that a subsequent ultrathin section, containing the same information as studied by light microscopy, can be analysed in the electron microscope. In the electron microscope, the label can directly and unambiguously be assigned to a defined cellular structure. Thus this approach allows correlative light and electron microscopy investigations: firstly, easy orientation in the tissue; secondly, wide-field evaluation of labelled cells; and thirdly, high-resolution identification of the labelled substructures.21,22-25 Now, the described concept of correlative microscopy is being applied for cryo-electron tomography. The GFP-labelled material, whole mount or CEMOVIS cryo-sections (see Chapters 11, 12) is cryofixed and mounted on a special cryostage attached to a light microscope. The position of the labelled structures are identified on a finder grid and recorded. Then the grid is transferred to the cryo-electron microscope and tomograms of the respective structure at high resolution are recorded (see Chapter 12).26 This concept for correlative light and electron microscopy is, of course, not limited to samples prepared by cryo-fixation, but can also be applied to chemically fixed samples processed at room temperature or, even better, to progressive lowering the temperature methods27 (PLT, see Chapter 18) or to cryo-sectioning according to Tokuyasu28 (see Chapter 19). The Tokuyasu technique often results in more efficient labelling than resinbased techniques.

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1. PRINCIPLES OF THE METHOD 1.1. Preparation  The biological specimen is, whenever possible, cryofixed, freeze-substituted and low-temperature embedded into methacrylate (or epoxy) resins.

 Excellent structural preservation close to the native state.  For morphology epoxy is the resin of choice.  For higher label efficiency, however, methacrylate resins are preferred.

 Alternatively, the biological specimen  Highly efficient immunolabelling. is chemically fixed (see Chapter 19) or even better prepared by the emerging cryo-fixation/rehydration method29 (see Chapter 14) followed by Tokuyasu cryo-sectioning.  After chemical fixation the sample  This is a good compromise when cryomay also be embedded into fixation or the Tokuyasu cryo-section methacrylates by standard procedures method cannot be applied. or preferentially by the progressive lowering of the temperature (PLT) method27,30 (see Chapter 18).  Ultrathin serial sections are cut and in  Observation of the same structure by sequence mounted for histology, light and electron microscopy. immunofluorescence and immunogold staining.

1.2. Imaging  The sections for light microscopy are  High z-resolution. even thinner than the depth-of-field of the high numerical aperture objectives. Further, on resin sections the label is restricted to the section surface.  Correlation of light and electron  Easy orientation and high-resolution microscopy. data.

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2. SUMMARY OF THE DIFFERENT STEPS

Figure 21.1 Flow diagram of the different preparation steps.  The sample is either embedded in methacrylate (or epoxy) for resin sections or prepared for Tokuyasu cryo-sections.  Subsequent sections are cut and collected for the different applications, one 500 nm for Toluidine blue staining and a few 50 to 100 nm for immunofluorescence and immunogold labelling.  Because these sections are thin and collected subsequently, the same organelles may be examined by immunofluorescence and electron microscopy.  If ultra-small gold particles are used for labelling, a silver enhancement step needs to be performed (see Chapter 23).

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3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Epifluorescence microscope  (Oil) immersion objectives with the highest numerical aperture  Band pass filter dedicated to the specific  High quality fluorescence filter fluorochromes:  Chroma Technology Corp., Bellows Falls, Vermont, USA.  Omega Optical Inc, Brattleboro, Vermont, USA.  In Europe, available from AHF Analysentechnik, Tübingen, Germany.  For ewample, Dumont #2a, #3, #4, #4N,  Forceps #7, Dumont & Fils, Switzerland.  Self-made loops from platinum wire or  Loop perfect loop, EMS, Fort Washington, Pennsylvania, USA.  Round glass coverslips, 12 mm in diameter  To mark the position of the sections on  Scratching diamond the microscope slide.  Moist chamber  Copper or nickel grids for electron  In general, copper grids can be used for microscopy, 50 to 200 mesh. The immunolabelling. grids are preferentially covered with For long-term incubation (overnight) or if carbon-coated Formvar or Pioloform the copper grids have no or a perforated support film or if they are used in support film.31 combination with azide, however, they will be oxidised.  Copper grids may impair silver enhancement (see Chapter 23).  Nickel grids are magnetic, therefore demagnetised forceps have to be used.  In addition, nickel grids may influence the astigmatism in the electron microscope, resulting in blurred images close to the grid bars.  Because of their magnetic properties, nickel grids are essential in combination with the automatic labelling apparatus from Leica (IGL, Leica Microsystems, Vienna).  Can be, 50, 100 or 200 W depending on  Mercury lamps the fluorescence microscope used and the manufacturer.

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 Precentred mercury lamps  Liquid light guide

 For optimal illumination in fluorescence microscopy Osram HXPTM R 120W/45C, Osram GmbH, Munich, Germany.  For optimal illumination in fluorescence microscopy, EXFO Life Sciences Group, Ontario, Canada.

3.2. Products  Toluidine blue O [C.I.52040]

 Serva 36693.02; Fluka 89640; Sigma T 0394.  4-88 Hoechst; Calbiochem 475904. 1,4-Diazabicyclo[2.2.2.]-  Antifading agent.

 Mowiol  DABCO, octane  PPD, p-Phenylene diamine  DAPI

 Propidium iodide

 Antifading agent.  4´,6-diamidine-2-phenylindole; for DNA staining.  Alternatively, Hoechst 33342 and Hoechst 33258 can be used for DNA staining.  3,8-Diamino-5-(3-diethylaminopropyl)6-phenanthridinium iodide methiodide to stain DNA and RNA.

 Gelatine  BSA, bovine serum albumin, Fraction V  Coldwater fish skin gelatine  Sigma G 7765.  Skim milk powder  BSA-c™  Acetylated BSA; Aurion, Wageningen, The Netherlands.  Poly-L-lysine  Sigma P 1274; MW 70,000-150,000 Da.  0.1% ready-to-use solution, Sigma P 8920.  Primary antibodies  Against target protein.  Fluorescent secondary antibodies  Against the species in which the primary antibody was produced.  Gold-labelled secondary antibodies  Against the species in which the primary antibody was produced (for details, see Chapter 23).  Gold-labelled protein A  Binds preferentially to the Fc region of rabbit, pig or human antibodies (for details, see Chapter 23).

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 (biotinylated) Lectins

 Uranyl acetate

 Lead citrate  Methyl cellulose  Sodium tetraborate

547  Mouse IgG2a, IgG2b and IgG3 can also bind to protein A under the standard labelling conditions (pH 7.4). For mouse IgG1, however, the pH must be higher, ~ 8.5.  Can easily be homemade or purchased from Dr. G. Posthuma, Department of Cell Biology, Utrecht Medical Centre, Utrecht, The Netherlands.  Detection of specific sugar moieties. Lectins are also successfully applied on epoxy sections; Vector Laboratories, Burlingame, California, USA.  Stain for electron microscopy. SPI Supplies, West Chester, Pennsylvania, USA.  Stain for electron microscopy.  Tokuyasu cryo-section embedding.28  Stable alkaline buffer solution; Merck 106308.



3.3. Solutions     

PBS buffer (pH 7.4) 137 mM NaCl 2.7 mM KCl 8.1 mM Na2HPO4 1.5 mM NaH2PO4

 Phosphate buffered saline, a standard buffer.

   

TRIS (pH 7.4) 150 mM NaCl 50 mM TRIS pH adjusted with HCl

 Label buffer; at pH 8.5 this buffer may be used to detect mouse IgG1 antibodies with protein A gold complexes.

    

PHEM buffer (pH 6.9) 60 mM PIPES 25 mM HEPES 10 mM EGTA 2 mM MgCl2

 Cytoskeleton buffer.32 This buffer can be helpful to suppress persistent background (E. van Donselaar, personal communication).

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 Blocking solution  Blocking protein dissolved in PBS or TRIS. Every lab uses its own recipe for blocking solutions. In distinct cases, specific blocking proteins, to be found empirically, are required. We routinely use 0.045% (w/v) coldwater fish skin gelatine and 0.5% (w/v) BSA in PBS.  Centrifuge prior to use.

 0.045% (w/v) coldwater fish skin gelatine and 0.5% (w/v) BSA, or 0.2% (w/v) gelatine, 0.5% (w/v) BSA, Fraction V 0.5%1% (w/v) coldwater fish skin gelatine33 1% (w/v) skim milk powder 1% BSA 0.1% BSA-c (BSA-c should not be used in combination with gelatine!)

 Toluidine blue O  Histological stain for orientation in the  Dissolve 0.5% (w/v) Toluidine blue O light microscope. in an aqueous solution of 1% sodium tetraborate, pH 9.2.  Filter and store as aliquots, e.g., in microtubes.  Centrifuge prior to use.  Mounting solution for fluorescence  Preparation, see Section 4.1.2. microscopy  Mounting solutions are also commercially available (e.g., Vectashield, Vector Laboratories or ProLong and SlowFade, Molecular Probes, Invitrogene).  Note: Vectashield is incompatible with Cy fluorochromes and DAPI staining.  Uranyl acetate  2% (w/v) in double distilled water  2% (w/v) in 1.5 M oxalate pH 7.028    

 See Chapter 19.

Lead citrate34 0.04 g lead citrate 10 mL H2O 100 μL 10 M NaOH

 Poly-L-lysine stock solution  Alternatively, the 0.1% ready-to-use  Dilute 2 mg/mL (w/v) in double solution, Sigma P 8920 may be used. distilled water.  Methyl cellulose

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 To embed Tokuyasu cryo-sections,28 see Chapter 19.

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4. PROTOCOLS 4.1. Protocols for Materials Needed for On-Section Immunolabelling 4.1.1. Poly-L-lysine coating of coverslips 1. Clean round glass coverslips with  For cleaning, 1% (v/v) hydrochloric acid ethanol. in 70% (v/v) ethanol or soap may also be used. 2. Rinse with double distilled water. 3. Dry coverslips separately on a filter paper in a large dish. 4. Place a 30 to 50 µL drop of poly-Llysine solution in the centre of the coverslip. 5. Allow the coating to settle for at least 30 minutes in a moist chamber. 6. Rinse briefly with double distilled  Coverslips can be coated in large batches water and dry face up on a filter paper and stored indefinitely in dust-free dishes at for one hour at 60°C or overnight at room temperature. ambient temperature.

4.1.2. Mounting solution for fluorescence microscopy 1. Dissolve 5 g Mowiol in 20 mL buffer (100 mM TRIS/HCl; pH 8.0) and stir for 16 hours. 2. Add 10 mL glycerol and stir again for 16 hours. 3. Remove undissolved centrifugation.

Mowiol

by  Aliquots can be stored at –20oC.

4. Add 20 to 50 mg/mL DABCO or  DABCO and PPD are antifading agents 1 mg/mL p-phenylenediamine (PPD) and reduce photobleaching of the fluorescence signal. to the final Mowiol solution.  DABCO is dissolved preferentially at 60°C.  PPD is more efficient in preventing bleaching, but turns a brown colour within one or two days, resulting in orange background fluorescence. DABCOcontaining solutions, however, can be stored for months in a freezer.

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4.2. Sample Preparation For our approach of correlative light and electron microscopy, any electron microscopic preparation technique suited for thin sectioning can be used. Note that in the following protocol all types of sections are treated in exactly the same way. 1. The specimen is cryofixed, freeze-  For optimal preservation of the cellular substituted and low-temperature ultrastructure (see Chapter 13).  Alternatively, chemically fixed samples embedded into a methacrylate. are conventionally dehydrated or dehydrated by PLT before embedding into a methacrylate (see Chapter 18). 2. Sample is chemically fixed and  In most cases, the label efficiency is prepared for Tokuyasu cryo- higher on Tokuyasu cryo-sections than on resin sections (see Chapters 14, 19). sectioning. 3. Sections are cut on a (cryo-) ultramicrotome: one 500 nm section to be mounted on a coverslip and a few 50– 100 nm sections to be mounted alternatively on coverslips and EM grids.

 The 500 nm section will be used for histological staining, e.g., toluidine blue for orientation.  The ultrathin sections on coverslips will be used for fluorescence labelling and those on EM grids for gold labelling.

4. Resin sections are picked up from the water trough with a loop and transferred onto the coverslip or EM grid. Cryo-sections are picked up from the dry knife edge with a loop containing a drop of, e.g., 2.3 M sucrose solution and after thawing  See Chapter 19, for alternative picking transferred onto the coverslip or EM up solutions grid. 5. Touch the loop edge with a pointed  The dried resin sections can be stored for piece of filter paper to remove the years without loss of antigenicity. water droplet. 6. The section will attach to the surface. 7. Cryo-sections are picked up from the  Cryo-sections, without removing the dry knife edge with a loop containing a drop of the transfer solution, can be stored up to one year at 4oC. drop of sucrose solution.  See Chapter 19.

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4.3. Toluidine Blue O Staining 1. 500 nm thick resin sections are put on a droplet of water on a poly-L-lysine coverslip. 2. They are dried on a hot plate (60 to 80oC) and immediately incubated with toluidine blue for 30 to 60 sec. 3. The sections are destained using a  Overstained sections can be recovered stream of (tap) water. by destaining in ethanol. 4. For high-resolution light microscopy  Do not use embedding media containing and long-term storage, the stained apolar solvents as they will destain the sections can be embedded in a thin sections. layer of Epon under a coverslip.

4.4. Protocol for On-Section Immunolabelling 1. The coverslips are put section side up  Note: For on-section labelling of resin on a piece of parafilm, mounted in the embedded material, the same rules as for solid-phase immunoadsorption apply. The moist chamber. reaction is restricted to the surface, therefore, not diffusion-dependent. This implies that the time for the washing steps is not critical.

Figure 21.2 Rehydration of sections mounted on 12 mm glass coverslips with buffer.

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2. For EM, drops of buffer are put on a piece of parafilm and the grids on the drop sections side down.

3. The sections are incubated with PBS  Resin sections are rehydrated and the for 10 minutes. sucrose drop on cryo-sections is removed.

4. Then they are incubated 2 × 10  The blocking proteins cover nonminutes with blocking buffer. specific binding sites and reduce background labelling due to charge or hydrophobic interactions.  We routinely use 0.045% (w/v) cold water fish skin gelatine and 0.5% (w/v) BSA in PBS. 5. Incubation with the primary antibody diluted in blocking buffer for 30 minutes to 2 hours at room temperature.

 For an unknown antibody, it is preferable to first make serial dilutions in blocking buffer: e.g., 1/10, 1/30, 1/100, 1/300, 1/1000, etc., to evaluate the concentration at which a clear signal is visible without background.  As a rule of thumb, the appropriate concentration of reacting antibody is in the range of 0.1 to 1 μg/mL.  To save precious primary antibodies, the grids may be put on the very same drop on the coverslip.

Figure 21.3. The coverslips with the sections facing upward and the grids with the sections facing downward are incubated in the same drop of primary antibody.

6. Wash six times for about two minutes with blocking buffer.

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 The drops on the coverslip are removed with a pipette and a fresh drop is immediately added. Take care that the sections never dry.

Figures 21.4 and 21.5 The drop of buffer or antibodies is aspirated with a pipette tip connected to a vacuum flask hooked to a vacuum pump.

Figure 21.6 A fresh drop of buffer or antibody is immediately added on the coverslip to prevent drying of the section.

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 The grids are lifted off the drop with forceps, blotted with a tissue or filter paper and immediately transferred on a fresh drop of blocking buffer.

Figure 21.7 During transfer from one drop to the other, the remaining drop of liquid on the grid is drained off by a short touch to a piece of tissue. Take care that the grids do not dry.

Figure 21.8 Coverslips (left) and grids during the label process.

7. Incubation with the secondary  For light microscopy, fluorescent antibody diluted in blocking buffer for antibodies are used.  For electron microscopy, gold one hour at room temperature. conjugated secondary antibodies or protein A gold is used. 8. Wash two times about two minutes with blocking buffer. 9. Wash four times about two minutes with PBS. 10. For light microscopy, DNA may be counterstained with 0.1 to 0.4 μg/mL DAPI or any nucleic acid may be stained with 0.1 to 0.4 μg/mL propidium iodide.

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 DAPI and PI penetrate methacrylate sections and, therefore, the signal intensity reflects the thickness of the section. They do not react with epoxy resin embedded nucleic acids.

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11. For electron microscopy, the  For light microscopy, the label should sections on the grids are fixed with 1% not be fixed, as glutaraldehyde at this glutaraldehyde in PBS. concentration may cause autofluorescence! 12. Wash four times with double  All salt ions, especially Cl- and PO43-, distilled water. have to be removed as they will interfere with silver enhancement and uranyl staining. 13. Silver enhancement, if ultra-small  See Chapter 23. gold particles were used as a marker. 14. Wash extensively with double distilled water. 15. The fluorescently labelled sections  Use a very small droplet of mounting medium. The medium must not be are mounted with Mowiol. squeezed out from the coverslips. Excess Mowiol will pollute the front lens of the microscope objectives. 16. The sections for electron microscopy are counter-stained with uranyl acetate and if desired with lead citrate. 17. Tokuyasu cryo-sections are stained  Methyl cellulose must always be kept in with uranyl acetate and embedded in the cold because it dissolves better in the methyl cellulose. cold.  The embedding is done on ice (see Chapter 19). 18. The fluorescently labelled prepar-  DAPI or PI counter stains may be lost ations can be stored for months in a during storage and PPD-containing media refrigerator or freezer. turn brown. For restoration, the coverslips can be removed after submerging in PBS and thereafter again stained and mounted.  Even additional immunolabelling or “section recycling” is possible (bound antibodies can be released as described for immunoadsorption techniques, e.g., with 4 M magnesium chloride, MgCl2).

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5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  Excellent structural preservation in light and electron microscopy.  Ease of orientation in stained histological tissue sections.  Fast overview on labelled structures.  High z-resolution in light microscopy.  Correlation of label and cellular ultrastructure.

 Application of cryo-electron microscopy techniques.  Large overview.  Which cells are labelled, what is the level of expression, evaluation of labelling conditions (label efficiency, background)?

5.2. Disadvantages  No live imaging possible.  Dedicated instrumentation for preparation is needed.  The preparation is rather time consuming.

 

 Limited access to antigens.

 Only the antigens on the section surface are accessible to antibodies. 

 Standard preparation for microscopy is comparably faster.

light

6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Specimen Preparation  Cryofixed, freeze-substituted and  See Chapter 13. resin-embedded samples in general have a better morphology.  Resin-embedded samples are easy to store and antigenicity is retained for many years.  Resin sections are easier to handle.  Large, undistorted sections are easier to obtain than with Tokuyasu’s cryosectioning.

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 The cellular morphology is preserved much better by epoxy embedding; however, epoxy sections are less suited for immunolabelling of protein epitopes.  Carbohydrate chains, such as sugar moieties, nucleic acids, biotin and digoxigenin, are much less affected.  Methacrylate-embedded samples are better suited for immunolabelling but the cellular morphology is inferior.  Cryo-sections of chemically fixed samples are optimal for immunolabelling.

 The epoxy resin covalently binds to the biological structures, masking protein epitopes.  Sugar moieties can be labelled with specific (biotinylated) lectins.  Biotin and digoxigenin can be used as tags in combination with epoxy embedding.  Methacrylates do not covalently bind to biological structures, therefore, the cellular morphology suffers, but the protein epitopes are more accessible.  During dehydration for resin embedding the tertiary structure of proteins may be changed resulting in reduced or even loss of antigenicity.  The proteins are not denatured by organic solvents and the matrix of the section is less dense allowing easier access for antibodies (see Chapter 19).

 Cryofixed, freeze-substituted rehydra-  See Chapter 14. ted samples further prepared for Tokuyasu cryo-sectioning may combine good structural preservation and antigenicity.29

6.2. Marker Systems  Fluorescent labels are preferred to  Fluorescence microscopy is a dark-field coloured enzyme reaction products. imaging mode that excels in contrast compared to bright-field imaging, therefore, the labelling is more intensive.  In fluorescence microscopy, double or  There is a wealth of fluorochromes multiple labelling is easy. available. In contrast to gold particles, the fluorochromes do not affect the binding properties of the antibodies.  Protein A gold is used in combination  At neutral pH, protein A has a very low with primary antibodies from rabbits, binding affinity to mouse or rat monoclonal pig or man. antibodies. It is, however, successfully used with a bridging antibody produced in rabbit or pig.  At pH 8.0 or higher, protein A can bind to mouse IgG1 antibodies. Here it is absolutely necessary to fix the protein A to the antibodies with glutaraldehyde before the sections are stained with aqueous (acidic!) uranyl acetate.

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 Protein A gold complexes generally have a higher label density and do not aggregate.  Gold-tagged secondary antibodies are used in combination with all other primary antibodies.  For electron microscopy, single labelling is preferred to double or even multiple labelling.

 This is only true for homemade protein A complexes or those supplied by the UMC Utrecht.

 Valid controls for multiple labelling are laborious and often difficult to design, while the extra information is minimal.  We prefer labelling of subsequent serial sections for different antigens.

6.3. Light Microscopy  Even illumination of the field of view  Centre the light source meticulously. without a light gradient is a pre-  A simple and more efficient approach is requisite. using precentred mercury lamps (Osram HXPTM R 120W/45C) coupled to the microscope via a liquid light guide. The system is available as X-Cite 120 from EXFO Life Sciences Group, Ontario, Canada.  Optimal alignment of the microscope  Remember, even an epifluorescence is essential. microscope must be aligned according to Köhler.  For fluorescence microscopy, oil  To collect as much signal as possible, a immersion objectives with a high large opening angle of the objective is numerical aperture (NA) must be used essential. for all magnifications.  The medium between the glass coverslip and the objective should have the same refraction index as glass to avoid loss of light by reflection at the glass surfaces.  The deflected light can also cause background noise because it does not contain any object information.  Though phase contrast objectives prolong the exposure time (by 10 to 15%) they are still suitable for fluorescence imaging. They offer the advantage of correlative phase contrast imaging for better orientation, e.g., phase contrast image overlaid with fluorescence signal. http://www.micro.magnet.fsu.edu/optics/ind ex.html

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 Sections may be searched with a low magnification phase contrast objective fitted with a wrong phase ring (e.g., Ph 3) to obtain pseudo dark-field illumination.  Sections may be found using DAPI or PI staining with a low magnification objective.  As a general hint to find the focal plane of the object, close the illumination diaphragm and focus the rim.  Close the illumination diaphragm so that only the field of imaging is illuminated.  Further closing results in an even better signal-to-noise ratio.

 The edges of the sections are highlighted.

 The DAPI and PI signals in general are very bright.  Under these conditions, the objective is focussed in the plane of the sections.  This operation requires an optimally aligned microscope.  Only the light directly emitting from the object contains image information.  Light outside the field of view only increases the background noise.

6.4. Staining for Electron Microscopy  The gold-labelled resin sections are stained with aqueous uranyl acetate and then examined. Only if the contrast is too weak, a second staining step with lead citrate follows.  Alcoholic uranyl acetate solution may cause dirty grids.  There are several methods to stain and embed Tokuyasu cryo-sections: a) Staining for 10 minutes with neutral uranyl oxalate followed by 5 minutes with acid uranyl acetate before methylcellulose embedding. b) Embedding in methyl cellulose containing 0.2 to 0.4% uranyl acetate.

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 The contrast of the gold label, especially of smaller gold particles, e.g., 5 nm, is quite weak and is easily overlooked on heavily stained sections.  The remaining blocking proteins may be precipitated by the alcohol.

 See Chapter 19.

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7. OBSERVED RESULTS  Figure on Chapter’s title page

 Localization of β-catenin in the adherens junctions, but also in the Z-line of guinea pig heart muscle.23

 Figure 21.9

 A 500 nm-thick section of Lowicrylembedded rat duodenum is stained with Toluidine blue. The bright field mode offers a good overview and orientation within the tissue.

 Figure 21.10 (see colour insert following page.)

 Multiple exposure of a rat duodenum section stained with actin (FITC), β-catenin (Cy3) and nucleic acids  Actine = FITC, green  β-catenin = Cy3, orange  Nucleic acids = PI, red

Bar = 20 µm

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 At low magnification the cellular morphology can directly be related to the light microscopy images, the cell–cell interphase in this case (see Figure 21.10).

 The same area at an intermediate magnification.

 Step-by-step zooming in on the position located by fluorescence microscopy the labelled structures can be analysed at high resolution.  Inset: β-Catenin is predominately located at the cell membrane between adjacent cells (arrows). Note that there is no β-catenin in the desmosomes.

Top bar = 20 µm Middle bar = 5 µm Bottom bar = 1 µm

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8. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17. 18.

Dubochet, J. et al. Cryo-electron microscopy of vitrified specimens, Q. Rev. Biophy., 21, 129, 1988. Fernández-Morán, H. Low-temperature preparation techniques for electron microscopy of biological specimens based on rapid freezing with liquid helium II, Ann. NY. Acad. Sci., 85, 689, 1960. Gilkey, J.C. and Staehelin, L.A. Advances in ultrarapid freezing for the preservation of cellular ultrastructure, J. Electr. Microsc. Tech., 3, 177, 1986. Humbel, B.M. and Schwarz, H. Freeze-substitution for immunochemistry, in Immuno-Gold Labelling in Cell Biology, Verkleij, A.J. and Leunissen, J.L.M., eds., CRC Press, Boca Raton, FL, USA, 1989, 115. Kaneko, Y. and Walther, P. Comparison of ultrastructure of germinating pea leaves prepared by high-pressure freezing-freeze substitution and conventional chemical fixation, J. Electr. Microsc., 44, 104, 1995. Matsko, N. and Müller, M. Epoxy resin as fixative during freeze-substitution, J. Struct. Biol., 152, 92, 2005. Müller, M., Marti, T., and Kriz, S. Improved structural preservation by freeze substitution, in Proc. 7th Eur. Congr. Electron Microsc., Brederoo, P. and de Priester, W., eds., The Hague, The Netherlands, 1980, 720. Müller, M. and Moor, H. Cryo-fixation of thick specimens by high pressure freezing, in Science of Biological Specimen Preparation 1983, Revel, J.P., Barnard, T., and Haggis, G.H., eds., SEM Inc., AMF O'Hare, IL, USA, 1984, 131. Schwarz, H., Hohenberg, H., and Humbel, B.M. Freeze-substitution in virus research: a preview, in Immunoelectron Microscopy in Virus Diagnosis and Research, Hyatt, A.D. and Eaton, B.T., eds., CRC Press Inc., Boca Raton, FL, USA, 1993, 97. Steinbrecht, R.A. and Müller, M. Freeze-substitution and freeze-drying in Cryotechniques in Biological Electron Microscopy, Steinbrecht, R.A. and Zierold, K., eds.. Springer-Verlag, Berlin, Heidelberg, Germany, 1987, 149. Van Harreveld, A. and Crowell, J. Electron microscopy after rapid freezing on a metal surface and substitution fixation, Anat. Rec., 149, 381, 1964. Brakenhoff, G.J. et al. 3-dimensional imaging of biological structures by high resolution confocal scanning laser microscopy, Scan. Microsc., 2, 33 1988. Petran, M. et al. Tandem scanning light microscopy, in Science of Biological Specimen Preparation 1985, Müller, M., et al., eds., SEM Inc., AMF O'Hare, IL, USA, 1986, 85. White, J.G., Amos, W.B., and Fordham, M. An evaluation of confocal versus conventional imaging of biological structures by fluorescence light microscopy, J. Cell Biol., 105, 41, 1987. Wilson, T. Scanning optical microscopy, Scanning, 7, 79, 1985. DeBiasio, R. et al. Five-parameter fluorescence imaging: wound healing of living Swiss 3T3 cells, J. Cell Biol., 105, 1613, 1987. Tsien, R.Y. The green fluorescent protein, Annu. Rev. Biochem., 67, 509, 1998. Gaietta, G. et al. Multicolor and electron microscopic imaging of connexin trafficking, Science, 296, 503, 2002.

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19. 20. 21. 22. 23. 24. 25.

26. 27. 28. 29. 30. 31. 32. 33. 34.

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Schwarz, H. Immunolabelling of ultrathin resin sections for fluorescence and electron microscopy, in Electron Microscopy 1994, ICEM 13, Jouffrey, B. and Coliex, C., eds., Les Editions de Physique, Les Ulis, France, 1994, 255. Schwarz, H. Correlative immunolabelling of ultrathin resin sections for light and electron microscopy, in Electron Microscopy 1998, ICEM 14, Calderón Benavides, H.A., et al., eds., Institute of Physics Publishing, Bristol, PA, USA, 1998, 865. Tonning, A. et al. Hormonal regulation of mummy is needed for apical extracellular matrix formation and epithelial morphogenesis in Drosophila, Development, 133, 331, 2005. Bierkamp, C. et al. Desmosomal localization of β-catenin in the skin of plakoglobin null-mutant mice, Development, 126, 371, 1999. Kurth, T. et al. Fine structural immunocytochemistry of catenins in amphibian and mammalian muscle, Cell Tissue Res., 286, 1, 1996. Nica, G. et al. Eya1 is required for lineage-specific differentiation, but not for cell survival in the zebrafish adenohypophysis, Develop. Biol., 292, 189, 2006. Schwarz, H., Müller-Schmid, A., and Hoffmann, W. Ultrastructural localization of ependymins in the endomeninx of the brain of the rainbow trout: Possible association with collagen fibrils of the extracellular matrix, Cell Tissue Res., 273, 417, 1993. Sartori, A. et al. Correlative microscopy: Bridging the gap between fluorescence light-microscopy and cryo-electron tomography, J. Struct. Biol., 160, 135, 2007. Carlemalm, E., Garavito, R.M., and Villiger, W. Resin development for electron microscopy and an analysis of embedding at low temperature, J. Microsc., 126, 123, 1982. Tokuyasu, K.T. Immunocytochemistry on ultrathin frozen sections, Histochem. J., 12, 381, 1980. Van Donselaar, E. et al. Immunogold labelling of cryo-sections from high-pressure frozen cells, Traffic, 8, 471, 2007. Acetarin, J.D., Carlemalm, E., and Villiger, W. Developments of new Lowicryl resins for embedding biological specimens at even lower temperatures, J. Microsc., 143, 81, 1986. Hayat, M.A. Principles and Techniques of Electron Microscopy. Biological Applications. 4th ed., Cambridge University Press, Cambridge, UK, 2000. Schliwa, M., van Blerkom, J., and Porter, K.R. Stabilization of the cytoplasmic ground substance in detergent-opened cells and a structural and biochemical analysis of its composition, Proc. Nat. Acad. Sci. USA, 78, 4329, 1981. Birrell, G.B., Hedberg, K.K., and Griffith, O.H. Pitfalls of immunogold labelling: Analysis by light microscopy, transmission electron microscopy, and photoelectron microscopy, J. Histochem. Cytochem., 35, 843, 1987. Venable, J.H. and Coggeshall, R. A simplified lead citrate stain for use in electron microscopy, J. Cell Biol., 25, 407, 1965.

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CONTENTS GENERAL INTRODUCTION .................................................................................... 571 1.

PRINCIPLES OF THE METHOD .................................................................. 571 1.1. 1.2. 1.3. 1.4. 1.5.

Fixation of Tissue (optional) .................................................................... 571 High-Pressure Freezing of Specimen ....................................................... 572 Freeze-Fracture Replication...................................................................... 573 SDS-Treatment of Replicated Materials................................................... 574 Immunodetection of Target Molecules with Gold Particles ..................... 574

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 575

3.

MATERIALS/PRODUCTS/SOLUTIONS ...................................................... 576 3.1. 3.2. 3.3.

Materials ................................................................................................... 576 Products .................................................................................................... 577 Solutions ................................................................................................... 577

4.

PROTOCOL ...................................................................................................... 578

5.

ADVANTAGES/DISADVANTAGES.............................................................. 581 5.1. 5.2.

6.

Advantages ............................................................................................... 581 Disadvantages........................................................................................... 582

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 582 6.1.

6.2.

6.3.

Preparation of Tissue ................................................................................ 582 6.1.1. Fixation ....................................................................................... 582 6.1.2. Unfixed tissue ............................................................................. 583 Replication................................................................................................ 583 6.2.1. Fracturing and shadowing temperature....................................... 583 6.2.2. Shadowing .................................................................................. 583 6.2.3. Grid-mapping of the replicated tissue ......................................... 583 Tissue Removal ........................................................................................ 583

7.

OBSERVED RESULTS .................................................................................... 584

8.

REFERENCES .................................................................................................. 586

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GENERAL INTRODUCTION One of the questions to be answered in the post-genomic era, at a time when most proteins constituting living organisms have been identified, is what particular protein species and amount of each species is expressed in a particular cell type and in their subcellular domains. Such information is indispensable for understanding mechanisms involved in living cells. New techniques for molecular localization that combine immunofluorescent labeling or expression of fluorescent proteins with laser microscopy have been developed and have successfully revealed new aspects of protein logistics and its dynamic regulation in living organisms. However, for subcellular localization that requires nano-scale resolution, conventional immunoelectron microscopy (pre- and postembedding immunolabeling) has been widely used. Although these techniques, especially with immunogold particles, have provided precise details of molecular localization in different subcellular domains, reliable quantification of immunoreactivity has often been hampered by low sensitivity and limited accessibility of antibodies to target molecules buried in tissues. In 1995, Fujimoto developed an epoch-making technique for localization of plasma membrane molecules, termed sodium dodecyl sulfate (SDS)digested freeze-fracture replica labeling (SDS-FRL), by which molecules on the plasma membrane can be directly approached by antibodies and visualized in a two-dimensional manner with high sensitivity and high resolution.1,2 In collaboration with Fujimoto, we have applied this technique to brain tissue with some modifications and proved its high sensitivity and quantitative capability in visualizing molecules in the plasma membrane.3,4 Here, we introduce our current protocol of SDS-FRL, which could be further modified for various purposes and other tissues to bring out the full potential of this powerful technique.

1. PRINCIPLES OF THE METHOD 1.1. Fixation of Tissue (Optional) For fixation of brain tissue, animals are  transcardially perfused with formaldehyde (0.5 to 4%). Use of formaldehyde without glutaraldehyde (GA) allows better removal of tissue debris from replica membranes  Fixation of tissue with formaldehyde and denaturation of membrane-attached (FA) proteins by SDS treatment. Thereby the molecules present in the plasma membrane can be studied quantitatively.

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Figure 22.1 SDS treatment resulted in better dissociation of proteins in FA-fixed brain tissue than in GA-fixed tissue. A = Coomassie brilliant blue (CBB) stain. B = Western blot immunodetection of AMPA-type glutamate receptor subunit GluR2.  Lane 1: unfixed brain tissue  Lane 2: 4% FA fixed tissue  Lane 3: 4% FA plus 0.05% GA fixed brain tissue  Numbers indicate molecular weight in kDa. Note that a larger proportion of proteins and GluR2 immuno-reactivity are detected at high molecular weight ranges in GA-fixed brain tissue. It seems likely that fixation of tissue improves reproducibility of labeling intensities from one experiment to another.

1.2. High-Pressure Freezing of Specimen For tissue freezing, chemically prefixed or unfixed brain slices between 90 to 150 micrometer thick are cut with a vibrating microtome and are rapidly frozen with a high-pressure freezing machine (see Chapters 5, 6), which produces a series of uniformly frozen tissues in a consistent manner. This is important for quantitative analysis of protein localization.



 Consistent production of the same quality of frozen tissues is necessary for quantitative analysis.

Homogeneous freezing throughout the  Homogeneous freezing within the slice whole slice is also important because is also necessary for quantitative analysis. random fracturing through the slice occurs when using a double replica specimen table.

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1.3. Freeze-Fracture Replication High-pressure frozen tissues are transferred into a freeze-fracture replica machine, and the lipid bilayer of the plasma membranes is split into two pieces at the hydrophobic interfaces by fracturing. Proteins in the plasma membrane are allocated in either of two membranes, exoplasmic or protoplasmic membranes, and molecules in these membranes, including lipids and proteins, are then immobilized with a thin layer (< 5 nm thick) of carbon deposit. This material is further coated with a 2 nm-thick platinum/carbon layer for shadowing the membrane faces and then strengthened with a 15 nm-thick carbon deposit.



 Deposition of the first carbon layer onto exposed faces of plasma membrane halves ensures immobilization of membrane proteins and phospholipids on the replica.

                    Figure 22.2 The first carbon layer on the exposed faces retains molecules at the surface. The plasma membrane in frozen tissue is frequently fractured at its hydrophobic interface (B) and exposed faces of the membrane half are coated with evaporated carbon (gray) and platinum (black) (C).  

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1.4. SDS-Treatment of Replicated Materials After preparation of the replicas, they are  SDS treatment exposes molecules treated with SDS, which dissolves lipids immobilized on replicas. and proteins that are not immobilized in the  replica membrane.

Figure 22.3 SDS treatment of replicated tissue dissociates molecules that are not immobilized in the replica membrane.

SDS treatment also denatures immobilized molecules and unfolds secondary and tertiary structures of the protein, and thereby facilitates detection of epitopes in  SDS denaturation of molecules on target molecules by immunolabeling. plasma membrane.

1.5. Immunodetection of Target Molecules with Gold Particles SDS-treated replicas are subjected to immunodetection where specific primary antibodies for target molecules are bound with immunogold-coupled secondary  Quantification of immunogold particles antibodies, and then investigated with a under EM. transmission electron microscope (TEM). Quantification of immunoreactivity at a nanometer scale resolution can be achieved.

Figure 22.4 Localization of target molecules is visualized with immunogold particles, which coincident with platinum/ carbon (Pt/C) replica images of membrane faces.

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2. SUMMARY OF THE DIFFERENT STEPS 1: Brain slice preparation  Perfusion of animal with fixative (optional) A) Slicing B) Trimming regions of interest C) Cryoprotection in freezing medium 2: Freezing  Mounting trimmed slices within two specimen carriers with a spacer made of double-sided adhesive tape (a)  Freezing by high-pressure freezing machine  Storage of frozen sample in liquid nitrogen 3: Freeze-fracture replication  Equilibration of sample to target temperature  Fracturing of sample  Replication of exposed surfaces with C-Pt/C-C 4: Removal of tissue A) Transferring replica with tissue into SDS solution B) Incubation at high temperature 5: Immunolabeling  Removal of SDS  Blocking  Labeling  Mounting the labeled replica onto electron microscopy (EM) grid 6: LM observation of replicas  Identification of tissue architecture in replicas by light microscopy (LM) (stereo or upright microscope) 7: EM observation of replicas  Investigation of immunoreactivities. Figure 22.5 Summary of the different steps.

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3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Vibrating microtome

 For preparation of slices

 High-pressure freezing machine

 For rapid freezing of tissue  HPM010, BAL-TEC, Balzers, Principality of Liechtenstein. Now available from Boeckeler Instruments, Inc., Tucson, Arizona, USA or ABRA-Fluid AG, Widnau, Switzerland: www.abra-fluid.ch

 Specimen carriers with or without a  BAL-TEC, 4.6 mm diameter, 0.6 mm ring-shaped, double-sided adhesive thickness, LZ 02127 VN. Now available tape (1.5 mm diameter hole, 150 µm from Leica. thickness or less)  Cell locator

 For storage of frozen samples

 Freeze-fracture replica machine

 For freeze-fracture and replication of specimens  BAL-TEC, BAF060 Now available from Leica.

 Double replica specimen table

 BAL-TEC. Now available from Leica.

 Stereo microscope

 For trimming of slices, placing trimmed tissues into freezing carriers, and immunolabeling of replicas

 Hybridization oven or autoclave

 For SDS treatment of replicated samples

 Sealable glass vial (5 mL)  A glass rod with a platinum wire or a  For replica handling glass rod with a fine tip  Shaker with flat bottom  For immunolabeling  Porcelain spot dish  EM specimen grids coated with  For observation under the EM Pioloform film

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3.2. Products  Liquid nitrogen  Primary antibodies

 For storage and handling of frozen samples and for operation of HPM010 and BAF060  For immunolabeling

 Secondary antibodies coupled to 5, 10  British Biocell International, Cardiff and or 15 nm colloidal gold Amersham, Biosciences, Buckinghamshire, U.K.

3.3. Solutions  Fixation  Saline or phosphate-buffered saline (PBS)  0.5 to 4% formaldehyde (FA), 15% saturated picric acid (PA) solution in 0.1 M PB.

 Higher concentrations of formaldehyde generally result in fracturing of plasma membranes into smaller pieces, making interpretation of images difficult.

 Freezing medium  30% glycerol or 30% sucrose in  Stepwise treatment of tissues with 10, 0.1 M PB (pH 7.4) 20, 30% glycerol or sucrose solutions may work better in terms of preservation of morphology.  SDS solution  2.5% SDS, 20% sucrose in 15 mM  Solution at pH 6.8 can also be used. Tris-HCl (pH 8.3)  Sucrose can be replaced with 10% glycerol.  Buffers for immunolabeling  Washing buffer  0.05% BSA  Addition of BSA in buffers dramatically  0.1% Tween-20 reduces stickiness of the replica to  0.05% NaN3 glassware and platinum wire and facilitates in 50 mM Tris buffered saline the isolation of intact (large) replicas after (TBS, pH 7.6) immunolabeling.  Blocking buffer  5% BSA  0.1% Tween-20  0.05% NaN3 in TBS  Primary antibody solution  1% BSA  0.1% Tween-20  0.05% NaN3 in TBS

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4. PROTOCOL Standard protocol for SDS-FRL 1. Preparation of tissue slices  After brief cardiac perfusion with saline, animals are fixed with 0.1 M PB containing 2% FA and 15% saturated PA, and the brain is sliced at 150 m thickness with a vibrating microtome in 0.1 M PB.

  Dissected tissues must be kept cold (4 °C) throughout Step 1.  Glass rods or paint brushes can be used to handle the tissue slices.  Unfixed slices can also be used.  Preparation of fixative solution and perfusion of animals must be done under a chemical hood to avoid inhalation of formaldehyde.  FA ranging in concentration between 0.5 to 4% is used.  Thickness of the slice should be optimized by each user so that it fits into the space between specimen carriers (see below).  Use of stereo microscope is recommended for the following steps.

 Trimming of slice

 Region of interest in the slice is trimmed out with a razor blade to fit into the hole of the specimen carrier. To prevent tissue damage, trimming is carried out on a 1% agarose gel in 0.1 M PB.

 Cryoprotection

 The trimmed slices are immersed into freezing medium for at least three hours.  The order of trimming and cryoprotection is reversible.

2. Rapid freezing of the trimmed slices   Mounting trimmed slices onto  In the freezing media, the slice is placed specimen carriers into a hole of double-sided tape attached to a specimen carrier. The carrier with the slice is transferred onto filter paper and the excess solution in the hole is removed with a brush. The carrier is covered with another specimen carrier without the tape so that the slice is sandwiched between the two carriers. Figure 22.6 Tissue slice mounted on the specimen carrier.

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 The room must be ventilated to avoid oxygen shortage. Wear cryo-protection gloves when handling chilled equipment.  The carrier pair is rapidly frozen with the high-pressure freezing machine and stored in liquid nitrogen until use.

3. Freeze-fracture replication  Installation of frozen samples into a  The frozen sample inserted into a slot of replica machine. a double replica specimen table in liquid nitrogen is transferred into the freezefracture machine.  Freeze-fracturing of samples  Fracture temperature at 110 to 120°C is recommended for ease of interpretation of the images as fracturing at lower temperature results in smaller membrane fragments in the replicas.  Replication of exposed tissue surface  Indispensable. To have homogeneous immobilization of surface molecules, a thin layer of carbon coating as the first layer is recommended. Conditions for each layer are as follows:  First layer, 5 nm carbon from overhead with sample rotation at 30 rpm  Second layer, 2 nm platinum/carbon at 60° angle without sample rotation  Third layer, 15 nm carbon from overhead with sample rotation  Grid-mapping of the replicated tissue  Optional. For better orientation, the replicas are attached to EM finder grids before tissue removal and nuclear stain images on the EM grid are recorded by light microscopy. To keep replicas in their original shape throughout the labeling procedures, they are stabilized with Lexan resin (see Section 6.2.3). 4. Tissue removal from replica mem-  Replicas with tissues are transferred into brane by SDS treatment glass vials containing 0.75 mL of the SDS solution.  These samples are incubated at +80°C for 18 hours with continuous shaking.

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 All of the following steps are carried out at room temperature unless otherwise noted.  All of the following steps are carried out under mild shaking conditions.

5. Immunolabeling

 Wash  SDS solution  Wash buffer  Blocking non specific sites  Blocking buffer  Primary antibody reaction  Primary antibody solution

 Wash  Wash buffer  Blocking non specific sites  Blocking buffer

10 min  Optional. 3 × 10 min  Indispensable ! 1 hour  Overnight  30 L drops of the solution are prepared at 15°C on a Parafilm sheet and placed in a humid chamber. Blocked replicas are transferred onto these drops. The primary antibody concentration is between 1 and 5 g/mL in the primary antibody solution. 3 × 10 min  For reduction of background labeling, 30 min but not indispensable.

 Secondary antibody reaction  The secondary antibody is diluted at  Secondary antibody Overnight 0.5 ~ 1 g/mL in the blocking buffer. For in blocking buffer multiple labeling, several secondary antibodies coupled with different sizes of colloidal gold are mixed. In general, those with smaller gold show higher labeling intensity and those with > 20 nm gold give very low labeling intensity.  Wash  Wash buffer 3 × 10 min  Wash  Double distilled water

© 2009 by Taylor & Francis Group, LLC

1 × 3 min  In solution without BSA, the tissue side of replicas tends to stick onto glassware and platinum wire leading to damaged replicas. Handle them on the nontissue side.

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 Mounting replica membranes onto the  Replicas should be kept on the water surface and be unfolded with platinum wire EM grids under a stereo microscope.  At this step it is important to have large, intact replica membranes in which information about tissue architecture can be retrieved.  Practice is required in handling replicas in solution. 6. LM observation  Stereomicroscopy

7. EM observation

 Stereomicroscopic images of replicas with transparent illumination and/or reflecting illumination are useful to obtain information about tissue architecture in the replicas.  For investigation of immunoreactivity and morphology of plasma membrane faces.  For interpretation of images, please refer to original papers listed in the reference section.

5. ADVANTAGES/DISADVANTAGES 5.1. Advantages  Two-dimensional visualization of  Instead of reconstructing serial ultrathin protein localization in the plasma sections, SDS-FRL can directly visualize membrane at EM level two-dimensional distribution of membrane molecules. Localization of the immunogold particles coincident with Pt/C replica images of membrane faces are obtained by TEM.   Quantitative analysis of protein  SDS treatment dissociates proteins from localization target proteins in the protein complex and denatures their higher-order structure, resulting in an equal detectability by specific antibodies. Visualization of immunoreactivity with antibodies coupled to colloidal gold particles enables quantitative analysis of protein localization.

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 Highly sensitive detection and clear-  In the conventional pre-embedding cut distinction of the pre- or post- immunolabeling method, molecules in synaptically localized molecules synaptic sites are often undetectable because of poor penetration of antibodies into the densely packed protein matrix of the specialized synaptic membranes. On the other hand, in the post-embedding immunolabeling method, the deviation of immunogold particles (up to 30 nm) from the antigen site makes it difficult to distinguish pre- and post-synaptic location.   Wide applicability of specific  SDS-FRL uses similar antigen–antibody antibodies reaction conditions to those used for the Western blot immunodetection. Therefore, a large proportion of antibodies usable in the latter method may also be applicable to this technique.

5.2. Disadvantages  Difficult to identify cellular or sub-  Information about fine structure in the cellular origin of each replicated vicinity of replicated membranes is lost. membrane  Difficult to produce replicas com-  Fracture occurs in a random fashion prising specific cellular components within the frozen tissues. within a frozen tissue.  

6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Preparation of Tissue 6.1.1. Fixation



 0.5% FA + 15% saturated PA in 0.1 M  For more synaptic profiles in the replica PB membrane  4.0% FA + 15% saturated PA in 0.1 M  For finer structure of the plasma PB membrane and intramembrane particles (IMPs)

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6.1.2. Unfixed tissue  Artificial cerebrospinal fluid in which  For some target molecules that show low sodium chloride is substituted with immunoreactivity in the fixed tissue. 0.8% sucrose.   

6.2. Replication 6.2.1. Fracturing and shadowing temperature  Fracture and replication at 130°C or  For finer structure of the plasma membrane and IMPs. below. 

 6.2.2. Shadowing  Rotary shadow for the second layer, 2.5 nm platinum/carbon from 25° with sample rotation. 

 For finer structure of the IMPs.   

6.2.3. Grid-mapping of the replicated tissue  Attach an EM grid to an entire replica  To prevent replica membranes from membrane with Lexan resin before breaking into small pieces during tissue tissue removal. removal and immunolabeling procedures.  For detailed mapping of the cytoarchitecture within replicas. Detailed procedure is described in References 3,5.

6.3. Tissue Removal  Incubations  105°C for 10 min by autoclave

 60°C for one day and 37°C overnight

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 When the hybridization oven is not available.   For detection of membrane-associated proteins.3

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7. OBSERVED RESULTS  Figure on Chapter’s title page

 See Figure 22.7.

 Figure 22.7 1. Description

 Co-localization of AMPA- and NMDAtype glutamate receptors in the postsynaptic specializations in the plasma membrane of the dentate gyrus granule cell. The postsynaptic specialization was identified by a cluster of IMPs on the exoplasmic fracture face.

2. Method

 SDS-FRL

3. Tissue

 Adult rat hippocampus

4. Fixation

 2% FA, 15% saturated PA in 0.1 M PB

5. Fracture temperature

 120°C

6. Coating layers

 5 nm C, 2 nm Pt/C from 60°, 15 nm C

7. SDS treatment

 80°C for 18 hours

8. Antibodies

 Primary antibodies:  Rabbit anti-GluR1-4 antibody was kindly donated by Prof. E. Molnar at University of Bristol U.K. (3.3 g/L).  Mouse anti-NMDA receptor 1 antibody (Chemicon, 3.6 g/L).  Secondary antibodies:  Antirabbit and antimouse IgG coupled with 5 and 10 nm colloidal gold particles, respectively (0.67 g/mL)

9. Bar

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 200 nm

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8. REFERENCES

1. 2.

3. 4. 5.

Fujimoto, K. Freeze-fracture replica electron microscopy combined with SDS digestion for cytochemical labeling of integral membrane proteins. J. Cell Science, 108, 3443, 1995. Fujimoto, K. SDS-digested freeze-fracture replica labeling electron microscopy to study the two-dimensional distributioin of integral membrane proteins and phospholipids in biomembranes: Practical procedure, interpretation and application. Histochem. Cell Biol., 107, 87, 1997. Hagiwara, A. et al. Differential distribution of release-related proteins in the hippocampal CA3 area as revealed by freeze-fracture replica labeling. J. Comp. Neurol., 489, 195, 2005. Tanaka, J. et al. Number and density of AMPA receptors in single synapses in immature cerebellum. J. Neurosci., 25, 799, 2005. Rash, J.E. et al. Grid-mapped freeze fracture: Correlative confocal laser scanning microscopy and freeze-fracture electron microscopy of preselected cells in tissue slices, in Rapid Freezing, Freeze Fracture, and Deep Etching, Severs, N.J. and Shotton, D.M., eds., Wiley-Liss, New York, 1995, 127.

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CONTENTS GENERAL INTRODUCTION .................................................................................... 591 1.

PRINCIPLES OF THE METHOD .................................................................. 592 1.1.

1.2. 1.3.

Small Markers .......................................................................................... 592 1.1.1. Small colloidal gold markers ...................................................... 592 1.1.2. NANOGOLD markers ................................................................ 593 1.1.3. Quantum dot (QD) markers ........................................................ 593 Silver Enhancement.................................................................................. 594 Gold Enhancement ................................................................................... 595

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 596

3.

MATERIALS/PRODUCTS/SOLUTIONS ...................................................... 597 3.1. 3.2. 3.3.

4.

PROTOCOL ...................................................................................................... 602 4.1. 4.2. 4.3. 4.4.

5.

Labeling with Small Markers ................................................................... 602 Appendix 1: Repeated Enhancement/Enhancement after Staining........... 605 Appendix 2: Double Labeling .................................................................. 606 Appendix 3: Inactivation and Blocking Solutions .................................... 606

ADVANTAGES/DISADVANTAGES.............................................................. 608 5.1. 5.2. 5.3. 5.4.

6.

Materials ................................................................................................... 597 Products .................................................................................................... 598 Solutions ................................................................................................... 599

Advantages of Small Markers .................................................................. 608 Disadvantages of Small Markers .............................................................. 608 Advantages of Enhancement Procedures.................................................. 609 Disadvantages of Enhancement Procedures ............................................. 609

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 610 6.1.

6.2.

Small Gold and QD Markers .................................................................... 610 6.1.1. NANOGOLD.............................................................................. 611 6.1.2. Ultra-small colloidal gold ........................................................... 611 6.1.3. Small QDs................................................................................... 611 Silver Enhancement Techniques............................................................... 611 6.2.1. HQ SILVER................................................................................ 611 6.2.2. R-GENT SE-EM......................................................................... 612 6.2.3. Danscher solution ....................................................................... 612

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6.3.

6.4.

Gold Enhancement Techniques................................................................ 612 6.3.1. GoldEnhance-EM ....................................................................... 612 6.3.2. Published recipes ........................................................................ 612 Silver Stabilization/Gold Toning.............................................................. 612

7.

OBSERVED RESULTS.................................................................................... 613

8.

REFERENCES .................................................................................................. 615



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GENERAL INTRODUCTION Very small (1 nm) gold markers, such as ultra-small (colloidal) gold (Aurion) and the gold compound NANOGOLD™ (Nanoprobes), have become more important in electron microscopic immunocytochemistry due to considerably improved sensitivity (see Figure on Chapter’s title page).1,11,22 The main reasons for improved sensitivity are less steric hindrance and reduced electrostatic repulsion (the colloidal gold surface is negatively charged). These markers penetrate into permeabilized cells and tissues more readily than larger gold colloids and, in ultrathin resin and cryo-section labeling experiments, the density of the label is often considerably higher when compared to colloid sizes of 4 nm or larger. In contrast to permeabilized samples, ultrathin resin sections cannot be penetrated by gold markers whatever the marker size used20 (see Figure 23.1). Also in the case of well preserved thawed cryo-sections obtained according to Tokuyasu,25 gold labeling is restricted mainly to the section’s surface, but (local) low matrix density, either due to inherent specimen properties or section damage, may allow some penetration, especially of 1 nm gold markers24 (see Figure 23.2). Due to their small size, the electron density of 1 nm gold particles is low and their visualization in a conventional transmission electron microscope (TEM) is difficult.22 This also holds true for small semiconductor nanocrystals, so-called quantum dot (QDs, e.g., QDOT® (Invitrogen) markers that exhibit low intrinsic contrast.6 Therefore, these markers have to be enlarged, either by deposition of metallic silver (so-called silver enhancement1,4,5,21,22) or gold (so-called gold enhancement26). Both enhancement techniques can be a source of problems, e.g., due to the low stability of the silver layer.1,18,21,22 The aim of this chapter is to help to choose the most useful small marker and enhancement method for a specific application and to cope with the typical practical problems associated with the use of very small markers and the application of enhancement techniques.

Figure 23.1 Cross-section through a gold labeled ultrathin resin section. Due to the high matrix density (polymerized resin, hatched area) luminal (white stars) and cytoplasmic (black) epitopes of vacuolar membrane antigens are only accessible for immunoreagents at the resin section surface (spheres, gold marker). Antibodies are not visualized.

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Figure 23.2 Cross-sections through goldlabeled, ultrathin Tokuyasu cryo-sections.  (a) In the case of well-preserved sections, gold markers are preferentially bound to the cryosection surface. Section damage (arrow) and local low matrix density in the vacuolar lumen allow some penetration of immunoreagents into the thawed cryo-section.  (b) In the case of weakly fixed and, therefore, extracted sections, antibodies and especially 1 nm gold markers can permeate the thawed cryo-section to a certain extent due to its low matrix density.

1. PRINCIPLES OF THE METHOD 1.1. Small Markers The preparation of 1 nm gold markers and their conjugation to ligands such as antibodies, are considerably more difficult when compared to larger ones.1 Therefore, commercial products are preferred.

 For example, unconjugated 1 nm gold colloids are less stable than larger ones.  Several recipes (for review see Reference 1). 

1.1.1. Small colloidal gold markers The smallest gold colloids in use have sizes between 0.8 nm and ~ 3 nm.1,14,17,21-23 The colloid surface is hydrophobic and negatively charged (due to adsorbed ions). The actual particle size is larger due to an additional hydration shell. Antibodies or antibody fragments are noncovalently (but tightly) bound via hydrophobic and electrostatic interactions. 

 Few commercial suppliers (Aurion (Ultra-small); British Biocell International (2 nm gold); Amersham/GE Healthcare (AuroProbe One).  Relatively large size distribution, e.g., ultra-small gold (Aurion).  Gold colloids have a rigid and thick coat of water dipoles due to their negative surface charge.

Figure 23.3 Fab (antibody) fragment noncovalently bound to 1 nm colloidal gold (black). The Fab fragment is 5 to 6 nm in length. Antigen-binding site (bright ends), IgGs may have more than one colloid disulphide bridge (white rectangle) (drawn to scale). bound.

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1.1.2. NANOGOLD markers NANOGOLD (Nanoprobes) is a gold compound with a gold core diameter of 1 to 1.4 nm and including the organic shell, 2.7 nm.10,11 Antibodies or antibody fragments are covalently linked to the organic shell via a hinge region thiol (one gold particle per Fab fragment or IgG). 

 One commercial supplier.  There are also double labeled NANOGOLD-antibody (fragment) conjugates, which are additionally linked to fluorochromes like fluorescein or Alexa dyes 488 and 594.  Figure 23.4 Fab fragment covalently linked to the organic shell (grey) of NANOGOLD (gold core, black). Antigen-binding site (bright ends), disulphide bridges (white rectangles) (drawn to scale).

Undecagold (Nanoprobes) is even smaller (gold core 0.8 nm, organic shell 2 nm in  Undecagold can be used to label-specific diameter),11 but cannot be satisfactorily sites on a protein for single particle enhanced by silver deposition. analysis.

1.1.3. Quantum dot (QD) markers QDs are fluorescent semiconductor nanocrystals made of substances, such as cadmium selenide as the core, which is surrounded by a core shell. QDs offer unique optical properties like high quantum yield, resistance to photobleaching and size-tunable emission spectra.6 Core and core shell exhibit moderate electron density6 (see Figure 23.9 a-c).



© 2009 by Taylor & Francis Group, LLC

 Several commercial suppliers of antibody-conjugated nanocrystals, e.g., QDOTs (Molecular Probes/Invitrogen), or EviTags (Evident Technologies/Antibodies Inc.).  Important for immunofluorescence microscopy.  The relatively small, green fluorescent QDOT 525 (Molecular Probes) has an electron dense spherical core/core shell of about 3 to 5 nm in diameter. The whole particle is 11 to 14 nm in diameter including the organic layer and bound (Fab`)2 fragments (see Figure 23.9 a-c).  Figure 23.5 Model of QDOT 525 conjugate: The core (black) is surrounded by a core shell (grey) and an organic layer (black ring), which is covalently linked to several (Fab`)2 fragments. Antigen-binding sites (bright ends), disulphide bridges (white rectangles) (drawn to scale).

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 There are QDs conjugated to secondary antibodies, which may be smaller (e.g., EviTag Adirondack Green), but it is not known whether they can be silver-enhanced or not.

1.2. Silver Enhancement Gold colloids and organo-gold clusters as well as QDs can act as nuclei, comparable to silver nuclei, in photographic development. In the presence of a reducing agent, the gold surface acts as a catalyst for the reduction of silver ions to metallic silver. The metallic silver deposited on the gold surface itself serves as nuclei for further reduction of silver ions to metallic silver, resulting in a growing silver layer.3,4

 Silver enhancement for immunogold electron microscopy was introduced in 1983.15  The enhancer solution contains a silver salt, a reducing agent, a buffer and often the so-called protective colloid gum arabic, which improves the enhancer’s performance: it makes the reaction more efficient and reproducible.1,3,4,21-23 

Figure 23.6 Principle of silver enhancement. The gold surface serves as a catalyst for the reduction of silver ions, e.g., by hydroquinone. Whereas silver enhancement of larger gold particles is relatively unproblematic, it turned out that enhancement of 1 nm gold is associated with a number of problems, such as inefficient enhancement, uneven growth and increased auto-nucleation due to prolonged enhancement time.1,21-23 A general drawback of silver enhancement is the instability of the silver layer caused by oxidation, which requires additional treatment or special storage conditions for protection.1,18

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 As a consequence, not all enhancers are suitable for 1 nm gold markers and QDOTs.1,21-23  OsO4 dissolves the silver layer; electron beam and air humidity cause dislocation/loss of silver (see Figure 23.14).

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We will describe the use of two commercial  Nickel grids should be used because silver enhancers and one published method: nickel does not interfere with silver enhancement, in contrast to copper.  HQ SILVER (Nanoprobes) contains  See Figure 23.10a, b. the protective colloid gum arabic. Its pH is near-neutral, the viscosity is high.  R-GENT SE-EM (Aurion) is a low  See Figure 23.10c. viscosity enhancer with near-neutral pH.  The Danscher method3,4,23 is an example of a published recipe of high efficiency, which is suited for all small markers mentioned. The enhancer contains the protective colloid gum arabic. The pH is low, the viscosity high.

 See Figure 23.11.  Two other published recipes deserve mentioning: The first one,16 a fast-working enhancer, can be used for copper grids. The second one7 causes less structural damage on whole mount samples or Tokuyasu cryosections than the Danscher method, probably due to a near-neutral pH.

1.3. Gold Enhancement In principle, the deposition of gold instead of silver offers potential advantages: it confers stability to the enhanced particle, thereby making it resistant to oxidation.1,18,26

GoldEnhance™-EM (Nanoprobes) is the only commercial product available (Near neutral pH, low viscosity, less sensitive to buffer salts).26 Gold chloride treatment has been described as being able to enlarge small colloidal gold markers (for review see References 1 and 18).

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 A process similar to silver enhancement.  Higher particle density (improved contrast).  Improved backscatter detection in the scanning electron microscope (SEM).  Caused by OsO4 treatment, beam damage, and air humidity.   In our lab, gold markers bound to resin sections can be readily enhanced (see Figure 23.12), but treatment of labeled cryo-sections results in strong background.   In our lab, gold enhancement following published procedures leads to loss of gold rather than to gold particle growth1,18 (see Figure 23.13). 

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2. SUMMARY OF THE DIFFERENT STEPS Ultra(cryo)microtomy and mounting of sections:  Mounting ultrathin cryo-sections (swimming on the surface of the pickup drop; see Chapter 19) or resin sections on plastic- and carboncoated13 grids (Figure 23.7, top and middle).  Transfer of grids (upside down) to drops of buffer placed on a hydrophobic film in a dish covered by a glass plate (moist chamber) (Figure 23.7, middle and bottom).  Optional: Cryo- and resin sections mounted on grids can be stored prior to immunolabeling.8 Figure 23.7 Mounting of sections on grids (top, middle) and transfer of grids to drops of buffer (middle, bottom) for immunolabeling.

Figure 23.8 Labeling of grids in moist chamber. Grids can be transferred with a forceps or a loop (see also Chapter 21). 1: Inactivation of residual fixative molecules (mainly aldehyde groups) (optional) 2: Blocking unspecific protein binding sites 3, 4: Antibody labeling 5, 6: Marker incubation 7, 8: Fixation 10, 11: Silver or gold enhancement 12, 13: Stabilization of silver layer (optional) (gold toning) 14: Staining (heavy metals) 15, 16: Drying (resin sections) or methyl cellulose embedding (cryo-sections)

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3. MATERIALS/PRODUCTS/SOLUTIONS 3.1. Materials  Vortex  Centrifuge  Forceps (antimagnetic)  Petri dish (glass)  Dish (stainless steel) with glass plate  Hydrophobic film  Loops (stainless steel, 3 mm in diameter) attached, e.g., to pipette tips  Tissue paper/wipes  Pioloform or Formvar coated and carbon-covered nickel grids for silver enhancement, copper or nickel grids for gold enhancement  Pipettes (glass or plastic)  Pipette tips  Desiccator (made of glass or plastic)  Silica gel-desiccant  Black cardboard  Filter paper  Water bottle (containing distilled water)  H2O

double

 Microtubes (0.5 mL, 1.5 mL, 2 mL)

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 For antibodies, markers, and enhancers; e.g., Vortex-Genie 2, Scientific Industries Inc, Bohemia, New York., USA.  For all solutions used; e.g., Centrifuge #5415D, Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany.  Grid transfer (nickel grids!); e.g., # 5/45 or #7, Dumont & Fils, Switzerland.  For lead staining.  Moist chamber for labeling and enhancement.  For incubating grids on drops; Parafilm “M”, Pechiney Plastic Packaging, Chicago, USA.  Self-made loops from platinum wire for grid transfer and for drying cryo-sections.  For generating humid air; e.g., Kleenex®, Kimberly-Clark Corp, USA.  For supporting sections (100 to 200 mesh nickel grids), e.g., Stork Veco B.V, Eerbeek, The Netherlands; or Gilder Grids, Grantham, England.  For plastic and carbon covering of grids, see Reference 13.  For placing drops.  For dilution, placing drops, etc. Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany.  For storing silver-enhanced sections in dry air, nitrogen gas, or vacuum.  For dry storage of silver-enhanced sections.  For dark chamber for light sensitive reactions (e.g., silver enhancement).  For drying grids, usually Whatman 541, Whatman International Ltd, Maidstone, England.  For jet wash of grids.  Water, double distilled (on quartz) or Milli Q.  For mixing solutions, for storing frozen aliquots; Eppendorf-Netheler-Hinz GmbH, Hamburg, Germany or Sarstedt AG & Co, Nümbrecht, Germany.

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3.2. Products  Ammonium hydroxide (NH4OH)                    

         

 For example, Merck Eurolab, VWR International; Darmstadt, Germany. Sigma-Aldrich, Fluka, Buchs, Bovine serum albumin (BSA)  Switzerland. fraction V Bovine serum albumin, acetylated  Aurion, Wageningen, The Netherlands. (BSA-c)  Merck. Citric acid (C6H8O7 x H2O)  Aurion, Sigma. Cold water fish gelatin Disodium hydrogenphosphate  Merck. (Na2HPO4)  Merck. Ethanol  Gibco, Invitrogen, Eugene, Oregon, Fetal / new born calf serum USA.  Merck. Gelatin  Sigma #G-5882 (keep frozen). Glutaraldehyde (aqueous, 25%)  Merck. Glycine  See tetrachloroauric acid trihydrate. Gold chloride  Merck. Gum arabic  Merck. HEPES  Merck. Hydroquinone  Sigma #L-7771 (Caution: light L-(+)-lactic acid sensitive). Lead (II) citrate trihydrate  Fluka, Buchs, Switzerland; or Agar Scientific. (Pb3(C6H5O7)2 x 3H2O)  Serva 29834, Heidelberg, Germany. MES (C6H13NO4S)  Sigma M-6385. Methyl cellulose  Regular non fat milk powder. Milk powder (non fat, dried)  Nanoprobes (Stony Brook,New York, NANOGOLD™ conjugates USA).  Ultra-small gold and NANOGOLD conjugates can be stored at 20°C after mixing equal parts of marker and 99% glycerol.  Invitrogen. Nonimmune serum / normal serum  Merck. N-propyl-gallate Ovalbumin (albumin from egg white)  Sigma.  Merck. Oxalic acid (C2H2O4 x 2H2O)  Merck. Potassium chloride (KCl) Potassium dihydrogen phosphate  Merck. KH2 PO4  Molecular Probes, Invitrogen, Eugene, Quantum dot (QDOT®) conjugates Oregon, USA.  Merck. Sodium borohydride (NaBH4) Sodium chloride (NaCl)  Merck.  Merck. Sodium hydroxide (NaOH) pellets

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Immunolabeling of Ultra-thin Sections with Enlarged 1 nm Gold or QDots

 Sodium thiosulfate (Na2O3S2)  Tetrachloroauric(III)acid trihydrate (AuCl4H x 3H2O)  Trisodium citrate dehydrate (C6H5Na3O7 x 2H2O)  Tween-20®  Ultra-small gold conjugates  Uranyl acetate (UO2(CH3COO)2 x 2H2O)  Uranyl oxalate

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 Merck.  Merck #101582.  Merck.  Sigma, Serva.  Aurion.  Stain for electron microscopy. EMS, Fort Washington, Pennsylvania, USA or SPI Supplies, West Chester, Pennsylvania, USA.  See Staining solutions. 

3.3. Solutions 1. R-GENT SE-EM Activator, initiator,  Aurion, Wageningen, The Netherlands.  Shelf life ~ 18 months from date of enhancer production.  Preparation of developer:  Light insensitive.  Put 40 drops of activator (1385 µL) into the developer bottle  The concentrated initiator can be stored  Add 1 (36 µL) drop initiator to the in a freezer. developer bottle  Shelf life of developer is about one  Mix well on a vortex month.  Use nickel grids.  Preparation of final enhancer:  Mix 20 drops (870 µL) of enhancer with 1 drop (34 µL) of developer  Mix well on a vortex 2. HQ SILVER  Moderator, activator, initiator  Equal amounts of moderator, activator, and then initiator are thoroughly mixed before use Danscher (acidic) silver enhancer3,23 Stock solutions: Gum arabic: 33% (w/v) in H2O Hydroquinone: 0.57 g in 10 mL H2O (5.7%, 0.52 M)  Citrate buffer: 2.55 g citric acid (23.5%, 1.5 M) + 2.35 g trisodium citrate dehydrate (23.5%, 0.5 M), add H2O to make 10 mL (pH 3.53.8)  Silver lactate: 7.3 g in 10 mL H2O (0.73 %, 37 mM) 3.   

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 Nanoprobes, Stony Brook, New York, USA  Solutions should be kept frozen in small aliquots.  Caution: Light sensitive.  Shelf life ~ 30 months.  Use nickel grids.  Stir for one day, centrifuge (30,000 g, 2 h, 4°C).  Gum arabic, citrate buffer, and hydroquinone can be premixed and stored in a freezer. Silver lactate has to be stored separately in a freezer.  Caution: Light sensitive!  Caution: Silver lactate salt may age with time and change properties. If in doubt, buy a new package.

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 Preparation of final enhancer:  Mix 6 parts gum arabic stock with 1 part citrate buffer stock and 1.5 parts  Use nickel grids. hydroquinone stock. Add 1.5 parts  Caution: Moderate light sensitivity after silver lactate stock and mix mixing. thoroughly. 4. Fast working silver enhancer16  Suitable for copper grids.  See above, replace citrate buffer by  Caution: Light sensitive! 0.2 M HEPES buffer, pH to 6.8 with NaOH.  Reduced ultrastructure damage 5. Neutral silver enhancer7  Stock solutions: (probably due to near-neutral pH).  0.5 M MES adjusted to pH 6.15 with NaOH  Gum arabic: 33% (w/v) in H2O  N-propyl gallate: dissolve 10 mg NPG in 0.25 mL ethanol, then add 4.75 mL of H2O  Silver lactate: 36 mg in 5 mL H2O  Caution: Light sensitive!  Preparation of final enhancer:  2 parts MES stock, 5 parts gum arabic stock, 1.5 parts N-propyl gallate stock,  Use nickel grids. and 1.5 parts silver lactate stock.  The first three components are mixed 30 min before use, the N-propyl  Caution: Light sensitive (dark room)! gallate stock is added 2 min before use. 6. GoldEnhance™-EM  Mix equal amounts of enhancer, activator, initiator, and buffer.  Mix enhancer and activator, wait 5 to 10 min, then add initiator and buffer.

 Nanoprobes, Stony Brook, New York, USA.  Shelf life ~ 36 months.  Use copper or nickel grids.  For numerous modifications, see www.nanoprobes.com.

PBS buffer (pH 7.4)  Standard buffer. 137 mM sodium chloride (NaCl) 2.7 mM potassium chloride (KCl) 8.1 mM disodium hydrogen-phosphate (Na2HPO4)  1.5 mM potassium dihydrogen phosphate (KH2 PO4)

7.   

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8. Staining solutions  Uranyl oxalate or neutral uranyl acetate:25  2% (w/v) uranyl acetate in 0.15 M oxalic acid, pH 7.4

 Stains for resin and cryo-sections.  Stir a 0.3 M oxalic acid solution vigorously and add slowly the same volume of a 4% (w/v) aqueous uranyl acetate solution. Adjust pH to 7.4 with 10% ammonium hydroxide.25  Store in refrigerator.

 4% (w/v) aqueous uranyl acetate  1% (w/v) aqueous uranyl acetate

 Store in refrigerator.  Store in refrigerator.

9. Methyl cellulose  2% (w/v) methyl cellulose in H2O (25 centipoises).  Methylcellulose–uranyl acetate (UA) solution:  3% UA in H2O  2% aqueous methyl cellulose  Mix 1 (1.5) part UA with 9 (8.5) parts methyl cellulose.

 Embedding of cryo-sections.  Centrifuge at 100,000 g at 4°C.  Store in refrigerator.  See also Chapter 19.  Stronger contrast and darker structures with higher UA concentration. Too high UA concentration causes membrane blebbing.25

10. Lead citrate staining  Recipe

 Stain for resin sections.  See Chapter 21.

11.

 See gold toning.

Gold chloride

12. Gold toning  Protection of silver layer.  0.05% aqueous gold chloride (diluted  Store in darkness (at 4°C). from 1 to 2% stock solution of tetrachloroauric acid trihydrate) 13. Fixative  0.5 to 1% aqueous glutaraldehyde

 Stabilization of antigen-antibody-marker complex prior to enhancement.

14. Blocking buffer  See Appendix 3 for alternative reagents.  0.5% BSA plus 0.5% nonfat milk powder in PBS 15. Glycine-PBS  Inactivation  50 mM glycine in phosphate buffer aldehyde groups. saline (PBS)

of

residual

16. Silver fixer, e.g.,  To stop silver enhancement.16  250 mM sodium thiosulfate in 20 mM HEPES (pH 7.4)

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reactive

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4. PROTOCOL 4.1. Labeling with Small Markers Labeling with small markers

 Indirect labeling.  All of the following steps are carried out at room temperature (20 to 22°C) in a moist chamber.  The entire immunolabeling and silver enhancement of sections mounted on grids is carried out on 50 to 100 L drops of solutions placed on a hydrophobic film (in a dish covered by a glass plate) (see Chapter 21).  All solutions should be centrifuged before use (13,000 g/2 to 3 min) to pellet aggregates and debris and to remove bubbles.

Ultrathin resin/cryo-sections collected on  Nickel does not interfere with silver Pioloform and carbon-coated13 nickel grids enhancement, in contrast to copper.  Copper and nickel grids can be used for (e.g., 100 or 200 mesh) gold enhancement.  Formvar or collodium coating is also possible.13  1. Inactivation of residual fixative  Mainly aldehyde groups (see Section molecules 4.3) Optional.  Glycine (50 mM) in 510 min  See Section 4.4. PBS 2. Blocking unspecific protein-binding sites  Blocking buffer 2 × 710 min  See Section 4.4, for different recipes. 0.5% BSA, 0.5% milk powder in PBS 3. Primary antibody incubation   Antibody diluted in 10 µL/grid  Detection of antigens. blocking buffer 3060 min  Final (specific) IgG concentration 1 to 5 µg/mL.  Moist chamber.  For double labeling, see Section 4.2. 4. Wash  Blocking buffer

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6 × 35 min  Removal of unbound antibodies.

Immunolabeling of Ultra-thin Sections with Enlarged 1 nm Gold or QDots

5. Marker incubation  Marker conjugate diluted in blocking buffer 6. Wash  Blocking buffer  PBS 7. Fixation  0.5% glutaraldehyde in PBS 8. Wash  H2O

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 Detection of bound primary antibodies. 10 µL/grid  NANOGOLD conjugates: 1:50 to 1:100.  Ultra-small gold conjugates 1:20 to 1:50. 3060 min  QDOT conjugates: 1:20.  Moist chamber.  Removal of unbound markers. 2 × 35 min 4 × 35 min  To stabilize antigen-antibody-marker 5 min complexes during subsequent treatment.  To remove ions that could interfere with 5 × 23 min silver enhancement (e.g., chloride).

9. Drying (only resin sections)

 Optional.

10. Silver (a-c) or gold (d) enhancement

 To make small markers visible.  Caution: Speed of enhancement depends on temperature, higher temperatures will speed up deposition of silver or gold.  Enhancement of larger gold is less problematic. All the silver enhancers mentioned as well as GoldEnhance-EM can be used. Caution: Enhancement time is shorter due to larger colloid size.

10a. HQ SILVER  Incubation  NANOGOLD  Ultra-small gold  QDOT 525 10b. R-GENT SE-EM  Incubation  NANOGOLD  Ultra-small gold  QDOT 525 10c. Danscher solution  Incubation  NANOGOLD  Ultra-small gold  QDOT 525

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 Three solutions. 50100 µL/grid  Caution: Light sensitive. in darkness 89 min 56 min  (Inefficient enhancement). ~ 5 min  Four solutions. 50100  Light insensitive. µL/grid  Not recommended, no enhancement. 4050 min  Not recommended, inefficient enhancement.  Four solutions (or two solutions when stored frozen).

50100 µL/grid  Moderate light sensitivity. in darkness 3545 min 3035 min  Best enhancer for QDOT 525. 30 min

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10d. GoldEnhance-EM  Incubation  NANOGOLD  Ultra-small gold  QDOT 525 11. Wash  H2O

 Four solutions. 50100 mL/grid  Low light sensitivity. (in darkness) 3060 sec 3060 sec  Not recommended, inefficient enhancement.  On drops, to stop reaction (and remove 5 × 4 min gum arabic).  R-GENT SE-EM: 5 × 2 min is sufficient.  For more rigorous halt of reaction, if desired: fixation with 250 mM sodium thiosulfate, 20 mM HEPES, pH 7.4 (5 min).16

12. Stabilization of silver layer by gold  Optional, to prevent redistribution or toning loss of silver due to oxidation (not necessary for gold enhancement).  0.05% gold chloride 13. Wash  H2O 14.

50 µL/grid  Freshly diluted from stock solution. 15 min  To remove ions that could interfere with 5 × 2 min staining, e.g., phosphate.

Staining

14a. Resin sections  1% aqueous uranyl acetate (UA)  Wash  H2O  Drying  Lead citrate solution, in Petri dish with NaOH pellets  Wash  H2O  Drying

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20 µL/grid  See Step 15b. 15 min  Depending on desired contrast. 2 × 5 sec  Final washing in a jet of H2O (wash bottle).  Optional, e.g., to check labeling in TEM. 20 µL/grid 15 sec3 min  Depending on desired contrast.  Keep Petri dish closed to prevent contact with CO2. 2 × 1 sec  Final washing in a jet of H2O from a wash bottle.

Immunolabeling of Ultra-thin Sections with Enlarged 1 nm Gold or QDots

14b. Cryo-sections  Uranyl oxalate  Or 1% UA

 Wash  H2O

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20 µL/grid  Optional. 10 min  We have never observed deleterious effects of UA or neutral uranyl oxalate on the silver layer during resin or cryo-section staining or in preembedding labeling experiments when samples were treated with aqueous UA for one hour before dehydration. However, silver-enhanced gold in epoxy sections (if samples were enhanced before embedding) is sensitive to oxidation (electron beam, air humidity). 2 × 1 sec

15. Embedding (only cryo-sections) 50 µL/grid  Numerous recipes for staining and a. Infiltration with 0.3 10 min embedding, see Chapter 19. to 0.5% UA in on ice  Facilitates infiltration with methyl methyl cellulose cellulose. b Picking up grids with a loop and  Dry methyl cellulose film should display removal of excess methyl cellulose by an interference color between gold and touching the loop at an angle of 45° to blue, see Chapter 19. 90°C to a filter paper (section side down). 16.

Air-drying

 In loop; after drying, remove grids carefully (see Chapter 19).

17. Storage (only for nonstabilized silver)  In dry air, under nitrogen gas or under  To prevent redistribution or loss of silver vacuum (in desiccator). due to oxidation.

4.2. Appendix 1: Repeated Enhancement/Enhancement after Staining  Repeated enhancement is possible  Except for the area illuminated by the 21,22 even after inspection in the TEM electron beam. (labeled) Lowicryl sections that were already stained with aqueous UA and  After washing the sections on several Pb citrate can be silver-enhanced. drops of H2O.

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4.3. Appendix 2: Double Labeling 1. Simultaneous or sequential labeling and simultaneous enhancement: After completing the labeling procedure (sequential or simultaneous labeling of two antigens), both markers can be enhanced in one enhancement step.

 Simultaneous labeling: Two specific antibodies, and later two markers are applied simultaneously.  Sequential labeling: In a first round, the first specific antibody is applied, followed by the corresponding marker. In a second round, the second specific antibody is applied, followed by the corresponding marker. A fixation step (e.g., 0.5% glutaraldehyde) after the first labeling round is recommended to stabilize the antigen-antibody-marker complex, provided the “second” antigen tolerates this treatment. Sections should be washed and blocked again before labeling the “second” antigen.  Sequential enhancement: Only possible if the “second” antigen tolerates the enhancement procedure.

2. Sequential labeling and sequential enhancement: Sections can be enhanced twice; after labeling the first antigen and after labeling the second antigen. This means that the first gold marker is silver enhanced twice.

 It is possible to use 1 nm gold markers twice (only for sequential labeling/enhancement19).  Caution: Sizes of silver-enhanced particles are not uniform, so care has to be taken to ensure that there is no overlap in particle size distribution curves of the two enhanced markers.  Caution: Gold chloride treatment results in partial disintegration of the silver layer and, therefore, is not compatible with double labeling or quantification studies.

4.4. Appendix 3: Inactivation and Blocking Solutions Undesirable interactions between antibodies and specimen/resin can be a serious problem. Primary antibodies and secondary antibodies can contain antibodies that recognize epitopes, which differ from the epitope under investigation. Non-specific background can derive from reaction of antibodies with residual fixative molecules, mainly aldehyde groups.

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 Such antibodies can be eliminated, e.g., by immunoaffinity purification12 (column, beads, dot blot) or by absorption on specimens lacking the antigen under investigation.

Immunolabeling of Ultra-thin Sections with Enlarged 1 nm Gold or QDots

From a theoretical point of view, fixative inactivation should be done directly after completing fixation. Most probably, after resin embedding, but also after cryosectioning, there are no reactive fixative sites left. Nonspecific background can also derive from hydrophobic interactions between sample and antibodies, and from ionic interactions, e.g., from positively charged specimen compounds, such as histone proteins in the nucleus, starch grains in plant cells, or molecules like polycations and collagen. In contrast, negatively charged areas (phospholipids, aldehydefixed proteins) can repulse negatively charged antibodies, thereby reducing label density. In general, blocking should be done not only before but also during antibody and marker incubation because, with time, blocking molecules can also detach from the specimen. For a detailed discussion, see Reference 9 and Web site of Aurion (www.aurion.nl).

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 Useful inactivation strategies: Aldehyde groups can be inactivated by amino acids like glycine or lysine (50 mM) and molecules like ammonium chloride (NH4Cl; 50 mM).

 Useful protein blocking strategies: Gelatin (0.2 to 0.5%), liquid cold water fish skin gelatin (0.5% or 0.005%), ovalbumin (0.5%), bovine serum albumin (BSA) (0.5%), fetal or newborn calf serum (0.5%), nonimmune serum (0.5 to 5%), especially from species used for secondary antibody production), and nonfat dry skim-milk are the most common blocking agents.9 BSA (negatively charged) is often used together with (cold water fish) gelatin or nonfat dry skimmilk powder. In our lab, it turned out that milk powder is the strongest blocking agent. Acetylated (linearized) BSA-c (0.1%, used during antibody and marker incubation) displays increased negative charge and hydrophobicity (www.aurion.nl). It is thought to mimic gold surface properties, therefore, it competes with gold colloids during binding to unspecific sites. Caution: Do not mix BSA-c with gelatin as gelatin exhibits a high tendency (low gold number) to adsorb to gold surfaces. Electrostatic interactions can be also influenced by buffers displaying increased ionic strength, e.g., 0.5 M NaCl or KCl. Hydrophobic interactions can be also influenced by detergents added to the antibody solution, e.g., 0.05% Tween-20, and are also used to reduce background. It should be kept in mind that detergents also extract lipids/cytoplasmic material and enhance wettability (i.e., accessibility of antigens, e.g., in entire mount labeling experiments).

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5. ADVANTAGES/DISADVANTAGES 5.1. Advantages of Small Markers  Often considerably higher label density and efficiency when compared to larger gold markers.  NANOGOLD seems to be the most sensitive gold marker available for labeling resin sections, cryo-sections or permeabilized specimens.  NANOGOLD (with bound fluorochrome) and fluorescent QDs can be used for correlative light/electron microscopy.  NANOGOLD conjugation kits available.  Custom labeling service for colloidal ultra-small gold and NANOGOLD.

 Necessary to detect scarce antigens.  Most probably, fluorochrome-coupled IgGs are even more sensitive (for LM).  Due to less electrostatic repulsion and smaller hydration shell when compared to colloidal gold (see above).  Useful for permeabilized samples, but also for resin and cryo-sections, which can be labeled in parallel for LM and TEM.  Covalent conjugation to thiols or primary amines of antibodies.

5.2. Disadvantages of Small Markers  Low intrinsic contrast, requires additional enhancement step.  Only a few commercial suppliers, quality of conjugate should be checked before use.  Risk of increased background.      

 Enhancement is a source of additional problems.  Use test sample with known label density; check every batch for aggregates, label efficiency, unspecific background.  For example, due to higher sensitivity (see Section 4.3). After resin labeling, unenhanced  Enhancement should be performed NANOGOLD particles are unstable. directly after the immunolabeling procedure. NANOGOLD and Qdots are degraded  Silver or gold enhancement should be by some chemicals and high done directly after immunolabeling. temperatures. No protein A-1 nm gold conjugate.  Due to inactivation of protein A by coupling.  Due to difficulty in obtaining uniformly Quantitation is difficult. sized enhanced particles and unknown stochiometry. Double/multiple labeling is difficult,  Due to difficulty in obtaining uniformly but possible. sized enhanced particles (see Section 4). QDs are relatively large.  Due to the “coating” of the nanocrystal core necessary for stabilization and coupling to antibody (fragments).  Reduced penetration requires strong permeabilization for whole mount labeling.

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5.3. Advantages of Enhancement Procedures  Small sensitive markers with low intrinsic contrast can be visualized in LM, conventional TEM, STEM, and SEM.21,22

   

 Final marker size can be freely chosen  Suitable for overview and detailed by varying enhancement time. images.   Gold-enhanced particles are stable  In contrast to silver-enhanced particles. against oxidation.  For example, by OsO4, beam damage, air humidity.   Gold chloride treatment (gold toning)  Enhanced gold markers are no longer stabilizes silver-enhanced particles. sensitive to oxidation. 

5.4. Disadvantages of Enhancement Procedures  The use of nickel grids is strongly  Exception: The recipe published by Lah 16 recommended for silver enhancement. et al is compatible with copper grids.   Especially true for the most efficient  Often long enhancement times. enhancers.   Not all commercial enhancers are  See 10, Section 4.1. useful for all markers.  Large size distribution of enlarged  Double (multiple) labeling difficult but possible (see Section 4.2). gold and QDs.  Quantitation difficult (unknown stochiometry).   Silver and gold enhancement products  Quality of enhancer should be checked before use, e.g., by using a test sample can be a source of problems. (sections with known label density); check on silver precipitates (unspecific background), enhancement efficiency, enhancement time.   Silver is sensitive to OsO4 treatment  Requires gold toning before OsO4 treatment, otherwise OsO4 treatment should (oxidation). be reduced in time and concentration (e.g., 0.1%; 15 min) or omitted; or gold has to be “over” enhanced.2

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 Silver layer is sensitive to oxidation  Requires gold chloride treatment or when sections have been exposed to special storage conditions for long-term electron beam and/or air humidity storage (e.g., in nitrogen gas or in vacuum). (storage) (see Figure 23.12).  Some enhancers are harmful to  Use enhancer with neutral pH or with ultrastructure (especially important for short enhancement time (R-GENT SE-EM, cryo-section, preembedding, and HQ SILVER, or see references 2, 7, 16.). whole mount labeling).2,7,16  Gold enhancement (GoldEnhance-EM) may be problematic with respect to enhancement efficiency, reproducebility, and background. Published enlargement procedures using gold chloride do not work in our lab.1,18

 Use test sample with known label density; check on enhancement time, enhancement efficiency and gold precipitates (unspecific background).  Gold markers become even smaller18 (see Figure 23.13).  The silver layer decomposes in several smaller silver colloids18 (see Figure 23.13).

 Silver-enhanced gold is not compatible  Silver-enhanced gold emits fluorescent with fluorescence markers in light light when illuminated with a Hg lamp.21,22 microscopy.

6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Small Gold and QD Markers  High sensitivity markers should be  Reduced steric hindrance, reduced used for low copy number antigens. electric repulsion, improved penetration properties.  All markers require routine tests for quality.  Useful for resin section, cryo-section,  Limited suitability for double or multiple and preembedding/whole mount label- labeling due to large size distribution. ing.  All markers require additional  Susceptible to enhancement artifacts. enhancement for LM, and conventional TEM and SEM.  Suitable for high resolution studies as  well as for overview images.   

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6.1.1. NANOGOLD  NANOGOLD is probably the most  For low copy number antigens. sensitive gold marker available.  Not all enhancers work with NANOGOLD.  Best “prerequisites” for penetration  Especially for cryo-section and preinto sample. embedding/whole mount labeling.  Gold/fluorochrome conjugate.  For correlative light/electron microscopy

6.1.2. Ultra-small colloidal gold



 Sensitivity slightly lower compared to  For low copy number antigens. NANOGOLD markers.  Penetration properties slightly poorer  Relevant to cryo-section and prewhen compared to NANOGOLD embedding/whole mount labeling. markers.

6.1.3. Small QDs



 Sensitivity between 4 to 6 nm colloidal  For low copy number antigens. gold and 1 nm gold markers  For cryo-section and resin section labeling, limited suitability for whole mount labeling.  Fluorescent marker of low electron  For correlative light/electron microsdensity, which can be silver-enhanced. copy. 

6.2. Silver Enhancement Techniques  Necessary to visualize small markers  All enhancers require routine tests for in LM, conventional TEM and SEM. quality.  Nickel grids should be used (for TEM).  Suitable for overview and detailed  Caution: Silver is sensitive to oxidation; images. stabilization useful prior to OsO4 treatment and for long-term storage (see Section 6.4). 6.2.1. HQ SILVER  Suitable for all gold and QDOT  Tailored specifically for NANOGOLD  Slightly reduced efficiency for markers and all labeling techniques. QDOT 525.  Low/no interference with ultrastruc-  Important for cryo-section and preembedding/whole mount labeling. ture preservation.

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6.2.2. R-GENT SE-EM  Suitable for all colloidal gold markers  Tailored specifically for ultra-small gold. (all labeling techniques).  Not suitable for NANOGOLD and QDOT 525.  No interference with ultrastructure  Important for cryo-section and prepreservation. embedding/whole mount labeling.

6.2.3. Danscher solution  Suitable for all gold and QDOT  Easy to prepare. markers and all labeling techniques.  Relatively cheap.  Premixed aliquots can be stored frozen.  May be harmful to ultrastructure in cryosections and preembedding/whole mount labeling experiments.

6.3. Gold Enhancement Techniques  Gold enhancement results in stable  Gold-enhanced particles are not sensitive either to OsO4 treatment, electron beam or gold particles. air humidity.

6.3.1. GoldEnhance-EM  Suitable for all gold markers.

 Limited reproducibility?  Slightly reduced efficiency?  Not suitable for QDOT markers.  Strong background on labeled cryosections.

6.3.2. Published recipes  Not recommended.

 Treatment with gold chloride is not suitable for enlargement of small gold markers.

6.4. Silver Stabilization/Gold Toning  Recommended for stabilization of silver-enhanced gold particles for long term storage and against the action of OsO4.  Alternatively, storage in dry air, under nitrogen gas or vacuum (e.g., in desiccator).

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 Gold chloride-treated particles are no longer sensitive to oxidation.  Caution: Gold chloride treatment slightly reduces the size of silver-enhanced particles and leads to their disintegration (see Figure 23.15).

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7. OBSERVED RESULTS  Centriole labeling in ultrathin cryosections of U2OS cells fixed with 8% FA. C-Nap1 was detected at the proximal end with specific rabbit antibodies and protein A-15 nm gold (left) or silver-enhanced NANOGOLD coupled to goat anti-rabbit F(ab)2 fragments (right). Bar = 250 nm

 Figure on Chapter’s title page

Ultrathin resin sections of Escherichia. coli cells embedded in methacrylate Lowicryl HM20 were incubated with rabbit antibodies raised against the outer membrane protein OmpA. In this overproducing strain, OmpA is mainly found in the periplasmic space. Markers used were protein A-6 nm colloidal gold, ultra-small (colloidal) gold, NANOGOLD, or QDOT 525. UA and lead citrate staining were omitted.

            Figure 23.9 QDOT 525F(ab`)2 marker: (a) Without staining. (b) Negatively stained with uranyl acetate. Bar = 50 nm QDOT 525 marker after immunolabeling of E. coli sections: (c) Without enhancement. (d) For comparison, labeling with protein A-6 nm gold marker is shown. Bar = 100 nm   Figure 23.10 HQ SILVER enhancer applied to: (a) NANOGOLD marker (8 min). (b) Ultra-small gold marker (5 min). (c) R-GENT SE-EM enhancer applied to ultra-small gold marker (40 min). 

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Bar = 100 nm

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 Figure 23.11 Danscher enhancer applied to: (a) NANOGOLD marker (35 min). (b) Ultra-small gold marker (30 min). (c) QDOT 525 marker (30 min). Bar = 100 nm  Figure 23.12 GoldEnhance-EM enhancer applied to NANOGOLD marker (30 sec). Bar = 100 nm  Caution: High background on immunolabeled ultrathin cryo-sections  Figure 23.13 Gold chloride treatment reduces gold size rather than leading to enhancement: (a) Untreated control. (b) Protein A-6 nm gold treated with 0.05% gold chloride for 30 min. Bar = 100 nm  Figure 23.14 The silver layer is sensitive to oxidation, e.g., during storage of labeled sections: (a) Image was taken directly after silver enhancement. (b) Image was taken several weeks after silver enhancement (same section, stored in humid ambient air). Bar = 100 nm  Figure 23.15 Gold chloride treatment (0.05%, 10 min, 20°C) stabilizes silver but leads to partial disintegration of the silver layer: (a) Untreated silver-enhanced control. (b) After gold chloride treatment, the silver layer decomposes into several smaller silver particles. Bar = 100 nm

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8. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

18.

Baschong W. and Stierhof, Y.-D. Preparation, use, and enlargement of ultrasmall gold particles in immunoelectron microscopy. Microsc. Res. Techn., 42, 66, 1998. Burry, R.W. Pre-embedding immunocytochemistry with silver-enhanced small gold particles, in Immunogold-Silver Staining, Hayat, M.A., ed., CRC Press, Boca Raton, FL, USA, 1995, 217. Danscher, G. Histochemical demonstration of heavy metals: A revised version of the sulphide silver method suitable for both light and electron microscopy. Histochem., 71, 1, 1981. Danscher, G. et al. Trends in autometallographic silver amplification of colloidal gold particles, in Immunogold-Silver Staining, Hayat M.A., ed., CRC Press, Boca Raton, FL, USA, 1995, 11. Dettmer, J. et al. Vacuolar H+-ATPase activity is required for endocytic and secretory trafficking in Arabidopsis. Plant Cell, 18, 715, 2006. Giepmans, B.N.G. et al. Correlated light and electron microscopic imaging of multiple endogenous proteins using quantum dots. Nat. Methods, 2, 743, 2005. Gilerovitch, H.G. et al. The use of electron microscopic immunocytochemistry with silver-enhanced 1.4 nm gold particles to localize GAD in the cerebellar nuclei. J. Histochem. Cytochem., 43, 337, 1995. Griffith, J.M. and Posthuma, G. A reliable and convenient method to store ultrathin thawed cryo-sections prior to immunolabelling. J. Microsc., 212, 81, 2003. Griffiths, G. Fine Structure Immunocytochemistry, Springer-Verlag, Berlin, Germany, 1993. Hainfeld, J.F. and Furuya, F.R. A 1.4-nm cluster covalently attached to antibodies improves immunolabeling. J. Histochem. Cytochem., 40, 177, 1992. Hainfeld, J.F., and Furuya, F.R. Silver-enhancement of Nanogold and undecagold, in Immunogold-Silver Staining, Hayat, M.A., ed., CRC Press, Boca Raton, FL, USA, 1995, 71. Harlow, E. and Lane, D. Using Antibodies, Cold Spring Harbor Laboratory Press, New York, 1999. Hayat, M.A. Principles and Techniques of Electron Microscopy, Cambridge University Press, Cambridge, UK, 2000. Hermann, R., Schwarz, H., and Müller, M. High precision immunoscanning electron microscopy using Fab fragments coupled to ultra-small colloidal gold. J. Struct. Biol., 107, 38, 1991. Holgate, C. et al. Immunogold-silverstaining: New method of immunostaining with enhanced sensitivity. J. Histochem. Cytochem., 31, 938, 1983. Lah, J.J., Hayes, D.M., and Burry, R.W. A neutral pH silver development method for the visualization of 1-nm gold particles in pre-embedding electron microscopic immunocytochemistry. J. Histochem. Cytochem., 38, 503, 1990. Leunissen, J.L.M. and Van De Plas, P. Ultrasmall gold probes and cryoultramicrotomy, in Immuno-Gold Electron Microscopy in Virus Diagnosis and Research, Hyatt A.D. and Eaton B.T., eds., CRC Press Inc., Boca Raton, FL, USA, 1993, 327. Pohl, K. and Stierhof, Y.-D. Action of gold chloride (“gold toning“) on silverenhanced 1 nm gold markers. Microsc. Res. Techn., 42, 59, 1998.

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20. 21. 22. 23. 24. 25. 26.

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Sibon, O.C.M. et al. Ultrastructural localization of epidermal growth factor (EGF)receptor transcripts in the cell nucleus using pre-embedding in situ-hybridization in combination with ultra-small gold probes and silver enhancement. J. Histochem., 101, 223, 1994. Stierhof, Y.-D. et al. Yield of immunolabel compared to resin sections and thawed cryosections, in Colloidal Gold: Principles, Methods, and Applications. Hayat, M.A., ed., Academic Press Inc., San Diego, IL, USA, 1991, 87. Stierhof, Y.-D. et al. Direct visualization and silver enhancement of ultra-small antibody-bound gold particles on immunolabeled ultrathin resin sections. Scan. Microsc., 6, 1009, 1992. Stierhof, Y.-D. et al. Use of TEM, SEM, and STEM in imaging 1-nm colloidal gold particles, in Immunogold-Silver Staining. Hayat, M.A., ed., CRC Press, Boca Raton, FL, USA, 1995, 97. Stierhof, Y.-D., Humbel, B.M., and Schwarz, H., Suitability of different silver enhancement methods applied to 1 nm colloidal gold particles: An immunoelectron microscopic study. J. Electr. Microsc. Tech., 17, 336, 1991. Stierhof, Y.-D. and Schwarz, H. Labeling properties of sucrose-infiltrated cryosections. Scan. Microsc. Suppl., 3, 35, 1989. Tokuyasu, K.T. A study of positive staining of ultrathin frozen sections. J. Ultrastruct. Res., 63, 287, 1978. Weipoltshammer, K. et al. Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography. Histochem. Cell. Biol., 114, 489, 2000.

                       

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CONTENTS GENERAL INTRODUCTION .................................................................................... 621 1.

PRINCIPLES OF ELECTRON TOMOGRAPHY......................................... 622

2.

SUMMARY OF THE DIFFERENT STEPS ................................................... 624

3.

INSTRUMENTAL REQUIREMENTS ........................................................... 625 3.1. 3.2. 3.3. 3.4.

4.

PROTOCOLS .................................................................................................... 631 4.1. 4.2. 4.3. 4.4.

5.

Specimen Preparation ............................................................................... 631 Data Acquisition ....................................................................................... 633 Alignment, Reconstruction and Modeling................................................ 638 Short Protocol for Electron Tomography ................................................. 639

ADVANTAGES/DISADVANTAGES.............................................................. 641 5.1. 5.2.

6.

The Transmission Electron Microscope ................................................... 625 The Charge Coupled Device (CCD) Camera............................................ 627 High-Tilt Holders ..................................................................................... 628 Software.................................................................................................... 628 3.4.1. Data acquisition software............................................................ 629 3.4.2. 3-D data reconstruction software ................................................ 629

Advantages of 3-D Electron Tomography................................................ 641 Disadvantages of 3-D Electron Tomography ........................................... 642

WHY AND WHEN TO USE A SPECIFIC METHOD .................................. 644 6.1. 6.2.

Why Electron Tomography?..................................................................... 644 Why Electron Tomography on Resin Sections? ....................................... 644

7.

OBSERVED RESULTS .................................................................................... 646

8.

REFERENCES .................................................................................................. 648

 

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GENERAL INTRODUCTION One of the techniques in electron microscopy that evolved rapidly in the past decade is transmission electron tomography. The method was first applied in 1970;1 however, only with increased computer power and once several of the technical challenges of the method were worked out,2 did electron tomography became a valuable and powerful tool. In the meantime, many electron microscopists realized that conventional electron micrographs merely were two-dimensional projections of three-dimensional structures and, in fact, an oversimplification of the actual structures in a cell. During the last few years, methods were developed to overcome some of the remaining limitations of electron tomography and focused on developing methods that provide a better understanding of the actual processes within cells, between tissues and in organs. The use of 3-D electron tomography in life sciences can be separated into several major fields of application, which have different requirements with respect to specimen preparation, the microscope and subsequent image processing. A possible distinction can be made between 1) cryo-electron tomography, 2) cellular tomography, 3) scanning transmission electron microscopy (STEM) tomography, and 4) energy filtering (EF) tomography. They all have their advantages and disadvantages. Cryo-electron tomography aims at imaging cryofixed and unstained material with lowdose transmission electron microscope (TEM)3 (see Chapter 12). Cryo-electron tomography is based on optimal specimen preservation, but is restricted to 3-D imaging of macromolecular complexes (>200 kDa), isolated cell organelles (< 500 nm), thin areas of cells (< 500 nm) or thin sections of cryo-fixed material.4,5 To date, there are no possibilities for specific labeling in cryo-electron tomography, but localization of large proteins with a characteristic and distinguishable shape can be mapped in their cellular context by template matching.6 Cellular tomography aims at reconstructing relatively large, unique structures by preferentially using high-pressure freezing (see Chapters 5, 6) and freeze-substitution (see Chapter 13) for specimen preparation.7-13 Often, several tomograms are combined to build 3-D reconstructions of even larger areas.14 Because of the dehydration and staining step, the specimen may be less well preserved than frozen-hydrated samples (see Chapters 1, 11). The method has, however, the advantage that large samples of cells and tissue can be processed easily. Furthermore, cellular components can be localized by specific labeling and the sections are more stable in the electron beam. Cellular tomography can also be applied to chemically fixed material and even to Tokuyasu cryo-sections for combination with immunogold labeling; however, one has to realize that structural artifacts are introduced.12 STEM tomography is a relatively new type of tomography.15-18 The technique has been shown to be very powerful for reconstructing highly diffracting specimens (e.g., crystalline material) that are not suitable for TEM imaging. When using a high-angle angular dark-field detector (HAADF), HAADF-STEM imaging collects images for which the contrast is more sensitive to differences in atomic weight. Therefore, for stained sections and for detecting ultra-small (gold) labels,19,20 STEM tomography has potential applications.18 To date, however, HAADF-STEM tomography is not often used in the life sciences.

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Electron tomography using energy filtering16 is an approach for imaging very thick sections (up to 1 µm) or cryo-sections (see Chapter 12). Often zero-loss filtering is used for cellular tomography. In addition, with sufficient electron dose, it may be possible to acquire 3-D images of areas containing a high concentration of a particular element, e.g., calcium.21 In this chapter, we will introduce the basic procedures and exemplify the methods that are used in electron microscopy facilities to obtain 3-D information on unique subcellular structures with nanometer-scale resolution and overview the continuing development of automated data acquisition programs as well as the improved user interface procedures.

1. PRINCIPLES OF ELECTRON TOMOGRAPHY  Electron tomography is a method to computationally generate a threedimensional (3-D) digital volume (the tomogram) from multiple 2-D projection images of the 3-D structure.  There are two assumptions that have to be taken into consideration when electron tomography is performed. 1 The first assumption is that the specimen does not change (shape, density) during the recording of the tilt series.

 For this reason, stained resin-embedded specimens have to be preirradiated, ensuring that the amount of specimen shrinkage during data collection is minimal.22

2 The second assumption is that the image is a true projection of the densities inside the specimen (projection requirement).

 This assumption does not hold for highly diffracting specimens (for those specimens STEM tomography is the method of choice).

 These assumptions set a limit to the  The use of energy filtering helps to meet thickness of the specimen that can be the projection requirement. observed. For very thick specimens (multiple), electron scattering events within the specimen can become too dominant.  For optimal results, the object must be  This is usually between 65o and +65o projected under an as wide as possible with an increment of 1o, resulting in a tilt range of viewing directions. series of 131 images.

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 The images need to be aligned and the  There are several procedures to compute reconstruction (called tomogram) can the 3-D reconstruction. Currently, the most widely used approach is resolutionbe computed. weighted back projection (WBP).7 In addition to this, there are other methods like the simultaneous iterative reconstruction technique (SIRT)23), the algebraic reconstruction technique (ART) or a dualaxis iterative algorithm from Tong et Midgley24  Once the tomogram is computed, complex structures in the interior of the specimen can be viewed in thin digital slices (often as thin as 3 to 10 nm), much thinner than can be accomplished with physical sectioning (50 to 80 nm). The obtainable resolution can be calculated by the following rule of thumb: three times section thickness divided by the number of images.

 This means that in a 350 nm-thick section with a recorded tilt series of 141 images (70 to +70o), a resolution of about 7.5 nm can be achieved.  3 × 350/141 = 7.5 nm

 Furthermore, in the 3-D digital  In this way, the structure of interest can volume, the object can be sampled then be analyzed and modeled. with image processing tools, e.g., segmented in any direction one wishes without being hampered by information from overlying structures in the region of interest.  With the current type of specimen  New approaches to achieve full, 180o holders, it is not possible to tilt the tilt, angles are under investigation.25 specimen to angles higher than 70o; at higher tilt angles the material holding the specimen blocks the field of view.  Due to the limitations of the angular tilt range, there is a lack of information at higher tilt angles. In the tomogram, this missing information is clearly visible in the shape of reconstructed high-density particles (e.g., gold beads).

 The beads will not be completely round, but will appear elongated in Z-directions. In the Fourier transform of the tomogram, the missing information can be seen as a missing wedge of information corresponding to the missing angular tilt range (see Section 5.2, Figure 24.18).

 The missing wedge can be reduced to a missing pyramid by rotating the grid over 90o, recording a second tilt series of the same area and combining the two tomograms into a dual-axis tilt reconstruction.26,27

 The improvement in resolution (especially in Z-direction) is very significant and results in a more isotropic resolution throughout the tomogram.

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2. SUMMARY OF THE DIFFERENT STEPS       A: The thick resin section with the structure of interest.

B: The projection images of the thick section. After recording the projection, images need to be aligned with respect to each other.

C: Individual slices from the computed tomogram.             

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D: Segmentation and annotation of the optical slices.

E: 3-D model of the structure.

Figure 24.1 (A-E) Schematic representation of the basic goal of performing electron tomography, namely the reconstruction of 3-D information from an object. (Sketch courtesy of Misjaël N. Lebbink.)

3. INSTRUMENTAL REQUIREMENTS 3.1. The Transmission Electron Microscope  To acquire 3-D information, electron tomography is performed on relatively thick specimens.   For imaging, sections of up to 500 nm thickness and a TEM with an acceleration voltage of at least 200 kV are necessary.

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 Keep in mind that at a 60o tilt angle the apparent thickness of a 500 nm-thick specimen has doubled (1000 nm), and at tilt angles of 70o tripled (1500 nm).  Increase in specimen thickness results in increased scattering inside the specimen creating inelastic scattered electrons, which leads to blurring and reduced image contrast.

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 In many cases an imaging energy filter will be very helpful in generating highcontrast images. The images are taken at zero-loss, filtering out the inelastic scattered electrons that lost energy within the specimen.



 The goniometer should allow accurate  A goniometer that can tilt under cryo tilting of the specimen holder over a conditions is required for cryo-electron microscopy (see Chapter 12). large angular range.  For room temperature applications, dedicated tomography holders are available that allow tilting at high angles, sometimes in combination with dual-axis tilting.  Sophisticated acquisition packages (commercial and academic) are under constant development. They allow the automated tracking, focusing and recording of the area of interest.  The stability of the goniometer and the high-tilt holders allow holder calibration procedures28-30 to predict the movement of the holder at eucentric height in the microscope.

 Many of the automated tracking and focusing procedures are based on methods introduced in the early 1990s.31-33  Recording of a tilt series (131 images) can be performed within 45 minutes.

Figure 24.2 Tecnai-20 with LaB6 filament (FEI Company, Eindhoven, The Netherlands) at Utrecht University. This dedicated microscope is used solely for recording tilt series for 3-D electron tomography. The microscope is equipped with the automated data acquisition software Xplore 3-D (FEI Company, Eindhoven, The Netherlands). 

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3.2. The Charge Coupled Device (CCD) Camera  Before the advent of digital image recording, one of the most laborious and time consuming tasks of doing tomography was the processing and digitization of recorded tilt series from film. During the last decade, digital data acquisition using CCD cameras has become the most common type of data recording. The incoming electrons are converted to photons on a scintillator and then transmitted to the CCD chip either via fiber coupling or via a lens-coupled camera system.

 The size of the individual pixels on the CCD chip determines the final resolution (sampling distance) and the sensitivity of the camera.  The characteristics of the scintillator (type of material, thickness) in combination with the noise characteristic of the CCD chip determine the sensitivity of the camera system.  

 For cellular tomography on high contrast specimens it might be better to use a camera with smaller pixels and imperfect noise characteristics, whereas for cryo-electron tomography, with beam sensitive specimens and therefore low beam intensities, it is better to have a camera with larger pixels and very good noise characteristics. The quality of the digital cameras is improving continuously.

 Currently, CCD cameras for transmission electron microscopy will have between 10242 and 40962 pixels.  The increasing amount of pixels leads to large data sets close to 1 Gbyte in size.  Especially for iterative reconstruction algorithms this may lead to challenges with respect to computation and data storage.

Figure 24.3 The TemCam-F214 CCDcamera (TVIPS GmbH, Gauting, Germany) that records the projection images at the Tecnai-20 LaB6 microscope at the electron microscopy department of Utrecht University.

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3.3. High-Tilt Holders  The specimen grid holders must be able to tilt to high-tilt angles without obstructing the electron beam. The higher the achievable tilt angle the less loss in Fourier space and the smaller the so-called missing wedge artifact.  A second approach to reduce the size of the missing wedge is to rotate the grid by 90o and record a second tilt series of the same specimen area.26,27  To achieve these high-tilt angles, the material of the holder near the two sides of the grid is made as thin as possible to have a relatively large field of view, even at high-tilt angles (see Figure 24.4). Until recently, the in-plane rotation of the grid over 90o for recording the second tilt series had to be done manually. To facilitate the process of recording dual-axis tilt series dedicated specimen holders are now available, which allow in-plane rotation of the grid even when the holder is inserted into the electron microscope.

 Causing shadowing on the specimen.  These high-tilting angles are necessary to minimize the size of the missing wedge.  By combining the two tomograms, the missing wedge can be reduced to a missing pyramid. In our experience, it is more advantageous to record two tilt series from 60 to +60o and to combine them in one tomogram instead of recording one singletilt series from 70 till +70o.   Currently, we use the Fischione 2020 rotational tilt holder (Fischione Instruments, Pittsburgh, Philadelphia, USA) for this purpose.  They allow tilting angles up to +/80o.

Figure 24.4 The Gatan high-tilt holder (Gatan U.K., Abingdon, U.K.) with a clamping device for easy and firm clamping of the grid. 

3.4. Software  Not only hardware (see above) but  Three different types concerning the also software for automated data Three different steps: acquisition, data handling and data analysis is necessary. Several software packages are available for  Acquisition these purposes. Some are commercially available (meaning  Reconstruction expensive and without source code) and some are academic (meaning  Analysis and modeling much cheaper or even free and with  source code).

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3.4.1. Data acquisition software  The main goal of the data acquisition software is to accurately collect a tilt series of digital images from a fixed location on the specimen. Irregularities in stage movement (x, y) and focusing (z) during the recording of the tilt series should be corrected. Many data acquisition software packages are available for image acquisition.

 Some of them are commercial packages (e.g., Xplore 3-D, FEI Company, Eindhoven, The Netherlands; EMmenu, TVIPS GmbH, Gauting, Germany; TEMography™, JEOL System Technology Co, LTD, Tokyo, Japan).  others have an academic origin and are freely available (e.g., TOM toolbox, MaxPlanck-Institute for Biochemistry, Martinsried, Germany; Serial EM, University of Colorado, Boulder, Colorado, USA; and Priism/IVE, UCSF, San Francisco, California, USA).

 The commercially available packages are stable and supported by a company but do not allow users to make adjustments in the software. The packages available through academia are more flexible and under constant development by the user community.

 Currently, in our department the Xplore 3-D package and the TOM toolbox are used for data acquisition at the electron microscopes.

3.4.2. 3-D data reconstruction software  The 3-D reconstruction software  In most cases, it takes less then an hour performs two tasks: alignment and to compute a dual-axis tilt tomogram. reconstruction. Currently, we use the IMOD package34 from University of Colorado (Boulder) to align the recorded projection images using fiducial markers. The tomograms are computed by means of the resolutionweighted back projection method.  For those occasions when the resolution-weighted back projection is less optimal (e.g., for very low contrast images), we use the ART or the SIRT modules available in the Xplore 3-D package. The resolutionweighted back projection algorithm implemented in IMOD is relatively fast.

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 The advantage of a SIRT reconstruction algorithm is that the low frequency information is better resolved than with resolution-weighted back projection.

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 The ART and SIRT reconstruction algorithms have the disadvantage that they are computationally intensive and still lack the ability to generate dualaxis tilt tomograms.

 There is, however, a considerable reduction in ART and SIRT calculation times possible by using the computational power of graphical processing units or graphic cards on a standard desktop computer.

Figure 24.5 Two images of the user interface from the data reconstruction software package IMOD.34 This is one of the few packages that allow the reconstruction of double-tilt tomograms.

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4. PROTOCOLS 4.1. Specimen Preparation   





     Figure 24.6 Specimen preparation step scheme used for cellular tomography of cells and tissue.

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 The quality of the tomogram produced with electron tomography completely depends on the quality of the sample in the microscope. The cellular structures under investigation must be prepared in a way that they represent a cellular state that is as close as possible to the native state. In order to achieve this, several cryofixation protocols were developed.  Specimen preparation protocols are under constant development and alternatives for traditional chemical fixation are used to overcome problems, such as displacement of the cellular proteins, conformational changes and denaturation of proteins. Also the shrinkage of tissues and cells during the fixation step12,35 and the loss of lipid content36 are important issues in developing new fixation protocols (see Matsko and Müller37 for a recent report).

 For specimens prepared by freezesubstitution, several protocols have been developed that include staining of cellular structures with heavy metals, like uranyl ions, lead ions or osmium tetroxide36 (see Chapter 13). For this type of specimen, the contrast mechanism is mainly scattering contrast.

 In our laboratory, the cryo-fixation  In our approach, section thickness method of high-pressure freezing normally ranges between 250 nm to (HPF) followed by freeze-substitution 500 nm. (FS) and resin embedding is often used (see Geerts et al8 for a detailed description). Briefly, the cryofixed cellular materials are embedded in a resin and thick sections are cut with an ultra-microtome using diamond knifes. The thick sections are collected on Formvar (or Pioloform)/carbon-coated copper grids.  In most of our studies on cellular arrangement, e.g., membrane continuities between organelles or connections between cellular compartments, 6, 10 or 15 nm gold particles are added randomly to both sides of the section. These gold beads function as fiducial markers during the alignment step that precedes the generation of the tomogram.

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 In cases where specific immunogold labeling for the localization of particular proteins is performed, the specific gold label can sometimes be used also as a fiducial marker for the alignment step.

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 G = Golgi complex  ER = Endoplasmic reticulum  M = Mitochondria

Figure 24.7 Overview of a plasma cell in a 300 nm-thick section. The plasma cell was prepared by high-pressure freezing followed by freeze-substitution and embedding in Epon.   This procedure is especially suited to study membranes and their relationship.

Figure 24.8 Detail of the previous plasma cell showing an extensive endoplasmic reticulum (ER), a large Golgi complex (G) in which cisternae and the trans Golgi network (TGN), with numerous small vesicular structures, can be clearly recognized. 

4.2. Data Acquisition  The first prerequisite is a well aligned TEM with an accelerating voltage of at least 200 kV. The microscope alignment procedures are not described because they are outside the scope of this chapter.

 In our department we use a Tecnai 20 with a LaB6 filament equipped with a Tietz Temcam F214 CCD camera with 2048 × 2048 square pixels.

 Next, it is required that the CCD camera is properly installed and that the reference images (bias and gain reference images) are correctly recorded to produce high-quality digital images.

 For the installation and proper calibration of the CCD camera, we refer readers to the manuals provided by the manufacturer of the electron microscope and/or the camera.

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 When one acquires a manual tilt series, it becomes clear that the repetitive actions needed to keep the area of interest centered and focused are time consuming. Area selection, focusing and tracking has to be done by intense interaction between the user and the computer program controlling the microscope. Once digital imaging became a commodity on most electron microscopes, automation was developed that replaced the human operations to center and focus.  In 2001, it was noted that imperfections of the tilting movement of the goniometer (see Figure 24.9) were very reproducible.28

Figure 24.9 The holder calibration curves for the Gatan high-tilt holder that can be used for automatic recording of data sets. For visualization, purposes the thickness of the calibration line is adjusted in Adobe ®  Photoshop .

 Calibration curves were used to  Today automated tomography data predict image and focus shifts and to acquisition software packages are available compensate for them automatically on a variety of TEMs. during data acquisition. Variations and improvements on this basic idea were realized and further improved in several packages (UCSF package 2004,29 TOM toolbox, Martinsried, 2005, SerialEM.30) 

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



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

 An important concept in automated data acquisition is the “optimized position” concept. From a theoretical point of view, any point on the specimen undergoes a circular motion, with the center of the circle lying on the tilt axis (a line in horizontal direction determined by the goniometer) and the radius of the circle being the distance between the point and the tilt axis.

635  Theoretically, the radius of the circular movement is then minimal.  By setting the specimen at eucentric height, the position of the specimen within the goniometer is changed until the height of the specimen and the tilt axis are in the same plane.

 However, in practice there still will be  The distance between these two axes, the movement visible when the misalignment, can be several m. goniometer is tilted. This is due to the fact that the position of the tilt axis (horizontal) does not intercept with the optical axis of the microscope (vertical) that defines the area that is imaged on the center of the CCD image.  The major effects of this misalignment are a focus change and translational shift when the specimen is tilted.

 However, focus changes and translational shifts can be corrected by adapting the objective lens and the image shift controls of the microscope.

 Fortunately, automated correction of the misalignment is carried out by adjusting the optical axis of the microscope (vertical) to coincide with the tilt axis of the goniometer (horizontal). To carry this alignment through, a number of basic calibrations need to be performed.

 The purpose of these calibrations is to give a physical meaning to the pixel size of the CCD camera (the magnification) and to calibrate the image shift, stage shift and focus shift controls

 Apart from these general calibrations there are two tomography-specific calibrations, those of the tilt axis (measuring the optimized-position shift — the distance between the optical axis and the tilt axis) and measuring the remaining focus changes and translational shifts of the specimen when it is tilted.

 Used to predict the image shift and focus changes that are needed during tilt-series acquisition to keep the area of interest in the center of the image.  These calibrations have to be checked once a month.

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 When these prerequisites are fulfilled, recording a tilt series is a straight forward procedure that can be performed within an hour.  As stated before, in our laboratory we image specimens prepared with cryofixation and freeze-substitution. We use stained resin-embedded sections of 250 to 500 nm thickness, often decorated with fiducial markers (gold beads in the size range of 5 to 15 nm) on both sides of the specimen.

 For optimal alignment, we need 10 to 15 gold particles per image.  The required dilution of the gold particle solution can be checked by applying a droplet of a particular dilution on bare grids and examining them in the microscope at the specific magnification that will be used for recording the tilt series

 The specimen grid is tightly clamped  This is absolutely necessary into a high-tilt holder (single-tilt or recording proper tilt series. rotation holder) and carefully inserted into the goniometer of the microscope.

for

 Next, the area of interest has to be set  Eucentricity has to be checked every at eucentric height. time before recording a new tilt series, either by hand or with the data acquisition software.  Before recording the tilt series, it also has to be checked (by tilting the holder) on how far the area of interest can be tilted without any obstructions from the grid bars or the holder itself.  Next, the intensity of the beam on the  This is necessary to prevent overspecimen is checked at the zero illumination of the area of interest during degree tilt angle. the recording of the tilt series.  Keep in mind that the light intensity on the CCD screen depends on the tilt angle of the sample; at high-tilt angles the section thickness increases considerably resulting in much weaker image intensity on the CCD chip.  Finally, the actual recording of the projection images can start. In general, it takes about 45 minutes to record a complete series of projection images.

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Figure 24.10 User interface of the FEI automated data acquisition software where data acquisition parameters can be adjusted.

Figure 24.11 User interface showing the tracking and focusing images and the corresponding cross-correlation images that are used for automated focusing and tracking of the area of interest.

Figure 24.12 The user interface that allows visualization of the recorded tilt series. The scale bar, the tilt angle and the tilt axes are viewed and stored as tiff images.

Figure 24.13 The goniometer of the Tecnai-20 (FEI Company).

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4.3. Alignment, Reconstruction and Modeling  The series of projection images (tilt  For the reconstruction process, several series) are stored for subsequent academic and commercial packages are computation of the tomogram. available.  The resolution-weighted back project-  WBP is a fast reconstruction procedure. tion algorithm,23 which is also implemented in the IMOD package is a widely used method for computing the tomogram. For a more detailed description of this package, we refer to Kremer et al.34  In short, the individual projection images of the recorded tilt series show small image shifts from one projection image to the next. The first alignment step in IMOD reduces the image shifts from one image to the next by means of a cross correlationbased alignment of the individual images.  In the second alignment step, fiducial markers present in the projection images (of the gold particles) are selected and used for a more accurate alignment of the projection images. Then the tomogram can be computed using the resolution-weighted back projection method. Also available in IMOD is the possibility to generate dual-axis tilt tomograms. After the tomogram is calculated, the analysis of the recorded data set can start.

 For a more elaborate description we refer to the excellent manual and the Web site of the University of Colorado (http://bio3d.colorado.edu/imod/).

 In the IMOD package, several options are available for modeling. There is a contour drawing mode (most often used), a thresholding tool for automatic selection and drawing of contours and contour volume rendering options.

 So far, all the steps in the acquisition process can be performed without specific knowledge of the object under study.  However, for interpretation of the tomogram and modeling, specific knowledge of the object is absolutely necessary.  In many studies, using the contour mode, the extent to which membranous compartments are connected to other membranous compartments is investigated, both in the exocytic as well as in the endocytic pathways.38,39

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 An illustrative example is the connection between the ER and the newly originating peroxisomes.40  The contour drawing mode is, however, prone to the interpretation by the investigator. Therefore, efforts are now being undertaken to find computational methods for automated pattern recognition by means of thresholding and template matching by computers.18,41

Figure 24.14 Projection image of a 300 nm-thick plastic-embedded section with randomly applied fiducial gold markers (10 nm), which can be seen as dense black dots all over the section.

4.4. Short Protocol for Electron Tomography 1. Before starting data acquisition check  Also the TEM basic calibrations and the the general microscope conditions: TEM tomography calibrations must have Beam tilt pivot points, tomo rotation been carried out. center, beam tilt calibrations, image (beam) calibration, image shift pivot points, image shift for the magnification range. 2. Check the CCD dark image and gain  For the dark image correction, close the correction. The fully corrected image screen and record the dark image. should appear homogenously grey.  For the gain correction, remove the specimen and illuminate the CCD evenly with a number of counts not too different from the working conditions later. 3. Load the sample and locate the area of interest. 4. Adjust eucentric height manually.

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5. Focus. 6. Center the condenser aperture. 7. Adjust the gun conditions. 8. Align rotation center. 9. Correct objective astigmatism and focus image.

Figure 24.15 User interface of Xplore 3-D for checking light intensity and astigmatism. 10. Check for the maximum tilt angles without blocking the area of interest. 11. Adjust the CCD settings for search, focus, exposure and tracking. 12. Set the parameters for recording a tilt series.

Figure 24.16 User interface of Xplore 3-D for setting the data acquisition parameters for automated recording and storage of the tilt series.

13. Provide a unique name for storage of the image series. 14. Check if the focusing and tracking steps are performed well. 15. Check the recorded tilt series in visualization mode.

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16. Align the individual images with IMOD.

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17. Calculate the tomogram with the weighted back projection method in IMOD. 18. Analyze the tomogram. 19. Perform the contouring and/or surface  Amira is available from Mercury rendering in IMOD or Amira. Computer Systems SAS, 33708 Merignac Cedex, France.

5. ADVANTAGES/DISADVANTAGES 5.1. Advantages of 3-D Electron Tomography  The produced dataset contains  To achieve the best possible Z-resolution projection images of the same in transmission electron microscopy position under different angles. studies, sections need to be as thin as possible. In the recorded projection image, however, structures obscure each other and the achieved Z-resolution is never better than the section thickness.  The third dimension in the section is  By the use of the back projection restored. method. In this way a “virtual” 3-D block is created and individual images can be retrieved from this block and used for analysis.  The Z-resolution obtained is  In general, the Z-resolution of a determined by the number of recorded tomogram is approximately 10 times better projection images and the section then in a single projection image. thickness.  The 3-D digital volume: The object can be sampled with image processing tools, e.g., segmented in any direction one wishes without being hampered by information from overlying structures in the region of interest.

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Figure 24.17 A. The information in a projection image of a 300 nm-thick plastic section displays low Z-resolution and is obscured by overlying structures and, therefore, difficult to interpret.  Note: The increased resolution in all directions and the amount of detail present in the slice. B. Single optical slice extracted from the 3-D reconstruction of the thick section (300 nm) shown above. N = Nucleus ER = Endoplasmic reticulum with ribosomes on the membranes G = Golgi S = Spindle

5.2. Disadvantages of 3-D Electron Tomography  The current types of specimen holders  Due to this limitation of the angular tilt prevent tilting the specimen to angles range, there is a lack of information at the higher than +/70o. higher tilt angles.  In the Fourier transform of the tomogram, the missing information can be seen as a missing wedge of information corresponding to missing angular tilt range.

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Figure 24.18 Schematic drawing of the missing wedge information in Fourier space. The gray area contains information that is not present in the reconstruction due to the limited tilt angles within an electron microscope.

 Have to combine two perpendicular  The improvement in Z-direction is tomograms into a dual-axis tilt significant and results in a more isotropic reconstruction.26,27 resolution throughout the tomogram.  No real 3-D labeling is possible.

 Many EM studies are performed on resin-embedded samples. This usually provides good morphological quality, but prevents specific localization studies because the penetration of antibodies into the section is not possible. So immunolabeling can be performed only at the surface of the resin-embedded section.

Figure 24.19 Comparison of the same area (X/Z) in a single and double-tilt tomogram. In the single-tilt tomogram (top), a clear distortion/elongation in the Z direction is visible. The double-tilt tomogram (bottom) shows improved Z-resolution.

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6. WHY AND WHEN TO USE A SPECIFIC METHOD 6.1. Why Electron Tomography?  With transmission electron microscopy of thin sections (40 to 70 nm), unique subcellular structures are studied and high X, Y resolution can be obtained. However, many fine details (especially in Z-direction) are obscured by overand underlying structures in the section.  3-D transmission electron tomography can provide unmistakable answers to these questions with a resolution especially in Z that is 10 times better then obtainable with the classical methods.  By performing 3-D tomography, it was possible to gain new insight and generate new hypotheses for many biological processes, such as the generation of the peroxisome,9 COP II localization,42 connections between cisternae in the Golgi39,43 transport in the endo- and exocytic pathways, nuclear organization and many more.

 This makes it difficult to give unambiguous answers to certain questions, like, for instance, is there a connection between organelles (direct open contact or MCS (membrane contact sides) or between subcompartments of an organelle (direct connections between cisternae of a Golgi system)?

 Resolution with light microscopy is at best, 200 nm.

6.2. Why Electron Tomography on Resin Sections?  Performing tomography on resin sections has a number of advantages.  First, the specimen preparation steps  Many EM labs can perform the basic are relatively simple. steps of fixing (either chemical or the more advanced high-pressure freezing followed by freeze-substitution), resin embedding, and sectioning and contrasting of sections.  Second, the presence of abundant amounts of heavy metals (uranium, osmium, lead) in the section results in a high signal-to-noise ratio (good contrast), making it easy to recognize structures and focus on the specimen.

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 Third, it is even possible to determine  When enough specific gold labels are protein localization on metacrylate- present on the section surface, they can be used for alignment of the projection images embedded material. and tomograms were specific labeling on top of the section can be studied and analyzed.  Fourth, the presence of resin provides rigidity to the specimen. After the initial shrinkage of the resin under the electron beam, the section is rather stable allowing the recording of a large number of projection images with small incremental values. This results in a resolution that is impossible to obtain with cryotomographic methods. The total electron dose that can be used on the specimen is much higher than in cryotomography.

 Initial volume shrinkage can be up to 30%.

 Fifth, it is easy to reduce the so-called missing wedge artifact into a missing pyramid by rotating the grid by 90o and recording a second tilt series of the same area of interest. The stability of the specimen and the unique nature of the object allow the microscopist to find the region of interest after rotating the grid.

 The two computed tomograms can be combined into one double-tilt tomogram. The resolution (especially in Z-direction) benefits considerably from this. Rotating can be done in the microscope (rotation high-tilt holder) or outside the microscope.

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 In cryo-EM, the total dose must be divided over all the recorded images. This results in fewer images with a much lower signal-to-noise ratio (see Chapter 12).

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7. OBSERVED RESULTS  Figure 24.20

 The whole process of doing 3-D electron tomography is exemplified on a cellular organelle (a multivesicular lysosome).  A-C: Representative images of the first stack of projection images recorded from 65 to +65o with an increment of 1o Shown are the projection images at 60o (A), 0o (B) and +60o (C).  D-E: Images of the stack perpendicular to the first one from the same area at 60o (D), 0o (E) and +60o (F).  G: The combined tomogram. Because it is constructed from two independently constructed tomograms, there is a region that contains singletilt information (upper right part) and one that contains double-tilt information (lower left part).  H-I: Two representative optical slices from the double-tilt tomogram illustrating the increased resolution of the tomogram compared to the zero tilt images in B and E.

 Figure 24.21 (see colour insert following page )

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 A: Optical slice with some contours of the different suborganellar structures in the multivesicular lysosome. The contouring was done with the IMOD software package.  B: A surface rendered model that was made from the contouring of the multivesicular lysosome. For demonstration, only a few of the subcellular structures are visualized.

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8. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Crowther, R.A., DeRosier, D.J., and Klug, A. The reconstruction of a threedimensional structure from its projections and its applications to electron microscopy, P. Roy. Soc. Lond. A Mat., 317, 319, 1970. Koster, A.J. et al. Perspectives of molecular and cellular electron tomography, J. Struct. Biol., 120, 276, 1997. Medalia, O. et al. Macromolecular architecture in eukaryotic cells visualized by cryoelectron tomography, Science, 298, 1209, 2002. Al-Amoudi, A., Norlen, L.P.O., and Dubochet, J. Cryo-electron microscopy of vitreous sections of native biological cells and tissues, J. Struct. Biol., 148, 131, 2004. Leis, A.P. et al. Cryo-electron tomography of biological specimens, IEEE Signal Proc. Mag., 23, 95, 2006. Frangakis, A.S. and Förster, F. Computational exploration of structural information from cryo-electron tomograms, Curr. Opin. Stuct. Biol., 14, 325, 2004. Frank, J. Electron Tomography: Three-Dimensional Imaging with the Transmission Electron Microscope. Plenum Press, New York, London, 1992. Geerts, W.J.C. et al. Electron microscopy tomography and localization of proteins and macromolecular complexes in cells, in Protein-Protein Interactions. A Molecular Cloning Manual, Golemis, E.A. and Adams, P.D., eds,. Cold Spring Harbor Laboratories Press, New York, USA, 2006, 715. Geuze, H.J. et al. Involvement of the endoplasmic reticulum in peroxisome formation, Mol. Biol. Cell, 14, 2900, 2003. Horowitz, R.A. et al. The three-dimensional architecture of chromatin in situ: Electron tomography reveals fibers composed of a continuously variable zig-zag nucleosomal ribbon, J. Cell Biol., 125, 1, 1994. Marsh, B.J. et al. Direct continuities between cisternae at different levels of the Golgi complex in glucose-stimulated mouse islet beta cells, Proc. Nat. Acad. Sci. USA, 101, 5565, 2004. Murk, J.L.A.N. et al. Influence of aldehyde fixation on the morphology of endosomes and lysosomes: Quatitative analysis and electron tomography, J. Microsc., 212, 81, 2003. Tomova, C. et al. New comprehension of the apicoplast of Sarcocystis by transmission electron tomography, Biol. Cell, 98, 535, 2006. Marsh, B.J. Reconstructing mammalian membrane architecture by large area cellular tomography, Methods Cell Biol., 79, 193, 2007. Midgley, P.A. et al. Probing the spatial distribution and morphology of supported nanoparticles using rutherford-scattered electron imaging, Angew. Chem. Int. Ed. Engl., 41, 3804, 2002. Midgley, P. and Weyland, M. 3D electron microscopy in the physical sciences: The development of Z-contrast and EFTEM tomography, Ultramicroscopy, 96, 413, 2003. Ziese, U. et al. Three-dimensional localization of ultrasmall immuno-gold labels by HAADF-STEM tomography, J. Struct. Biol., 138, 58, 2002. Yakushevska, A. et al. Comparison of TEM and STEM tomography in cell biology, J. Struct. Biol , 159, 381, 2007. Otten, M.T. et al. High-angle annular dark-field STEM imaging of immunogold labels, Scanning, 14, 282, 1992. Stierhof, Y.-D. et al. Use of TEM, SEM, and STEM in imaging 1-nm colloidal gold particles, in Immunogold-Silver Staining: Principles, Methods, and Applications, Hayat, M.A., ed., CRC Press, Boca Raton, FL, USA, 1995, 97. Leapman, R.D. Detecting single atoms of calcium and iron in biological structures by electron energy-loss spectrum-imaging, J. Microsc., 210, 5, 2003.

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22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

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Luther, P.K. Sample shrinkage and radiation damage, in Electron Tomography. Three-Dimensional Imaging with the Transmission Electron Microscope, Frank, J., ed., Plenum Press, New York, London, 1992, 39. Gilbert, P. Iterative methods for the three-dimensional reconstruction of an object from projections, J. Theor. Biol., 36, 105, 1972. Tong, J. and Midgley, P. A novel dual-axis reconstruction algorithm for electron tomography, J. Physics: Conf. Ser., 26, 33, 2006. Kamino, T. et al. Application of a FIB–STEM system for 3D observation of a resin-embedded yeast cell, J. Electr. Microsc., 53, 563, 2004. Penczek, P. et al. Double-tilt electron tomography, Ultramicroscopy, 60, 393, 1995. Mastronarde, D.N. Dual-axis tomography: An approach with alignment methods that preserve resolution, J. Struct. Biol., 120, 343, 1997. Ziese, U. et al. Automated high-throughput electron tomography by pre-calibration of image shifts, J. Microsc., 205, 187, 2002. Zheng, Q.S. et al. An improved strategy for automated electron microscopic tomography, J. Struct. Biol., 147, 91, 2004. Mastronarde, D.N. Automated electron microscope tomography using robust prediction of specimen movements, J. Struct. Biol., 152, 36, 2005. Dierksen, K. et al. Three-dimensional structure of lipid vesicles embedded in vitreous ice and investigated by automated electron tomography, Biophys. J., 68, 1416, 1995. Koster, A.J. et al. Automated microscopy for electron tomography, Ultramicroscopy, 46, 207, 1992. Fung, J.C. et al. Toward fully automated high-resolution electron tomography, J. Struct. Biol., 116, 181, 1996. Kremer, J.R., Mastronarde, D.N., and McIntosh, J.R. Computer visualization of three-dimensional image data using IMOD, J. Struct. Biol., 116, 71, 1996. Szczesny, P.J., Walther, P., and Müller, M. Light damage in rod outer segments: The effects of fixation on ultrastructural alterations, Curr. Eye Res., 15, 807, 1996. Humbel, B.M. and Schwarz, H. Freeze-substitution for immunochemistry, in Immuno-Gold Labeling in Cell Biology, Verkleij, A.J. and Leunissen, J.L.M., eds., CRC Press, Boca Raton, FL, USA, 1989, 115. Matsko, N. and Müller, M. Epoxy resin as fixative during freeze-substitution, J. Struct. Biol., 152, 92, 2005. Murk, J.L.A.N. et al. Endosomal compartmentalization in three dimensions: Implications for membrane fusion, Proc. Nat. Acad. Sci. USA, 100, 13332, 2003. Trucco, A. et al. Secretory traffic triggers the formation of tubular continuities across Golgi sub-compartments, Nat. Cell Biol., 6, 1071, 2004. Murk, J.L.A.N. 3-D analysis of endosomes, lysosomes and peroxisomes. PhD thesis, Utrecht University, Utrecht, The Netherlands, 2004. Lebbink, M.N. et al. Template matching as a tool for annotation of tomograms of stained biological structures, J. Struct. Biol., 158, 327, 2007. Zeuschner, D. et al. Immuno-electron tomography of ER exit sites reveals the existence of free COPII-coated transport carriers, Nat. Cell Biol., 8, 377, 2006. Polishchuk, R.S. and Mironov, A.A. Structural aspects of Golgi function, Cell. Mol. Life Sci., 61, 146, 2004.

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Final Considerations

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FINAL CONSIDERATIONS The publication of this handbook is a wonderful satisfaction for a scientist engaged throughout his long life in the development of instrumentation and methodologies for research in biological ultrastructure. It is proof that some dreams may become reality and that “cryo” is still in a dynamic and fruitful state. twenty years ago, Steinbrecht and Zierold10 edited the first compilation in this field. Their book already demonstrated the incredible advances since the early 1950s. At that time, high resolution transmission electron microscopes were available for research.3,7 The first chapters of this book document the current state of the art. It’s a story of success. A dream became reality; the dream to look directly by microscopic observation inside the basic structures of life. The way to do this starts with the proper vitrification of macromolecules, cells in suspension or tissues. Both instrumentation and methodology for freezing, post- and prefreezing steps of such preparations have now reached a state of perfection, which makes it almost an easy task and at the very least a feasible one. Progress in cryo-technology had an important side effect for science — the tiny water molecule was brought again into the forefront. It was astonishing that most scientists — even biologists — for a long period of time had no real interest in this simple compound, albeit with a complex behaviour, made of two hydrogen and one oxygen atom. Really astonishing, because it was obvious and well known that water molecules represent a major component of our huge universe. Astonishing also because more than half of our earth’s surface is covered by oceans. Astonishing with respect to the community of biologists because any student knows that cells, plants and animals, including man, contain more water than other components. Nevertheless, water was ignored successfully. It was hampering structural studies in the light microscope and had to be removed completely to allow wax (paraffin) embedding. A good penetration of the dehydrating agent was only possible after poisoning the cells to make their membranes leaky and penetration possible. Those were useful methods, but also a solid basis for plenty of artefacts. Concerning ultrastructural research, it was obvious that proper “vitrification” (see Chapter 1) within milliseconds was the only way to stabilise all components of the sensitive, mixed plasmatic phases, including also small molecules and rapidly moving ions. But there was a general scepticism by the experts in physical chemistry as to whether real vitrification of pure water and diluted aqueous solutions was actually possible. In 1980/81, two independent teams led by Brüggeller and Dubochet demonstrated this possibility.1,2 This was a breakthrough of great importance that opened the door to answering a lot of questions. If water has no time to aggregate into crystals, then all other components must also be held in their original position. Proper vitrification, therefore, justifies the considerable effort carried out to develop cryoultramicrotomy and perform diffraction analysis of ultrathin cryo-sections in the “frozen-hydrated state.” That now really makes sense.9

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Most progress discussed above was achieved against all odds. Similar obstacles were encountered in the development of the electron microscope. Gabor, as a member of the team around Knoll at the Techniche Hochschule Berlin, wrote the most impressive and competent report about the early days of this development.3 I cite in the original language: “Niemand konnte anhand der damals verfügbaren Unterlagen vorausahnen, dass das Elektronenmikroskop so erfolgreich sein würde, wie es sich später erwies”. He continued, “Wir wollen im Voraus die Moral aus dieser Geschichte ziehen: Selbst in der Wissenschaft ist es oft besser, Mut zu besitzen, als gescheit zu sein”. Translated into English: “At that time, nobody could according to the available data predict that the electron microscope would be such a success as it turned out later on” and “We draw in advance the conclusion from this story: even in science, it is often better to be courageous than to be very intelligent.” I noticed with much pleasure the cynical comments of all the honourable old professors in scientific journals at the time. They widely discussed this useless crazy new instrument. These comments would be worth a historical study — as an example for the old slogan, “errare humanum est.” I would like to recall a few other examples of such errors: a. Ultramicrotomy: No one believed it would be possible to reproducibly cut ultrathin sections in the nanometer-range suited for the EM. Neither precision mechanics nor knife materials nor fixatives nor embedding media seemed to be achievable. Nevertheless, among others, Porter and Blum6 did it. b. High-pressure freezing: Moor and Riehle5 were successful. I cite. Moor4: “The interest of the audience was not overwhelming because everybody thought that this approach is over sophisticated and unnecessary” – what an error! High pressure freezing provided the first pictures of well-frozen ultrastructures and a world of new information. c. Finally, my own experience in cryo-ultramicrotomy:9 Nobody wanted to believe that sectioning at 180°C would be successful. A group of smart, experienced cryotechnologists from industry visited our prototype FC4 and stated that such a set-up with direct LN2 cooling and an open top could never work (it was already running and not so badly). One of these fellows stated that we probably had manipulated the temperature indicators. He wet his fingertip and felt if the knife holder was really cold (it was approximately 150°C). I was silent and smiling when he yelled because he suffered a severe burn. I noticed something else, all these “crazy developments, which never would work” were achieved by rather young scientists, often students. The first EM was built by a “young crew” around the also youthful assistant. Knoll. His team was made up only of students of the TH Berlin. Jacques Dubochet2 was young as well as his team at the EMBO, Heidelberg. Nevertheless, they achieved and documented proper vitrification of pure water. Porter and Blum6 were young fellows when they built (at the Rockefeller Institute in New York), the first ultramicrotome, which was clearly suited for routine work. That was the Volkswagen in ultramicrotomy and served generations of scientists over decades, an astonishingly simple apparatus. Moor and Riehle5 as young scientists also did an excellent job at the ETH Zürich. I started as a young student in the third semester on an EM and built my first ultramicrotome.8 I wanted, together with my brother Peter (also a student), to have a look into cells. In general, youngsters have the courage necessary to enter into risky projects and have the drive needed for success. The slogan:“Doing

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Final Considerations

655

the Impossible” fits to their kind. One should be careful with this precocious resource and try to keep these youngsters instead of firing them after some years of successful engagement. The United States tenure track system seems a good solution, which we lack in Europe. I want to make some further comments on “cryo” analyses (and on expensive research in general): “Enthusiasm cannot substitute for Money.” This means that efficient cryowork in many —if not most — sectors needs some money. Electron microscopes (equipped with a cryo-stage, low dose, possibly filter lenses), cryo-ultramicrotomes (with cryo-diamonds, antistatic devices, etc.), a high-pressure freezer, cryotransfer units, automated freeze-substitution and freeze-drying units are not available for nothing. The same applies to running costs. This is a threat for universities and grant-giving organisations. Money is rare — especially in Europe and the Southern Countries. This is a severe problem for cryo. Our colleagues in the US and Canada are in a splendid situation. They also complain that the budget decreases every year. But a true comparison reduces my compassion immediately to nothing. Certainly there are some low-cost set-ups like home-built plunging systems working with a rubber belt like David’s catapult to kill Goliath. But such instruments are rather the exception. There are some examples in which lack of money may provoke inventions that end in a Nobel Prize award. The one most often cited is about the Austrian analytical chemist Fritz Pregl who earned the Nobel Prize for the foundation of quantitative organic microanalysis in 1923. It was said that he was forced to this discipline simply by lack of money. I do not believe this. The same rumour concerns the discipline of theoretical physics in Göttingen (Germany). Maybe, but I want to warn governments from reducing budgets of universities and research organisations to increase the number of Nobel Prizes that such an inverse relationship will certainly not work. It would also be dangerous to split the old fashioned universitas litterarum into expensive and inexpensive disciplines. Then cryo will come to an end together with other expensive disciplines. Markl, former president of the big German research organisation (Deutsche Forschungsgemeinschaft) stated in an official meeting about the financial situation, that we must avoid supporting, for financial reasons, more low-cost projects. Then we would provoke the degeneration of research into “Mickey Mouse research.” I guess personally that plenty of Mickey Mouses are already at work in our scientific community. These are scientists who calculate exactly the probability of success and the time needed. Only 100% success rates and short periods of time are wanted. They should leave the university and change over to a commercial employment. In my opinion, they will not do this and also will not be wanted in this alternative area — sorry. But, I again warn governments. There is a pitfall, a kind of sin. The “splitting of money into fractions of “useful” (applied) and “useless” (basic) research” is a horrible mistake. Useful research is most often light-weight research that serves commerce and governments. Useless research is heavy-weight research, which has no immediate visible and predictable influence on commerce and, therefore, income taxes for the government. The claim is that this useful research increases the welfare of all citizens. Most citizens feel that this is a striking argument. The truth is just the contrary.

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The economic success in the United States resulted to a great extent from the generous support of the so-called “useless” basic research. Only such basic research opens completely unexpected new areas for commerce, industry and other avenues in a country. Internationally, well-known scientists made very clear statements and warned the Austrian Government. I cite two colleagues working in Vienna in prominent positions. (1) Freissmuth, pharmacologist and director of a big Institute of the Medical University in Vienna, stated, “You cannot save (basic) research.” (2) Penninger, pathologist and director of the Institute of Molecular Biotechnology (Austrian Academy of Sciences) just recently stated in an official discussion of the broadcast ORF1, “If the government saves money on basic research it will change our university into a museum.” This is perfectly valid for cryo-work. With an old EM, a historical ultramicrotome and an old evaporation unit, you may impress plenty of visitors. But you are unable to carry out state-of-the-art cryo-work. You degenerate into a “Mickey Mouse,” according to Markl. Since I wish all young colleagues a satisfying and fruitful career in this fascinating field, I have made some comments concerning a sufficient amount of money for this type of research. I repeat: “Enthusiasm in most situations cannot substitute for money.” A strong financial basis is of key importance for success besides courage, intelligence and the always needed drive. In this sense, I wish all fellow workers, especially the young ones, “Good luck in the fascinating cryo-work.”

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Brüggeler, P. and Mayer, E. Complete vitrification of pure liquid water and diluted aqueous solutions, Nature (London), 288, 569, 1980. Dubochet, J. and McDowall, A.W. Vitrification of pure water for electron microscopy, J. Microsc. (Oxford), 124, RP3, 1981. Gabor, D. Die Entwicklungsgeschichte des Elektronenmikroskopes, Elektrotechn. Zschr., 78, 522, 1957. Moor, H. Theory and practice of high pressure freezing, in Cryotechniques in Biological Electron Microscopy, Steinbrecht, R.A. and Zierold, K., eds., Springer Verlag, Berlin, Heidelberg, Germany, 1987, 175. Moor, H. and Riehle, U. Snap-freezing under high pressure: A new fixation technique for freeze-etching, in Proc. 4th Europ. Reg. Conf. Electron Microsc., Bocciarelli, S., ed., 1968, 33. Porter, K.R. and Blum, J. A study in microtomy for electron microscopy, Anat. Rec., 117, 685, 1953. Ruska, E. Die frühe Entwicklung der Elektronenlinsen und der Elektronenmikroskopie, Acta historica Leopoldina Nr. 12. Deutsche Akademie der Naturforscher, Halle/Salle, 1979. Sitte, H. Ein einfaches Ultramikrotom für hochauflösende elektronenmikroskopische Untersuchungen, Mikroskopie (Wien), 10, 365, 1955. Sitte, H. Advanced instrumentation and methodology related to cryoultramicrotomy: A review, Scan. Microsc. Suppl., 10, 387, 1996. Steinbrecht, R.A. and Zierold, K. Cryotechniques in Biological Electron Microscopy. Springer Verlag, Berlin, Heidelberg, Germany, 1987.

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Glossary

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A  Algebraic reconstruction technique  A reconstruction algorithm that (ART) compares the differences between the reprojections of a tomogram and the measured data, one image at a time, and corrects the volume iteratively until a given stopping criterion is fulfilled.  Amorphous ice  Solidified water devoid of crystals, also called vitreous ice.  Analytical probes  Electron beam or ion beam that interacts with specimens are analytical probes.  Antibody  Immunoglobulins that recognize specific antigens.  Antifreezing agent  A substance that is added to water to lower its melting point.

B  Bar  Basic calibrations  Blocking buffer

 Blotting  Boltzmann constant, k

 Measure of pressure: 1 Bar = 105 Pa = 752.5 Torr = 0.99 atm.  Measured curves describing the movement of a high tilt holder in x, y and z during a tilt range.  Used to prevent undesired binding of antibodies to biological structures and resin (usually interactions of electrostatic and hydrophobic nature).  Removal of excess fluid from the grid prior to vitrification in liquid ethane.  The physical constant relating temperature to energy; experimentally determined as 1.38 × 10 to 23 joules/Kelvin.

C  Celsius

 Anders Celsius 1701–1744, Swedish astronomer, devised the Celsius (oC) temperature scale (Dictionary of Science and Technology, Academic Press, San Diego, CA, 1992).

 CEMOVIS

 Technique allowing observation of sections of fully frozen-hydrated samples under an electron beam (in a cryo-electron microscope).

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660  Charge-coupled device (CCD)

Handbook of Cryo-Preparation Methods for Electron Microscopy

 An image sensor comprising an integrated circuit and a linked array of radiation-sensitive capacitors.  Chatter  One of the major cutting artifacts. It is a periodic variation of section thickness at the µm scale. It may be due to vibration of the knife in respect of the specimen. In vitreous cryo-sections, it is generally due to variation of friction of the section on the knife surface.  Chemical mapping  Construction of 2– or 3–dimensional maps of the chemical composition of specimens for one or several elements.  Critical micelle concentration  The CMC corresponds to the minimum (CMC) concentration of detergent at which micelles form. The CMC is very sensitive to temperature and polarity of the medium.  Colloidal gold marker  Colloidal gold bound to antibody (fragments) via hydrophobic and electrostatic interactions.  Contouring  Manual drawing of contour lines in slices of a tomogram.  Contrast transfer function  Function associated to the electron magnetic lens and which describes the proportion of signal that is transferred by the imaging system for each spatial frequency. The CTF is an oscillating function that varies with defocus value and the spherical aberration of the objective lens.  Conventional  A useful term to designate anything except the present subject. In this book, conventional electron microscopy is noncryo-electron microscopy. More specifically, a conventional preparation method means chemical fixation and resin embedding.  Correlative light microscopy (LM)  Combination of observing and imaging and electron microscopy (EM) with light microscopy before detailed analysis at high resolution with electron microscopy.  One of the major cutting artifacts  Crevasse induced by cutting stress in vitreous samples.  A simple formula that defines the  Crowther criterion theoretical resolution of a tomogram only in terms of the total number of projection images and the tilt range, which by definition also defines the tilt increment.

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 Cryo-fixation or cryo-immobilization 







 Solidification of a biological specimen by cooling with the aim of minimal displacement of its components.  A liquid that boils at extremely low Cryogen temperature, e.g., liquid nitrogen (boiling point 196°C). Liquid nitrogen is a primary cryogen. Other cryogens, such as liquid ethane, are referred to as secondary cryogens because they are cooled by liquid nitrogen.  Pertaining to preservation and/or Cryogenic maintenance of molecules, cells or organisms at extremely low temperatures, typically in liquid nitrogen. Cryo-section according to Tokuyasu  Ultrathin thawed frozen section. Biological samples are weakly fixed with aldehydes, protected against freezing damage by infiltration with sucrose, frozen in liquid nitrogen and sectioned at 90°C to 120°C by cryo-ultramicrotomy (usually used for immunolabeling.  Production of frozen-hydrated sections Cryo-sectioning for observation in a cryo-electron microscope (see CEMOVIS).

D  Dehydration

 Freeze drying

 Freeze-substitution  Depth-of-field

 Detergent  Devitrification

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 Free water from the fixed sample must be removed when water immiscible resins are used for embedding.  Dehydration of cryofixed samples through the sublimation of ice (lyophilisation). Starting temperature of procedure: 100°C.  Dehydration of cryofixed samples by using an organic solvent carried out below 90°C.  The distance in object space over which the objective focused on the specimen can provide adequate definition or clarity (Dictionary of Science and Technology, Academic Press, San Diego, CA, 1992).  Amphiphilic molecules used to solubilize membrane proteins.  Crystallization of water by warming a vitrified sample. For pure water, the devitrification temperature is ca. 135°C. It is higher when the solute concentration is high.

662  Dialysis  DOGS-NTA-Ni  Dose fractionation

Handbook of Cryo-Preparation Methods for Electron Microscopy

 Mechanism allowing removal of some components from a solution.  Synthetic lipid formed by a hydrophobic domain and a hydrophilic domain containing chelated nickel.  The process of distributing the total electron dose that can be tolerated by a specimen between the individual projection images (cryo-electron tomography).

E  EELS

 EFTEM

 Elvanol/Mowiol  Energy filtered image  Epi-fluorescence microscope

 Epon  Epoxy resin

 ESI

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 Among other structural information, the electron energy-loss spectrum contains chemical information about the specimen in the form of specific edges (with specific shape and position in energy) superimposed on a decreasing background.  Energy filtered transmission electron microscopy can be used for the measurement of the elemental composition of irradiated specimens. This analysis is possible by the use of an imaging energy filter integrated into the microscope column or by using a prism spectrometer below the final screen.  Semisolid embedding medium for light microscopy.  See ESI.  The illumination light of the object is using the same light path through the objective as the emission signal.  Epoxy resin.  Chemically: polyaryl esters of glycerol with terminal epoxy groups and hydroxyl groups spaced along the chain. With addition of cross-linking agents, they are converted into an inert solid.  Generates images with electrons of selected energy loss. These data concern only element-specific electrons = imaging for one element.

663

Glossary

 Etching

 Sublimation of a solvent for a short period of time.  In a liquid form, ethane is the ideal vitrification coolant.

 Ethane

F  Fiducial slang)

marker

(or

“fiducial,”  An exogenous, inorganic colloidal particle, usually 10 nm colloidal gold, introduced into or onto a specimen for the purposes of facilitating subsequent alignment of projection images.

 Fixation

 The goals of fixation are to preserve the structure of samples with minimum alterations from the living state.

 Fluorochrome

 A chemical component, which upon illumination radiates light at a longer wave length.

 Formaldehyde

 Weak fixative that has little influence on immunolabeling efficiency.

 Fourier space

 Synonymous of reciprocal space; the “space” defining the Fourier transform of an object, i.e., its decomposition into a continuous spectrum of its component frequencies, as opposed to Euclidean or “real” space, where positions are defined in terms of an x, y, z coordinate system.

 Freeze-drying

 A technique by which the frozen water of a cryo-fixed specimen is removed by sublimation at low temperature in a vacuum chamber.

 Freeze-etching

 After breaking the specimen and before evaporating metal, the specimen undergoes a process of etching, i.e., ice sublimation, for a short period of time.

 Freeze-fracturing

 To break a frozen specimen into pieces and immediately make a heavy metal replica of the fractured plane.

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664  Freeze-substitution

Handbook of Cryo-Preparation Methods for Electron Microscopy

 Dehydration method by replacing water (in solid state) for a (fluid) solvent (mostly methanol, ethanol or acetone). After freeze substitution, the sample can be warmed up to room temperature (i.e., the process to dehydrate and chemically fix specimens at temperatures between 90°C and 30°C).

G  Gaussian denoising

 Glow discharge

 Glutaraldehyde  Gold enhancement

 Gold toning

 Green fluorescent protein

 Denoising of a signal with the assumption that the noise has a Gaussian distribution, or in other words, that it is “white noise.”  Procedure by which ionized atoms are deposited onto a carbon support to modify its surface properties. The carbon-coated grids are deposited onto an electrode that is used to ionize residual gas under vacuum.  Aliphatic dialdehyde, an efficient crosslinking fixative. It may considerably reduce immunolabeling efficiency.  Similar to silver–enhancement. Small gold particles increase size by gold atoms, instead of silver atoms, deposited onto the surface.  Treatment of silver–enhanced structures with gold chloride. Originally used to improve contrast in LM and to cover/enlarge silver enhanced gold.  Protein from the jelly fish Aequorea victoria. GFP is a marker of gene expression and protein targeting. It is used for live cell imaging.

H  H+-ATPase

 HC-Pro

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 An ion pump that actively transports hydrogen ions across lipid bilayers in exchange for ATP.  The helper component proteinase is encoded by plant virus of the genus

665

Glossary

Potyvirus. HC-Pro is involved in different steps of the viral cycle, aphid transmission, replication, and virus cell-to-cell movement and is a suppressor of posttranscriptional gene silencing.  High-pressure freezing

 Rapid cooling 204.8 MPa.

of

a

sample

at

I  Immunoadsorption  Indirect labeling

 Purification of antibodies by adsorption on the specific antigen often bound to a sepharose column.  The primary antibody is not coupled to a marker molecule. The secondary antibody, which is used to detect bound primary antibodies, is coupled to a marker molecule.

K  Kelvin

 Baron William Thomson Kelvin 18241907), British physicist and mathematician, devised the Kelvin (K) temperature scale (Dictionary of Science and Technology, Academic Press, San Diego, CA, 1992). The international standard unit of temperature where 0° Kelvin is equivalent to 273°C.

L  Lipid monolayer

 Layer of lipids formed at the interface buffer-air.

 Low-dose

 Microscope operation mode with a beam-deflection unit that allows the precise determination of the total exposure dose on the specimen during data collection. Typically between 10 to 20 electrons/Å2.

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 Lowicryl resin

 A mixture of acrylate-metacrylate resins with very low viscosity at low temperatures (up to 80°C).

 Low-temperature embedding

 Embedding in methacrylates, typically Lowicryls, at temperatures (below 0°C) by UV irradiation.  London Resin, brand name for methacrylates.

 LR White, LR Gold

M  MAPs

 Marker

 Methyl cellulose  Microtubules

 Missing pyramid

 Missing wedge

 Molecular distillation drying

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 Proteins that interact with microtubules in the cell, regulating their dynamic behavior or allowing them to build complex organelles, such as axonemes and centrosomes.  Necessary to visualize the antigenantibody reaction by light microscopy (fluorescent molecules and enzymes) or electron microscopy (usually gold particles).  Embedding medium for ultrathin Tokuyasu cryo-sections.  Polymers of the tubulin molecule associated with proteins (MAPs) in the cell, and involved in various functions, such as cell division through the mitotic spindle, vesicular traffic through the action of molecular motors, cell compartmentalization, or cell motility through the motion of cilia and flagella.  Term used to describe the region of Fourier space that remains unsampled in a dual-axis tilting experiment, defined by orthogonal tilt axes. By combining two perpendicular recorded tomograms of the same region, the missing wedge can be reduced to a missing pyramid.  Missing information in Fourier space, due to limited tilt angles. Results in anisotropic resolution in X,Y,Z.  A special method of freeze-drying.

Glossary

667

N  NANOGOLD marker  Neurospora crassa  Numerical aperture

 Commercially available gold compound that can be covalently bound to antibodies and other molecules.  A type of red bread mold of the phylum Ascomycota.  Measure for the resolution power of an objective:  NA = n × sin α  n = Refraction index of the medium between sample and objective lens  α = Half opening angle of the objective.  The correlation with the resolution d is:  d = 0.61λ/NA  λ = The wave length of the electromagnetic wave.

O  Optimized position

 Osmication

 Osmium tetroxide

 Theoretical positions in a perfectly adjusted specimen stage where the distance between the optical axis of the microscope and the tilt axis of the specimen is zero.  A method to stain and fix biological specimens with the vapors from osmium tetroxide crystals or with an osmium tetroxide solution.  Strong fixative, but also contrasting agent especially for membranes. It may degrade proteins and, therefore, has a strong effect on immunolabeling efficiency.

P  Phosphotungstic acid  Photobleaching

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 Heavy metal chemical used for negative staining.  Loss of intensity of the fluorescence signal during imaging. Fluorochromes are affected by radical oxygen species produced upon illumination.

668  Primary antibody  Protective colloid  Protein A

Handbook of Cryo-Preparation Methods for Electron Microscopy

 Antibody specific to the antigen/epitope under investigation (usually raised in rabbit or mouse).  Slows down silver enhancement and makes the reaction more efficient and reproducible.  In EM, often used instead of secondary antibody. Binds to a number of IgGs from different species.

Q  Quantum dot (QD) marker

 Fluorescent semiconductor nanocrystal covalently bound to antibodies. Most QDs exhibit relatively low electron density, but can be silver-enhanced.

R  Replica  Resin embedding

 Resin polymerization  Rotary shadowing

 The metal imprint or cast of the surface of an object.  Dry specimens are fragile and porous and must be permeated with a fluid resin that can polymerized resin for conservation and sectioning.  Resin can be polymerized by heat or by UV radiation with addition of accelerating agents: cross-linking and catalysts.  The specimen is tilted under a chosen angle and rotated while shadowing.

S  Secondary antibody

 Antibody (usually raised in goat) coupled to a marker molecule that detects the bound primary antibody.

 Segmentation

 The delineation of the features of a complex, three-dimensional image, either manually using a mouse or drawing tablet, or automatically by means of appropriate, e.g., edge-detection, algorithms.

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Glossary

 Shadowing

 Silver enhancement

 Silver stabilization

 SIMS (SIMS imaging)

 Simultaneous iterative reconstruction technique (SIRT)  Slam-freezing

 Spurr’s resin

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669  To evaporate a heavy metal from a point source at an oblique angle onto a specimen surface in order to produce a cast of the specimen.  Process similar to photographic development. In the presence of a reducing agent, the gold surface acts as a catalyst for the reduction of silver ions to metallic silver. The metallic silver deposits on the gold surface, resulting in a growing silver layer.  Protection of the silver layer that is sensitive to oxidation (OsO4, electron beam, air humidity), by gold chloride treatment.  SIMS is a mass spectrometric technique using a primary ion beam to analyze the specimen. Chemical and isotopic microanalyses can be carried out by focusing this primary beam to get a microprobe (diameter under 100 nm). SIMS imaging is obtained by scanning such a microprobe over the surface of interest of the specimen.  In TOF-SIMS, the primary ion beam is pulsed and a whole mass spectrum can be analyzed. This imaging method gives molecular information, but is less sensitive than dynamic SIMS imaging.  A reconstruction algorithm similar to ART (see above) where the reconstructed volume is updated only after all corrections have been performed.  Cryo-fixation technique where the surface of a biological sample is cooled down by the polished surface of a metal block, which, in turn, is cooled by the cryogen.  One of the most fluid resins used in electron microscopy (about 60 cP at ambient temperature for fresh mixture). Easily sectioned and resistant under the analytical probes.

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Handbook of Cryo-Preparation Methods for Electron Microscopy

T  Tomogram

 Tomography

 Torr

 Transmission fluid

 Triton X100

 Computed 3-D volume reconstruction of a specimen by using as many projection images as possible (usually between 121 and 141 images).  The process of obtaining a threedimensional image volume (tomogram) from a series of two-dimensional images, represented either by x-y slices, (e.g., confocal microscopy) or projections, (e.g., x-ray tomography or electron tomography).  Old unit for measuring vacuum still frequently used in electron microscopy. Derived from an Italian physicist and mathematician, Evangelista Torricelli (1608–1647), who invented the mercury barometer and was the first to create vacuum. 1 Torr = 1.33 × 102 Pa or N/m2 (SI unit for pressure).  A fluid used to mediate transfer of heat and pressure during high-pressure freezing without interacting with cellular specimens.  A nonionic surfactant that has a hydrophilic polyethylene oxide group and a hydrocarbon or hydrophobic group.

U  Uranyl acetate

 Fixative and contrasting agent. The divalent uranyl cation forms salt bridges with negatively charged groups, e.g., phosphate groups, hence, stabilizing membranes.

V  Vitreous

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 From the Latin, literally “glass-like”, often used interchangeably with “amorphous.”

Glossary

 Vitrification

671  The formation of a non-crystalline (vitreous) solid state.

W  Weighted back-projection (WBP)

 Synonymous of filtered backprojection; the standard method of reprojecting aligned, two-dimensional projection images into a three-dimensional volume; the weighting factor is used to account for differences in low- and highfrequency information in Fourier space.

 Well-freezing/well-frozen

 Term to describe the state of solid water in a biological object. A sample is said to be well frozen if ice crystal ramifications formed cannot be seen with the electron microscope.

 Whole mount labeling

 Immunolabeling of large samples that have been permeabilized and extracted (e.g., by detergent or solvent treatment) prior to antibody and marker incubation to allow their penetration into the sample.

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