Atlas of cell organelles fluorescence

ATLAS OF

CELL ORGANELLES FLUORESCENCE

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ATLAS OF

CELL ORGANELLES FLUORESCENCE Elli Kohen René Santus Joseph G. Hirschberg Nuri Özkütük

CRC PR E S S Boca Raton London New York Washington, D.C.

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COVER PHOTO CREDITS TOP THREE (FIGURE 102) A model of a multiparameter (multidimensional) system for tumor cell diagnostics, prognostics, and drug trials. Three organelle ßuorescence parameters are used: (A) mitochondria, (B) Golgi, and (C) lysosomes. It is highly desirable to add a nuclear DNA parameter, speciÞcally based on ßuorescence in situ hybridization (FISH). In the case of osteosarcoma with mitochondrial DNA-deÞcient mutations, imaging of the mitochondrial DNA would be a welcome development. BOTTOM LEFT (FIGURE 72) Fusion-deÞcient myoblasts stained with NBD ceramide. The Golgi apparatus is fragmented and smaller than in the fusion-competent myoblasts. This difference remains unexplained so far, and we do not know how it may affect the myoblast fusion function to construct a functional myocyte. BOTTOM MIDDLE (FIGURE 36) Same as Figure 35C with maximum autophagy of mitochondria resulting in heavy rhodamine loading of lysosomes. SP. BOTTOM RIGHT (FIGURE 53) Fluorescence in the Golgi apparatus of a Þbroblast maintained for 24 h in the presence of the carcinogen benzo(a)pyrene.

Library of Congress Cataloging-in-Publication Data Kohen, Elli. Atlas of cell organelles ßuorescence / Elli Kohen, Rene Santus, and Joseph G. Hirschberg. p. cm. Includes bibliographical references and index. ISBN 0-8493-1440-2 (alk. paper) 1. Cell organelles—Atlases. 2. Cytoßuorometry—Atlases. I. Santus, Rene. II. Hirschberg, Joseph G. III. Title. QH585.5.C98K64 2003 571.6′5—dc22

2003055696

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microÞlming, and recording, or by any information storage or retrieval system, without prior permission in writing from the publisher. The consent of CRC Press LLC does not extend to copying for general distribution, for promotion, for creating new works, or for resale. SpeciÞc permission must be obtained in writing from CRC Press LLC for such copying. Direct all inquiries to CRC Press LLC, 2000 N.W. Corporate Blvd., Boca Raton, Florida 33431. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identiÞcation and explanation, without intent to infringe.

Visit the CRC Press Web site at www.crcpress.com © 2004 by CRC Press LLC No claim to original U.S. Government works International Standard Book Number 0-8493-1440-2 Library of Congress Card Number 2003055696 Printed in the United States of America 1 2 3 4 5 6 7 8 9 0 Printed on acid-free paper

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Preface The idea of an atlas on ßuorescence of cell organelles was Þrst inspired by Professor Feroze Nevroze Ghadially’s Ultrastructural Pathology of Cells and Matrix. This unique book describes the ultrastructural makeup and alterations of cell organelles in the context of over a thousand pathological conditions. It became somewhat of a challenge to build on the foundations of images obtained from dead and Þxed cells an equivalent body of images obtained from living and functioning cells under various physiopathological conditions and in the context of treatment with carcinogens, xenobiotics, chemotherapeutic drugs, and photosensitizers. The idea of pursuing such studies was born out of the perspectives envisioned by another giant in the Þeld, Professor Albert Policard, one of the Þrst proponents of microcompartmentation. The links of the research expounded in this book are an odyssey of collaborative visits, travels, and interactions. Historically it passes from the thresholds of the Laboratoire de Pathologie Cellulaire, Kremlin Bicêntre run by Marcel Bessis to the Laboratoire de Photobiologie, Muséum National d’Histoire Naturelle in Paris, and then Þnally to the Papanicolaou Cancer Research Institute and University of Miami. In the historical context, Professor Bessis and Bo Thorell, my professor at the Karolinska, Stockholm, were quite good friends from their prime years. Another link in this chain of research is that Thorell and Britton Chance, my professor in Philadelphia, were lifelong friends. Thorell’s dream was a dynamic pathology of the living cell. Chance’s own dream, and the object of my work in his foundation, was the synthesis of biophysics and cellular pathology. In my sabbatical year I planned to retrace this parable which justiÞably should be called “My ScientiÞc Odyssey from Philadelphia to Miami through Paris and Stockholm.” The endpoint of the vision projected by Policard leads to the birth of three new disciplines in cellular biology: microecology, the science of microecosystems; microethology, behavior in microscopic systems; and microrheology, the science of microcurrents or microcirculation in living cells. All these interactions and visions are not without their symbolism. The chain extends from Policard to his student Bessis and Bessis’ students Giuliana Moreno and Christian Salet. I would like therefore to introduce some memorabilia of my French connection. In his conversations, Bessis often used to say “Mon maître Policard avait dit…” Thus I have found it appropriate to include here a facsimile of Policard’s book cover and his dedication to Moreno (Figure I and Figure II). In the last year of his life, Bessis attempted the realization of one of Policard’s dreams, the founding of a center for microecological studies, as documented by the letter announcing the news of his untimely death (Figure V). At this juncture it seems pertinent to recall a very special tradition in the life of French academicians: the award of an academic sword to new members of the French Academy of Science on the occasion of their joining this august assembly. As expressed in the address at the occasion of this award, the academic sword carries the aura of aristocracy: the aristocracy of talent and spirit. As the emblem of a certain spirit, the sword expresses ornamental prodigality, beauty of material, voluptuousness of form, and artistic creativity. ScientiÞc friends and colleagues of the recipient from all over the world are asked to contribute components that are then artistically combined in the design of the sword (Figure III, Figure IV).

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FIGURE I Front cover of Professor Albert Policard's book.

Policard has been the innovator and the promoter who started this unbroken chain of scientiÞc endeavors. The path goes from cellular ultrastructure to cellular pathology, imaging of organelles in living cells in the dynamic context of physiopathological processes, to the triple disciplines of microecology–microethology–microrheology. The current book of ßuorescent cell organelles is a precursor that should lead to further questions and works, leading to subsequent books on cellular pathology and cellular pharmacology.

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FIGURE II Professor Albert Policard's dedication to second-generation researcher Giuliana Moreno.

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FIGURE III Professor Marcel Bessis’ academic sword.

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FIGURE IV The book on Professor Marcel Bessis’ academic sword.

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FIGURE V Professor Bessis’ last stop, showing his dedication to research in the ecology of cancer cells.

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Authors Elli Kohen, Professor Emeritus of Biology at the University of Miami, Florida, is an anatomopathologist and cell biologist. He is the author of several books on the use of ßuorescence for the study of the living cell and its constituent structures, called organelles. He has a long career in teaching, and his research work is potentially of important beneÞt to the Þelds of cellular pathology and pharmacology. His diversiÞed cultural interests have motivated him to produce books in diverse areas. Dr. Kohen has for many years been a corresponding member of the European Academy of Science, Arts, and Letters. Joseph G. Hirschberg, Professor Emeritus of Physics, University of Miami, Florida, earned an A.B. magna cum laude from Dartmouth College, Hanover, New Hampshire, and a Ph.D. in physics from the University of Wisconsin, Madison. He served in the U.S. Army Air Force, active and reserve, with highest rank as Captain. He was a Fulbright Fellow at the École Normale Supérieure in Paris, France and a research associate at the University of Wisconsin. He was head of the Optical Group, Project Matterhorn, Princeton University, Princeton, New Jersey and was an exchange professor at Université de Paris, France. Hirschberg served as Physics Department Chair, University of Miami. He was a NATO Fellow at the French Atomic Energy Laboratory, Paris, France, a visiting astronomer at Sacramento Peak Observatory, Sunspot, New Mexico, and a visiting scientist at Oak Ridge National Laboratory, Oak Ridge, Tennessee, Princeton University, Institut d’Optique, and the Museum d’Histoire Naturelle, the last two in Paris. His research interests include atomic spectroscopy, solar astronomy, optical diagnosis of thermonuclear plasmas, development of interferometers, optical oceanography, microspectroßuorometers for the study of living cells, and clean energy methods. Hirschberg is a member of Phi Beta Kappa, Gamma Alpha, Sigma Xi, Omicron Delta Kappa, and the European Academy of Science, Arts, and Literature. He is a Fellow of the American Physical Society and the Optical Society of America and is an honorary member of the Florida section of the Optical Society of America. Professor René Santus completed his graduate studies at the University of Paris. He earned his Doctorat-es-Sciences Physiques in 1968 on the photophysics and photochemistry of aromatic amino acids. In 1969 he was hired as Assistant Director and Maître de Conférences of the Biophysics Laboratory at the Museum National d’Histoire Naturelle in Paris. In 1971 he received a 1-year NATO fellowship to work on newly developed fast kinetic spectroscopies at the Biophysics Laboratory of the Illinois Institute of Technology, led by Professor L.I. Grossweiner. Professor Santus was appointed visiting scientist in 1974 by Professor J. Ovadia at the Department of Medical Physics of the Michael Reese Hospital of the University of Chicago, working on the pulse radiolysis and radiation biology of proteins. In 1978 he became head of the photobiology team at the Museum National d’Histoire Naturelle, where he developed a program on molecular and cellular photobiology and application of ßuorescence techniques to cell photobiology in relation to photochemotherapies and phototoxicity of drugs. This was the beginning of his long-lasting collaboration with Professor Elli Kohen. In 1988, in addition to his research and teaching duties at the museum, Professor Santus became a member of a French National Institute of Health and Medical Research Unit in photodermatology, directed by Professor L. Dubertret, a dermatologist at the Hospital Saint-Louis in Paris. From 1996 to 2002 Professor Santus served as director of the photobiology laboratory at the Museum National d’Histoire Naturelle. He is a member of several

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learned societies in Europe and the United States. He has authored or coauthored over 260 publications in peer-review journals, and has coauthored two monographs, Photobiology (Academic Press, 1995) and Fluorescence Probes in Oncology (Imperial College Press, 2002), with Professors Elli Kohen and Joseph G. Hirschberg. Nuri Özkütük earned a doctorate in medicine from the School of Medicine, University of Uludag, Turkey, in 1988. He earned his Ph.D in microbiology and clinical microbiology from the University of Celal Bayar, Turkey, in 2002, where he went on to become an assistant professor in biology on ßuorescence detection methods, working with Dr. Elli Kohen. He then returned to Celal Bayar where he is an assistant professor in the Faculty Medicine Department of Biology and Clinical Microbiolgy.

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Acknowledgment The authors are extremely grateful to Amado Salazar, Information Systems, University of Miami, Coral Gables, Florida, who deserves a special commendation for his remarkable contribution in helping to scan and reformat the images. Another commendation is due to Marco Monti, systems analyst, Department of Physics, University of Miami, for his outstanding help with the color Þgures. A special thank you is deserved by volunteer intern Dalgis Mesa for her unending help in the compilation and sorting of images, as well as processing of the accompanying text. Another thank you goes to Francesca Marrero for help in sorting and compiling. The additional help of undergraduate students Vera Lafosse, Johanna Lopez, Margaretta Watkins, Tanya Kanarek, and graduate student Ceren Örnek is acknowledged.

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Introduction This atlas represents an attempt to promote the development in cell biology of three new disciplines of microecology, microethology, and microrheology, almost prophetically foreseen by the pioneer Albert Policard, and also to a great extent by the pioneers of ultrastructural anatomy and pathology of cell and matrix. The atlas will strive to further the foundations of a good understanding of cellular metabolism, biochemistry, physiopathology and pharmacology, and intra- and extracellular communication and signaling. It will have succeeded in its purpose if every image and its associated legend helps to guide researchers in cell biology by pointing to critical points of attack and suggesting possible strategies for investigation. As the conclusion points out, a great deal resides in future accomplishments in real-time imaging tuned to the real timing of intra-intercellular processes and probe/drug reactivity. There, progress in detector quality for microscopic imaging is intimately tied to and may follow progress in space research and astronomy. Unless indicated otherwise, for trials with ßuorescent probes, metabolic inhibitors, and xenobiotics a good starting point is the range of 10 µM added to the cell incubation medium for ßuorescence imaging. Incubation times vary from acute shock treatment to 10−30 min, 1−3 h, overnight, up to 7 days. The determination of drug penetration and action times, as well as action sequence at the level of different cell organelles, is crucial, whether it takes seconds, minutes, hours, days, or weeks, often accompanied by xenobiotic metabolization, detoxiÞcation, extrusion, or even conversion to more toxic compounds (erroneous detoxiÞcation). Fluorescence images are computerscanned ßuorescence microphotographs, pseudocolored as speciÞed. Fluorescence images are also obtained through the unicolor (black and white) CR-300 CCD (Dage, MTI, Michigan City, Indiana) used in conjunction with an “InvestiGater” (sic) by which two-dimensional video images, each scanned in 32 msec, are summed. For very bright images, the system may also be used in the live (real-time) mode. The images obtained through the CCD camera instead of conventional microphotography are specially identiÞed. For these images, data recorded on the videotape are digitalized by the use of a frame-grabber program (SNAPPY); the computer Þle so obtained allows pixel-bypixel quantitative image and a printout of the topographic distribution of the ßuorescence. Compared to the ßuorescence image observed in the VCR monitor, there is some unavoidable image degradation in the obtained printout.

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FIGURE A Ultrastructural map of L cell Þbroblast, which serves for guidance in planning the strategy of ßuorescence studies (see Figure B). Such images are used for organelle morphometry. Although preliminary topographic microßuorometric studies were limited to unidimensional scan, the goal of excitation/emission ßuorescence imaging is to record pixel-by-pixel (two-dimensional scan) and voxel-by-voxel (three-dimensional scan). Thus the time course of metabolic reactions and the alterations, or metabolization, of introduced xenobiotics are to be followed site-by-site within the living cell’s organelles and microcompartments. The ultrastructural map is the guiding basis for the cellular physiopathology and pharmacology to be established.

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Strategic Map of Research Areas Covered by Atlas of Cell Organelle Fluorescence Lysosomes: Lysosomotropic ßuorochromes [quinacrine (Atabrine), quinacrine mustard] Lysosomotropic carcinogens [benzo(a)pyrene] Lysosomotropic cancer chemotherapeutic agents (adriamycin and other anthracyclins) Fluorogenic and ßuorescent probes of lysosomal dehydrogenases: the entire family of genetic lysosomal diseases; so far only glucoceramidase-deÞcient Gaucher disease studied: same methodology is applicable to probing of Tay-Sachs, glycogen storage disease, mucopolysaccharidose, pseudo-Hurler polydystrophy, Schindler disease, mannosidosis, fucosidosis, Wolman disease, Farber lipogranulomatosis, Niemann-Pick, Krabe disease, metachromatic leukodystrophy, Fabry disease, gangliosidosis, galactosidosis, and other lysosomal hydrolase deÞciencies Formation of phagolysosomes, myelinosomes, i.e., giant lysosomes in response to cytotoxic agents [quinacrine, benzo(a)pyrene, porphyrins] Golgi • Participation in multiorganelle detoxiÞcation complex [quinacrine, benzo(a)pyrene] in correlation with ER, lysosomes, and nuclear membrane • Golgi changes (dispersed Golgi vs. enlargement of the organelle complex) in myoblast maturation (fusion competent vs. fusion deÞcient: relevant to research on muscular dystrophy • Golgi changes during in vitro spontaneous transformation of hepatocytes

Intracellular Enzymology Mitochondria A typical prototype includes the • Cell energy metabolism [coenzyme intracellular study of glutathione ßuorescence response to perfused dehydrogenases and its or injected metabolites: NADH, compartmentation highly relevant to NAD(P)H] the oxydo-redox changes in exchange • Vital ßuorescence probes of with the NAD(P)H complex mitochondria: DASPMI, (monochlorobimane probing) rhodamine 123, tetramethylrhodamine, MitoTracker Green • Probing of mitochondrial membrane potential (rhodamine123, TMRE); study of agents effecting mitochondrial charge (magainin) • Mitochondrial reaction to metabolic inhibitors (dinitrophenol, oligomycin) • Mitochondrial reaction to antipsoriatic agents (anthralin) • Mitrochondrial reaction to topical dicarboxylic acids, used in treatment of melanoma (azelaic acid, sebacic acid) • Mitochondrial reaction to photosensitizers (hematoporphyrin and other porphyrins) • Mitochondrial morphology changes between globoid and Þlamentous (cellular transformation, e.g., Nucleus hepatocytes, keratinocytes; • Response to cytotoxic agents: myoblast maturation: fusion association of nuclear membrane compatible, fusion deÞcient) to multiorganelle detoxiÞcation • Mitochondrial changes in genetic complex disease: giant mitochondrial • Multi-drug resistance to anticancer disease agents: daunomycin, adriamycin • Mitochondrial structural and The postulated existence of a functional changes: cystic Þbrosis nuclear pump coupled to a cell membrane pump (Westerhoff’s hypothesis of a “vacuum cleaner” effect for the injection of therapeutic agents) • Formation of nucleolar channels joining the cytoplasmic multiorganelle complex for detoxiÞcation and ejection of cytotoxic agents (e.g., quinacrine mustard)

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Strategic Map of Research Areas Covered by Atlas of Cell Organelle Fluorescence (Continued) Endoplasmic Reticulum (ER) In correlation with xenobiotics (cytotoxic agents) formation of a multiorganelle complex associated with Golgi and lysosomes plus intercommunicating channels Study of Schiff base (imino–amino propenes) accumulation spontaneously, and in UV-irradiated cells (in presence and absence of porphyrins). This study is at the convergence of cellular senescence, transformation and differentiation (all An entirely new Þeld will be the three conditions include concomittant application of vital mitochondrial variations in Schiff base indicative of ßuorescence probes for detecting the cell peroxidation potential). The structural and membrane potential investigation of Schiff bases can be changes in cells with genetically extended to the family of genetic altered mitochondria: diseases known as porphyrias. • Mitochondrial DNA-deÞcient osteosarcoma lines produced by repeated culture in ethidium bromide • Mitochondrial DNA-deÞcient neuroblastoma culture fused with thrombocytes from patients (platelets are literally bags of mitochondria) to form CYBRIDS • Cells with genetically altered enzymes (defective subunits coded by nuclear DNA) The Atlas aims to be the equivalent of N.F. Ghadially’s Ultrastructural Pathology of the Cell and Matrix (Butterworth’s, London, UK), a pioneering electron microscopy study of over a thousand cellular pathological conditions.

FIGURE B Strategic map for studies on the structure and physiopathology of living cell organelles.

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FIGURE C Metabolic map of the living cell. At the center top is a schematic of the microtrabecular network, a postulated system of microcirculation in the living cell.

REFERENCES Ghadially, F.N., Ultrastructural Pathology of the Cell and Matrix, Butterworths, London. Haugland, R.P., Handbook of Fluorescent Probes and Research Chemicals, Spence M.T.Z., Ed., Molecular Probes, Eugene, OR, 1996. Johnson, L.V., Walsh, M.L., and Chen, L.B., Localization of mitochondria in living cells by rhodamine 123, Proc. Natl Acad. Sci. USA, 77, 990–994, 1980. Kohen, E. and Hirschberg, J.G., Histological correlates of cellular detoxiÞcation, Microscopy Research and Technique, 36, No. 4, Johnson, E., Ed., 1997, Wiley-Liss, NY. Kohen, E., Kohen, C., Hirschberg, J.G., Prince, J., Santus, R., Morlière, P., Schachtschabel, D.O., Shapiro, B.L., Mangel, W.F., and Grabowski, G., The spatiotemporal organization of metabolism in living cells, in Fundamentals of Medical Cell Biology, Vol. 3B (Chemistry of the Living Cell), Bittar, E.E., Ed., 1992, pp. 561–606, JAI Press, Greenwich, CT. Kohen, E., Santus, R., and Hirschberg, J.G., Fluorescence Probes in Oncology, Imperial College Press, London, 2002. Lakowicz, J.R., Fluorescence Spectroscopy, 2001. Slavik, J., Fluorescent Probes in Molecular Biology, CRC Press, Boca Raton, FL, 1994.

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Table of Contents Chapter 1

Vital Fluorescence Probes of Cell Organelles ........................................................... 1

Mitochondrial Probes ...............................................................................................................1 Golgi Probes .............................................................................................................................8 Lysosomal Probes...................................................................................................................13 Nuclear Probes........................................................................................................................18 Chapter 2

Metabolic Probes ...................................................................................................... 23

Chapter 3

Cytotoxic Drugs ....................................................................................................... 33

Lysosomotropic Agents ..........................................................................................................33 Mitochondria-Toxic Agents....................................................................................................52 Mitochondrial Inhibitors.........................................................................................................68 Photosensitizers ......................................................................................................................72 Carcinogens and Cancer Chemotherapeutic Drugs: Agents Stimulating the Proliferation of the Endoplasmic Reticulum and Golgi Together with the Loading of Lysosomes ...........................................................................................................86 Chapter 4

Genetic Diseases....................................................................................................... 99

Chapter 5

Cell Differentiation and Cell Pathology ................................................................ 113

Chapter 6

Cell-to-Cell Communication .................................................................................. 129

Chapter 7

The Study of Microecosystems.............................................................................. 141

Chapter 8

Biotechnology......................................................................................................... 145

Chapter 9

Instrumentation ....................................................................................................... 151

Chapter 10 Novel Methods and Instrumental Designs............................................................. 159 Two-Photon Excitation Microscopy.....................................................................................159 Fourier Interferometry for Excitation–Emission Fluorescence Spectral Imaging ..............163 Excitation–Emission Fluorescence Imaging Combined with Photoacoustic Microscopy. A Combined Fluorescence Imaging and Photoacoustic Microscopy Design for Studies in Cancer Cells......................................................................................168 Is the Study of Nanocampartments in Living Cells Feasible?............................................171 Chapter 11 Conclusion .............................................................................................................. 173 Index ..............................................................................................................................................175

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Fluorescence Probes 1 Vital of Cell Organelles MITOCHONDRIAL PROBES In addition to the ancient dimethylaminostyrylpyridiniummethyl iodine (DASPMI) and somewhat more recent rhodamine 123, there is a broad variety of new probes: tetramethylrhodaminemethylester (TMRM), tetramethylrhodamine-ethylester (TMRE), and MitoTracker Green (Molecular Probes, Eugene, Oregon). All these probes are positively charged and they accumulate by over a thousandfold within the negative microenvironment of the mitochondrial matrix, thus representing mitochondrial membrane potential probes. DASPMI is also retrieved within the nucleus, particularly within the nucleoli. Rhodamine 123 leads to some mitochondrial swelling; thus, mitochondrial fine structure such as beading is better recognized and the fine details of filamentous mitochondria are better visualized with DASPMI. Practically all mitochondrial probes, except for the ultraviolet (UV)-excited natural probe NAD(P)H, are excited in the blue (436 nm mercury line) and emit in the green-yelloworange. Probe treatment is accompanied by some fragilization of mitochondria. In TMRE-labeled yeast mitochondria, microexplosions with disintegration of mitochondria are observed following 30 sec or longer, depending on the fluence rate of continuous blue irradiation with the mercury line (436 nm). A probe of mitochondrial oxygen concentration, pyrenebutylrhodamine (PRE4), synthesized by Ribou, Vigo, and Salmon in 2002. The probe’s positively charged rhodamine moiety is attracted to the negative internal microenvironment of the mitochondria, and the oxygen-sensitive pyrenebutyl moiety follows. Rhodamine is blue excited; pyrenebutyl is UV excited (366 nm). Energy transfer occurs between the two moieties. PRE4 illustrates the advantages of two- to multiwavelength excitation and the recording of fluorescence-excitation emission in cell fluorescence imaging. The search with probes is for high–low accumulation; filamentous, globoid, beaded, or branched structures, fragmentation, swelling, or disintegration. In the case of mitochondrial fragmentation or disintegration with cytotoxic agents, the probe fluorescence is retrieved within lysosomes as a result of autophagocytosis.

REFERENCES Bereiter-Hahn, J., Dimethylaminostyrylpyridiniummethyliodine (DASPMI) as a fluorescent probe for mitochondria in situ, Biochim. Biophys. Acta, 423, 1-14, 1976 Johnson, L.V., Walsh, M.H., and Chen, L.B., Localization of mitochondria in living cells by rhodamine 123, Proc. Natl. Acad. Sci. U.S.A., 77, 990-994, 1980. Ribou, A.-C., Vigo, J., Salmon, J.M., J. Photochem. Photobiol. A., 151, 49, 2002.

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FIGURE 1 In these fibroblasts stained with DASPMI, mitochondria are filamentous and they form a network. Several questions are raised by the image seen. a) Could the network seen imply that instead of mitochondria, there is a single branched mitochondrion? b) The individual segments of the network show zones of spindleshaped nodulation. Could these nodules represent mitochondrial microcompartmentation? If so, do they represent structural only or both structural and functional compartmentation? Are these hypothetical microcompartments identifiable by different fluorescent probes? Can they be selectively stained at localized sites, and is it conceivable that such selectivity may be determined by metabolic modifiers or mitochondria-active agents? c) Could one relate changes in mitochondrial compartmentation to physiopathological conditions? d) What are pathological conditions leading to disruption of the network, and what are the effects of mitochondria toxic agents on the integrity of the network? The original microphotograph has been scanned and pseudocolored (SP) in green.

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FIGURE 2 Characteristic mitochondrial network in a DASPMI-stained fibroblast. This figure indeed raises the point that the network might correspond to a single-branched mitochondrion. This cell is a good candidate for treatment with mitochondria-toxic agents; disruption and incorporation of probe-stained mitochondrial fragments into autophagocytosing lysosomes is certainly to be expected. Could long-term cell viability beyond this stage be maintained, especially after recovery from cytotoxic damage and reconstitution of the mitochondrial network? Does such a network represent a certain stage of mitochondrial function and regulation? Is it conceivable that under physiological conditions there could be cycles of network formation? If that is the case, is it possible to find metabolic or functional conditions, including cytoskeletal reorganization, which might provoke the changes from network constitution to network fragmentation, or subsequent reconstitution? SP of original microphotograph. SP = scan and pseudocolored. (See color figures following page 42.)

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FIGURE 3A Network of DASPMI-stained mitochondria. Pseudocolor green.

FIGURE 3B Network of DASPMI-stained mitochondria. Pseudocolor with gray scale. (See color figures following page 42.)

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FIGURE 4 Fibroblast mitochondria labeled with rhodamine 123. In contrast to images with DASPMI, the nucleus is free of probe. SP of micrograph. (See color figures following page 42.)

FIGURE 5 Positive and negative contrast confocal imaging of mitochondria in hepatocytes. Rat hepatocytes were loaded with 1 mM calcein acetoximethylester at 37oC for 15 min, followed by 250 nM tetramethylrhodamine methylester (TMRM). Cytosolic esterases release and trap green-fluorescing calcein in the cytosol and nucleus (left panel). Dark voids in the green fluorescence image correspond mostly to mitochondria, which exclude calcein. TMRM is a red-fluorescing mitochondrial marker. Each TMRM-labeled mitochondrion in the red fluorescence image (right panel) corresponds to a dark void in the calcein image (left panel). Courtesy of Dr. Ting Qian and John J. Lemasters. (See color figures following page 42.)

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FIGURE 6 Mitochondria stained with DASPMI viewed by image intensifier. The image quality and resolution is somewhat less than obtained in microphotographs. The image is transferred from the intensifier to a videocassette. Further image deterioration occurs in the frame grabber SNAPPY, in the attached personal computer and printer.

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FIGURE 7 Mitochondrial DNA probe. Viewing mitochondrial DNA. Fluorescence micrograph of DAPIstained mtDNA of living CV1 cells (green monkey kidney) in phosphate buffered saline + 1g/L glucose, irradiated with UVA light (340 to 380 nm). Fluorescence was observed at wavelengths greater than 420 nm. The mtDNA appears as bright fluorescent spots due to DAPI in the filamentous structures typical of mitochondria delineated by their weak autofluorescence. The viewing of mtDNA with a vital fluorescent stain raises the possibility of detecting mtDNA structural changes under metabolic control of mitochondrion function, or detecting mtDNA damage during deleterious stresses inflicted on mitochondria by reactive oxygen species produced during drug-induced perturbation of oxidative phosphorylation by the systemic subcellular action of photoactive drugs. Cells grown in monolayers were stained with DAPI (0.1 mg/ml) for 90 min in culture medium and then washed with phosphate-buffered saline. Bar = 10 mm. (Courtesy of M. Dellinger and M. Gèze, Muséum National d’Histoire Naturelle, Paris.)

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GOLGI PROBES One of the most effective is NBD-ceramide. With Golgi probes it is possible to observe the organelle structure under various physiopathological conditions, such as glandular secretion, malignant transformation, and differentiation abnormalities. Other interesting Golgi probes are cytotoxic agents such as carcinogens, i.e., benzo-(a)-pyrene, quinacrine, and quinacrine mustard, which accumulate in the organelle, and cancer chemotherapeutic agents, i.e., adriamycin. In hepatocytes, Golgi images obtained with benzo(a)pyrene or adriamycin are practically indistinguishable from Golgi images obtained with NBD-ceramide. The search with probes is for Golgi gigantism, atrophy, or dispersion.

REFERENCES Chen, C.S., Martin, O.C., and Pagano, R.E., Changes in the spectral properties of plasma membrane lipid analog during the first seconds of endocytosis in living cells, Biophys. J., 72, 32-50, 1997. Pagano, R.E., A novel fluorescent ceramide analog for studying membrane traffic in animal cells; accumulation at the Golgi apparatus results in altered spectral properties of the sphingoid precursor, J. Cell Biol., 113, 1267-1279, 1991.

FIGURE 8 Fibroblasts cultivated for six days in the presence of NBD-ceramide (1.7 mM), a fluorescent probe of the Golgi apparatus. A leopard-skin appearance of fluorochrome-loaded Golgi sacks is obtained. Similar fluorescence images may be recorded when the cell is treated with fluorescent cytotoxic agents (see Chapter 3). The Golgi image obtained may be a prelude to detoxification. SP micrograph. (See color figures following page 42.)

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FIGURE 9 Fibroblast treated with NBD-ceramide for six days. Illustrates the polymorphism of Golgi images obtained in cells preincubated with probe fluorochromes. SP micrograph.

FIGURE 10 Golgi apparatus in fibroblast cultivated in the presence of NBD-ceramide. The Golgi apparatus is seen in the form of a large paranuclear body, which is network-like with highly fluorescent microcorpuscles. This Golgi apparatus is part of a multiorganelle detoxification network, which in other cells appears complemented by, and connected to, other probe- or toxic fluorochrome-loaded organelles such as lysosomes, ER, and even nuclear membrane. SP micrograph. (See color figures following page 42.)

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FIGURE 11A, B, C, D, E Illustrative visualization of Golgi using the probe NBD-ceramide. (Courtesy of Richard F. Pagano, Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, Minnesota.) See also Chapter 3, Cytotoxic Drugs, sections on lysososomotropic agents and carcinogens. (See color figures following page 42.)

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FIGURE 11C, D

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FIGURE 11E

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LYSOSOMAL PROBES These probes are primarily of two kinds: • •

Lysosomotropic agents and cytotoxic agents that localize specifically in the lysosomes such as blue-excited, yellow-emitting quinacrine (atebrine) Fluorogenic substrates of lysosomal hydrolases which release their fluorochrome label, primarily UV-excited, blue-fluorescing methylumbelliferone

Atebrine and similar compounds often result in the formation of giant lysosomes, also called megalysosomes, phagolysosomes, and myelinosomes, as a result of autophagocytosis. The penetration of fluorogenic substrates is facilitated by: • • •

Fragilization of mitochondrial membrane by photosensitizers Use of detergents such as N-dodecyl imidazole Incubation with apolipoprotein E to facilitate penetration of the fluorogenic substrate through the cholesterol pathway

If the fluorogenic substrate is not channeled through the cholesterol pathway, it is treated by the cell as a xenobiotic and accumulates within a multiorganelle paranuclear detoxification complex often constituted by the Golgi and the endoplasmic reticulum (ER) with participation of lysosomes.

REFERENCES Biberich, E. and Legler, G., Intracellular activity of lysosomal glucoceramidase measured with 4-nonylmethylumbelliferyl-beta-glucoside, Biol. Chem. Hoppe-Seyler, 370, 809-817, 1989. Kohen, E., Kohen, C., Hirschberg, J.G., Santus, R., Grabowski, G., Mangel, W., Gatt, S., and Prince, J., An in situ study of beta-glucosidase activity in normal and Gaucher fibroblasts with fluorogenic probes, Cell. Biochem. Func., 11, 167-177, 1983. Richards-Czernicki, T., A method for analyzing the subunits of membrane-bound and soluble enzymes in a heterogenous detergent solution using fluorogenic substrates. Doctoral dissertation, Carnegie-Mellon University, Pittsburgh, PA, 1990.

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FIGURE 12 Lysosomes with lysososomotropic agent quinacrine. Color image; microphotograph. (See color figures following page 42.)

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FIGURE 13 Quinacrine imaging of lysosomes. SP micrograph.

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FIGURE 14 Quinacrine imaging of lysosomes. SP. (See color figures following page 42.)

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FIGURE 15 UG9, nonylmethylumbelliferylglucoside. While the lysosomotropic agent quinacrine can be considered the equivalent of a fluorescent probe, UG9 is a fluorogenic probe of the lysosomal hydrolase b-glucosidase. In UG9 the fluorescence of the umbelliferyl group is masked by the glucoside and remains so until the masking glucoside group is digested by the glucoceramidase. SP. See also Chapter 3, Cytotoxic Drugs, section on lysosomotropic agents.

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NUCLEAR PROBES From the two Hoechst dyes for live and dead cells to the many generations of nuclear probes produced by molecular probes, there is a very broad armamentarium. Amsacrine, an inhibitor of topoisomerase, has been tried. However, its excitation wavelength and its emission in the UV make it impractical for current studies. To this may be added the extensive fluorescence in situ hybridization (FISH) methods of DNA in situ hybridization. Until now, the FISH procedure remained largely inapplicable to living cells. However, the complementation of FISH with spectral imaging has led to the development of spectral karyotyping (SKY). A normal human metaphase spread follows hybridization with a cocktail of 24 painting probes for all human chromosomes. Pseudocolors are assigned to each chromosome according to the spectral signature of the chromosome. Using this method of rearrangements, translocations are easily detected in a chromosome from tumor cells and identified according to their origin. This development has spearheaded a new era in human genetics with respect to aberrations in human diseases and general genetics. SKY has provided additional information that could not have been detected using the conventional banding technique, such as identification of marker chromosomes, detection of subtle chromosome translocations, and clarification of complex karyotyping. In addition to hematological diseases, SKY promises to be very useful in the analysis of specific chromosomal arrangements in carcinomas, sarcomas, and melanomas, about which little is known. Until now it has been very difficult to analyze such material with complicated karyotypes and often suboptimal morphology of the chromosomes. SKY can help to overcome these limitations and can contribute to the recognition of specific chromosomal aberrations, which can be of diagnostic and prognostic value. In clinical cytogenetics, SKY can be of great importance for the identification of the precise breakpoint of chromosomal rearrangements. A true breakthrough will be provided by the use of maximum resolution fluorescence excitation-emission imaging for DNA typing. The acquisition of dynamic functional nucleic acid probes which go beyond simple staining and can seize the actual kinetics of DNA changes is a difficult undertaking that would revolutionize nuclear probing. As an example of dynamic changes, NAD(P)H fluorescence transients make possible the rapid followup of metabolic activity. This would be even more so if nuclear matrix probes could be developed such as exemplified by the glutathione dehydrogenase (GDH) probe monochlorobimane. It is also desirable that amsacrine-like probes will emerge, but in more convenient regions of the excitation and emission spectra, which would then substantiate the high hopes placed on this kind of probe. There are also promising developments in the area of optical sensing beyond the diffraction limit (near-field scanning optical microscopy (SNOM). Near-field optical probes (NFOs), i.e., single-molecule probes, allow the imaging of living cells down to the molecular level and their spectroscopic analysis at that resolution. Thus DNA could be sequenced in situ and even repaired by the right probe at the right site.

REFERENCES Rhotmann, C., Malik, Z., Bar-AM, I., and Cabib, D., Spectral imaging of fluorescence in cell biology, cancer therapy, and cytogenetics. In: Applications of Optical Engineering to the Study of Cellular Pathology, Vol. 2, Kohen, E. and Hirschberg, J.G., Eds., 1999, pp. 121-132, Research Signpost, Trivandrum, Kerala, India. Tan, W., Gallion, V., and Kopelman, R., Intracellular sensing and optical imaging beyond the diffraction limit. In: Applications of Optical Engineering to the Study of Cellular Pathology, Vol. 2, Kohen, E. and Hirschberg, J.H., Eds., 1999, pp. 47-65, Research Signpost, Trivandrum, Kerala, India.

For details on DNA probes the reader is referred to E. Kohen, J.G. Hirschberg, and R. Santus, Fluorescent Probes in Oncology, Imperial College Press, London and World Scientific Publishing, Inc., Singapore, 2002.

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FIGURE 16 Fibroblast treated with calcium probe Fura-2. It is generally noticed that the probe accumulates at higher level within the nucleus. It is not, however, known whether the high nuclear fluorescence is only dependent on calcium or whether there are other unknown components. Microphotograph scanned.

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FIGURE 17A Cells treated with topoisomerase probe amsacrine. It has been speculated that amsacrine would have been a very sensitive monitor of the enzyme's activity because its emission spectrum would be altered by binding to DNA. However, the excitation and emission spectra of amsacrine, particularly excitation, happen to be in a region difficult to exploit under the standard fluorescence microscopy conditions. Quartz optics are necessitated for excitation in the short UV, and even a significant portion of the emission would be lost in the absence of such optics. Also, the penetration and targeting of the probe are not as expected; the micrograph here is after overloading with amsacrine in dimethyl sulfoxide (DMSO) solution (3 mg amsacrine in 1 ml DMSO; 50 ml 0.8 ml Eagle’s medium with 10% calf serum). Such conditions are incompatible with cell viability. Therefore, the exploitation of amsacrine remains an open question. While there are considerable limitations to what information may be derived from amsacrine imaging, rapid imaging with a UV laser emitting in the 250 nm region seems quite promising and may help to detect the kinetics and spectral features of the amsacrine–topoisomerase interaction. In such a case, amsacrine would be used as a dynamic probe of intranuclear dynamic reactions. It is also highly suitable to develop fluorescent DNA probes, which eventually may be used for in situ DNA sequencing or to follow carcinogen metabolite–DNA adducts and single molecule–DNA interactions. SP. (See color figures following page 42.)

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FIGURE 17B Nuclei of yeast Saccharomyces cervisae stained with Hoechst dye. The image is obtained with a black-and-white charge couple device (CCD) camera, with some loss of image quality in transfer to videocassette. The image observed on the VCR monitor had significantly higher quality and resolution than the final image obtained in the printout, due to some image deterioration in the frame grabber (SNAPPY), PC, and printer. (See color figures following page 42.)

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2 Metabolic Probes Since the description of the Krebs cycle over half a century ago, it has been known that the addition of glycolytic substrate results in the reduction of mitochondrial NAD(P); however, the actual demonstration by fluorescence imaging of such response to glucose challenge is quite recent. NAD(P)H formation in metabolic activity is coupled to glutathione dehydrogenase (GDH) activity. Monochlorobimane has been used as a specific probe of GDH-produced thiol (RSH) groups for the actual localization of the enzyme. A preponderent localization within the nucleus has been observed, but it is not a constant finding because GDH distribution may be also quite diffuse, since many proteins involved in intracellular signal transduction or iron metabolism use the redox complex RSH/(RS.) as a regulator.

REFERENCES Bellomo, G., Palladini, G., and Varietti, M., Intranuclear distribution, function, and fate of glutathione and glutathione-S-conjugate in living rat hepatocytes studied by fluorescence microscopy. In: Histological Correlates of Cell Detoxification, Kohen, E., Hirschberg, J.G., Guest Eds., Johnson, J.E., Editor-inChief, Thematic issue of Microscopy Research and Technique, 1997, 36, No. 4. Bennett, B.D., Jetton, T.L., Ying, G., Magnuson, M.A., and Piston, D.W., Quantitative subcellular imaging of glucose metabolism within intact pancreatic islets, J. Biol. Chem., 271, 3647–3651, 1996. Dellinger, M., Gèze, M., Santus, R., Kohen, E., Hirschberg, H.V., and Monti, M., Imaging cells by autofluorescence: a new tool in the probing of biopharmaceutical effects at the intracellular level, Biotechnol. Appl. Biochem., 28, 25–32, 1998. Kohen, E., Kohen, C., Hirschberg, J.G., Wouters, A.W., Thorell, B., Westerhoff, H.V., and Charyulu, K.K., Metabolic control and compartmentation in single living cells, Cell Biochem. Funct., 1, 3–16, 1983. Patterson, J.H., Schroeder, S.C., Yu Bai, P., Weil, A., and Piston, D.W., Quantitative imaging of TATA-binding protein in living yeast cells, Yeast, 14, 813–825, 1998. Patterson, J.H., Knobel, S.M., Arkhammar, P., Thastrup, G., and Piston, D.W., Separation of the glucosestimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet b cells. Proc. Natl. Acad.Sci., USA, 97, 5203–5207, 2000. Piston, D.W., Knobel, S.M., Postic, C., Shelton, K.D., and Magnuson, M.A., Adenovirus-mediated knockout of a conditional glucokinase gene in isolated pancreatic islets reveals an essential role for proximal metabolic coupling events in glucose-stimulated insulin secretion, J. Biol. Chem., 274, 1000–1004, 1999.

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A

B FIGURE 18A, B, C Osteosarcoma, glucose response. (A) Phase image of wild-type osteosarcoma. (B) Background natural fluorescence image. (See color figures following page 42.)

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C FIGURE 18C NADH response in the mitochondria a few seconds after addition of glucose. The response of mitochondria to glucose challenge was certainly to be expected, since the activation of the glycolytic chain is followed by the activation of the Krebs cycle pathways linked to mitochondrial NAD reduction. However, this image represents one of the first direct visual observations as recorded by the CCD camera-integrater system, and as such it is a striking in situ demonstration of the mitochondrial metabolic function. The effect is quite impressive when one sees the mitochondria suddenly light up. The image observed in the VCR monitor after videocassette recording from the CCD camera-integrater had significantly higher resolution than the printed final image obtained through the Frame grabber (SNAPPY)–PC–printer sequence. Under exactly the same experimental condition, the best state-of-the-art image would require the use of an intensified charge couple device (ICCD) instead of the black-and-white CCD–integrater combination. (See color figures following page 42.)

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FIGURE 18D The time course of the NADH transient (oxidation–reduction transient) after microinjection of glycolytic substrate (Glucose-8-phosphate).

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FIGURE 18E The lag preceding coenzyme reduction, the rise time, t1/2off the rise and decay halftime, and the reoxidation time are indicated. One-dimensional topographic scan of NADH transients in the cytoplasm and nucleus of L cell fibroblast under three conditions: untreated control, azaleic acid treated, and sebacic acid treated. Both azaleic acid and sebacic acid are dicarboxylic acids used in the topical treatment of melanoma, and they are toxic to mitochondrial structure and function.

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FIGURE 19A Serial images showing the temporal responses of cells visible in an optical section of intact islet of Langerhans. Two-photon excitation of cellular autofluorescence is shown in A–H. After measuring the dynamic changes, b and a cells (see I green for insulin and red for glucagon) were identified by immunofluorescence staining and confocal microscopy. The NAD(P)H images shown (time course from 0 to 140 sec) begin one scan before any increase is detected and continue at 8-sec intervals thereafter. NAD(P)H fluorescence began to increase in peripheral cells 32 sec after glucose addition (B) and then spread inward toward the center of islet (C–H). Peak NAD(P)H autofluorescence in central b cells lagged that of the periphery by about 40 sec, consistent with glucose diffusion into the islet. (Bennett, B.D., Jetton, T.L., Ying, G., Magnuson, M.A., and Piston, D.W., J. Biol. Chem., Figure 3, 271, 3647–3751, 1996. Courtesy of Journal of Biological Chemistry.) (See color figures following page 42.)

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FIGURE 19B NAD(P)H autofluorescence measured by two-photon-excitation fluorescence (TPEM) in isolated b cells. The response of these dissociated cells as glucose is increased from 1 (A) to 30 mM (B) is more heterogeneous than those of b cells in intact islets. The scale bar is 12 mm. (Bennett, B.D., Jetton, T.L., Ying, G., Magnuson, M.A., and Piston, D.W., J. Biol. Chem., Figure 3, 271, 3647–3751, 1996. Courtesy of Journal of Biological Chemistry.)

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FIGURE 19C NAD(P)H response to glucose of control and Cre recombinase (AdenoCre)-treated gklox/lox islets containing glucokinase (GK)-inactivated gkdel cells. The relationship between GK and glucose-stimulated metabolism and the potential for metabolic coupling between b cells was examined in isolated pancreatic islets by using a recombinant adenovirus that expresses AdenoCre to inactivate a conditional GK allele (gklox). This results in a heterogeneous population of b cells where 30% of these were converted to a gklox non-expressing variant gkdel. (A) Autofluorescence images of both islets [control islet (left) and adenovirus-treated islet (right)] under 1 mM glucose perfusion. Both islets show a low, but fairly uniform signal level. (B) Same two islets after 5 min of 25 mM glucose in the perfusion medium. In the control islet, the NAD(P)H signal is greatly elevated in all cells, but in the AdenoCre-treated islet many cells show a weak response. (Piston, D.W., Knobel, S.M., Postic, C., Shelton, K.D., and Magnuson, M.A., J. Biol. Chem., Figure 2, 274, 1000–1004, 1999. Courtesy of Journal of Biological Chemistry.)

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FIGURE 20 This micrograph shows the autofluorescence of vesicles and mitochondria in living CV1 cells (green monkey kidney) grown in monolayers and exposed to monophotonic excitation with UVA light (340 to 380 nm) in phosphate-buffered saline plus glucose (1g/L). The emission is recorded at wavelengths greater than 420 nm and therefore mainly includes the fluorescence of NADH in mitochondria, or extramitochondrial NAD(P)H. In lysosomes, in addition to NAD(P)H fluorescence of autophagic debris, the contribution of fluorescent lipofuschin-like pigments is possible. Lipofuschins are fluorescent materials of complex and large chemical structure formed by the reaction of lipid peroxides with reactive free amino and sulphydryl (SH) groups of cell constituents such as proteins and lipids. (A) Gray scale 0–10,000. (B) Gray scale 0–3000. (C) Crop of B with gray scale 0–150, showing details of NADH fluorescence in mitochondria. The linearity of the scale is not guaranteed by the reproduction. Bar = 10 mm. (Courtesy of M. Dellinger and M. Gèze, Muséum National d’Histoire Naturelle, Paris.)

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FIGURE 21 Preferential intranuclear location of glutathione dehydrogenase probe monochlorobimane. The availability of fluorescent probes to detect soluble and protein-bound thiols has made it possible to investigate some aspects of reduced glutathione (GSH) metabolism and function in intact living cells. The first demonstration was by Bellomo in rat hepatocytes. Monochlorobimane (BmCl) has been used to study the subcellular compartmentation of GSH and the formation and fate of the BmCl-GSH conjugate. The occurrence of relatively high concentrations of GSH within the intranuclear matrix has been inferred from fluorescence imaging studies. Concomittant biochemical analysis in hepatocytes by Bellomo’s group has revealed the presence of a GSH-stimulated ATP hydrolysis and an ATP-stimulated GSH accumulation in isolated nuclei. This provides the molecular basis for nuclear glutathione compartmentation. The use of fluorescent probes to label nuclear-free sulfhydryl groups and chromatin status has led to the demonstration that intranuclear accumulation of glutathione may modulate the thiol/disulfide redox status of nuclear proteins and control chromatin compacting and decondensation. In this case monochlorobimane has been used as a probe of the nuclear matrix. The development of fluorescent nuclear matrix probes in addition to fluorescent DNA probes would be a significant development in terms of cellular physiopathology.

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3 Cytotoxic Drugs LYSOSOMOTROPIC AGENTS Cytotoxic lysosomotropic drugs such as quinacrine (atebrine) show extreme accumulation within the lysosomes together with formation of gigantic phagolysosomes. These represent a model for the study of lysosomal storage diseases. The formation of phagolysosomes could be viewed as a Þrst step toward the elimination of such overloaded structures and their extrusion. This is a very challenging path for research in the treatment of lysosomal diseases, but at present it is in a rudimentary stage.

REFERENCES Kohen, E., Kohen, C., Prince, J., Schachtschabel, D.O., Hirschberg, J.G., Morlière, P., Santus, R., Dubertret, L., and Shapiro, B.L., Bioregulatory mechanisms at the level of cell organelle interactions: microspectroßuorometric in situ study, J. Biotech., 13, 1–28, 1990. Kohen, E., Kohen, C., Hirschberg, J.G., Prince, J., Santus, R., Morlière, P., Schachtschabel, D.O., Shapiro, B.L., Mangel, W.P., and Grabowski, G., The spatiotemporal organization of metabolism in living cells. In: Fundamentals of Cell Biology, Vol. 3B (Chemistry of the Living Cell), Bittar, E.E., Ed., 1992, pp. 561–606, JAI Press, Greenwich, CT. Kohen, C., Kohen, E., and Hirschberg, J.G., in Fundamentals of Medical Cell Biology, Vol. 4 (Membranology and Subcellular Organelles), Bittar, E.E., Ed., 1992, pp. 465–497, JAI Press, Greenwich, CT.

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FIGURE 22A Human Þbroblast incubated for 1 h in the presence of 2.8 µM quinacrine (minimum essential medium [MEM]). In this cell treated with quinacrine, the cytotoxic drug has accumulated in lysosomes because of its Lewis acid-base properties characterized by a pKa close to neutrality. The acid-base equilibrium is established in the cytosol, but basic quinacrine species are continuously pumped into the strongly acidic lysosome by the pH gradient. Various sizes of organelles are recognized, from tiny ones to phagolysosomes. One of the challenges for the future is the possibility of relating intralysosomal storage of a cytotoxic drug as a possible model for the storage of various lipids, glucolipids, and ceramides within these organelles, as seen in over 100 hereditary lysosomal storage diseases. There is a compelling need to investigate whether one could go beyond storage toward the ejection of lysosomes from the cell. In parallel to the gene correction of lysosomal disease, is it possible to mobilize the cell energy metabolism and activation of the cytoskeleton for the extrusion of overloaded lysosomes from the cell? SP. (See color Þgures following page 42.)

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FIGURE 22B Quinacrine-loaded Þbroblast. Most of the ßuorochrome is retrieved in lysosomes. Interestingly, such ßuorescent organelles are also found within outward extending cell annexes. Formation of phagolysosomes is noticeable. The ultimate fate of such organelles is unknown. Extrusion from the cell is as yet a remote possibility. In some way, the quinacrine loading of lysosomes could represent a model of lysosomal storage disease in which an analogous loading of undigested glycolipid is evident. SP.

FIGURE 22C Electron micrograph of 1439 rat liver cell submitted to shock treatment with quinacrine (0.4 mM, 30 min) and then washed for 3 min, left overnight in quinacrine-free Eagle MEM medium with 10% calf serum. A giant lysosome (phagolysosome, myelinosome), reminiscent of lysosomal structures observed in storage diseases and pneumocytes type II, is seen in the vicinity of the nucleus. 35,000×.

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FIGURE 22D A cisterna of the rough endoplasmic reticulum outlines the electron transparent space of a myelinosome or penetrates into this area. The portion of the RER cisterna that penetrates into the myelinosome contains material of low electron density. 61,000×.

FIGURE 22E A multicentric myelinosome possibly being released from a cell near a gap junction. The plasma membrane appears to be continuous around the outer membrane of the myelinosome. An electron transparent area of a myelinosome contains material of low electron density. 79,000×.

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FIGURE 22 F, G, H Lysosomotropic quinacrine. Pseudocolor, green. Fluorescent cytoplasmic channels are seen in Figure 22H. For the connection of endoplasmic reticulum cisterns to lysosomes (myelinosome), see Figure 22D.

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FIGURE 23A Fibroblast with quinacrine in lysosome and microcanal-like structure reminiscent of nucleolar channel. The role of nucleolar channels is enigmatic. It is obvious that accumulation of cytotoxic compounds in lysosomes is part of an overall detoxiÞcation mechanism, which often results in a multiorganelle complexing with the Golgi apparatus and ER. The nucleolar channel should be investigated as a means of intranuclear-to-cytoplasmic communication. The activation of such a channel may be involved in protecting the nuclear genetic apparatus from cytotoxic compounds. It is noteworthy that the nucleus, except for staining of the nucleolar channel, appears completely free of quinacrine. This does not exclude the possibility that at the molecular level small amounts of quinacrine or its metabolites may still be bound to the genetic apparatus, even leading to alterations and mutations in such apparatus with serious consequences for the cell on the road to chemical transformation. In this regard, quinacrine is a positively charged molecule that can bind to the negatively charged DNA phosphate backbone. This issue has been brought up repeatedly in connection with ßuorescent carcinogens. The researcher Abraham Stein (Biochemistry, Florida International University, Miami), speciÞcally asked this question: “Are we able to detect a single molecule of carcinogen attached to the genetic apparatus?” The answer is not yet resolved, but studies by Hirshfeld (Hirschfeld, T. in Cell Structure and Function by Microspectroßuorometry, Kohen, E. and Hirschberg, J.G., Eds., 1989, Academic Press, San Diego, pp. xxiii-xxiv) point at least to the theoretical possibility of such a determination. Another topic for exploration is the cell energy metabolism correlate of nucleolar channel activation. SP. (See color Þgures following page 42.)

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FIGURE 23B Quinacrine-loaded Þbroblast. A few paranuclear microchannels (see Color Figure 23C). SP.

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FIGURE 23C Fluorescence micrograph of human Þbroblast following approximately 2 h incubation in MEM with 2.8 µM quinacrine, followed by washing in quinacrine-free medium. The ßuorochrome is accumulated in lysosome-like vesicles and a paranuclear network of microchannels. Massive quinacrine loading of another Þbroblast with formation of phagolysosomes in large number. Another striking feature is the visualization of ßuorescent microchannels that may connect to the nucleus or nuclear membrane on one side and possibly to lysosomes on the other side. Studies with ßuorochromes other than quinacrine, such as the anticancer agent adriamycin, suggest that in an earlier phase the compound may have penetrated into the nucleus and later be extruded. An interesting comparison could be made with cell lines expressing the multidrug resistance phenotype. The existence of a nuclear pump activated by nuclear energy metabolism has been raised. In other cases, channels extending from within the nucleus or nucleolus toward the nuclear membrane have been observed, which could be involved in the extrusion mechanism from the nucleus. Material extruded from the nucleus may then be relocated in the lysosomes. A further step might follow signiÞcantly: the until-now unseen self-healing of lysosomal storage disease. This is a matter of thought for future research and therapeutic programs. SP. (See color Þgures following page 42.)

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FIGURE 24 Quinacrine treatment of Þbroblast. In addition to lysosomes, the ßuorochrome is found accumulating in Golgi clusters and interconnecting channels; there is also suggestion of a short connecting channel between a lysosome and the Golgi apparatus. The ultrastructural electron microscopy equivalent of such interconnecting channels has been researched, but so far it has not been conÞrmed. These channel images, however, raise interesting questions about the formation and organization of a multichannel detoxiÞcation complex, following the addition of cytotoxic compounds. Further studies are needed on the timetable of such complexing and eventual dissociation of the system. Another region of the Þbroblast shows a tendency to giant phagolysosome formation. Could this be a Þrst step in the ultimate expulsion of these organelles loaded with cytotoxic agents from the cell?

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FIGURE 25 Another micrograph of quinacrine-loaded Þbroblast. The whole cell, except for the nuclear region, is Þlled with lysosome-like structures having incorporated the ßuorescent cytotoxic agent. This image is almost like that of a cell from any one of the lysosomal storage diseases. Of the natural cell constituents, primarily lysosomes are invariably loaded with xenobiotics. Loading within these organelles may be preliminary to the detoxiÞcation possibly by conjugation or extrusion from the cell. The fate of the cytotoxic agent after it is incorporated within lysosomes needs further research. SP.

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FIGURE 26A Massive accumulation of ßuorescent cytotoxic compounds in NmuLi (nude mouse liver) hepatocyte after treatment for 3 min with nitramine plus quinacrine.

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FIGURE 26B Aged NMuLi hepatocyte. Quinacrine + nitramine (N-Methyl-N-2,4,6-tetranitrobenzamine, Nmethyl-N, 2,4,6-tetraaniline, picrylmethylnitramine, picrylnitromethylamine, tetralyte, tetryl). There is extreme vesiculation of the cytoplasm. These vesicles may be interpreted as tertiary lysosomes loaded with the two cytotoxic agents. Scanned microphotograph.

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FIGURE 26C Quinacrine + nitramine. Aged hepatocyte NMuLi cells maintained for 30 min in the presence of the cytotoxic compounds nitramine and quinacrine. There is extensive formation of large vesicles, possibly converted from lysosomes, and quinacrine + nitramine loaded. SP.

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FIGURE 27 Quinacrine + nitramine. Aged hepatocyte NMuLi cells maintained for 30 min in the presence of the cytotoxic compounds nitramine and quinacrine. There is extensive formation of large vesicles, possibly converted from lysosomes, and quinacrine + nitramine loaded. Phase image.

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FIGURE 28A, B Pseudocolor. Other examples of lysosome-loading with quinacrine.

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FIGURE 29 Pseudocolor. Quinacrine-loaded lysosomes, also in cytoplasmic extensions.

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FIGURE 30 Quinacrine-treated, DASPMI stained. Patches of DASPMI staining in the nucleus, especially in the nucleoli. Usually dual probe staining is avoided for toxicity reasons. However, it was used in Figure 30 and Figure 31 to reveal mitochondrial status in cells loaded with the lysosomotropic agent quinacrine. SP. (See color Þgures following page 42.)

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FIGURE 31 Fluorescence micrograph of quinacrine-treated Þbroblasts. Cells stained with DASPMI. The mitochondria show spindle-shaped lateral extensions; this image has been rarely noticed. In addition, the mitochondria are segmented and some of these organelles show fusiform dilatations along their full length. Superimposition of DASPMI makes possible visualization of mitochondria and nucleus, especially nucleoli. The mitochondria appear as tailed structures with fusiform dilatations at one or two ends, which make these organelles look like spermatozoa. Interpretation of these morphological changes and knowledge about their reversibility require further study. SP. (See color Þgures following page 42.)

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FIGURE 32 Phenobarbital. Fluorescence micrograph of human Þbroblast grown for 6 days in the presence of 4 mM phenobarbital sodium and subsequently treated with the vital Golgi probe [N-(7-nitrobenzo-2-oxa1,3diazole)-6-amino-caproyl sphingosine (NBD-ceramide) (1.7 µM)]. The prominent paranuclear structure visualized is strongly reminiscent both in location and morphological detail of Golgi images obtained from cells stained for thiamine pyrophosphatase, and also of the ßuorescence images observed in human Þbroblasts grown in the presence of benzo(a)pyrene. Scanned microphotograph.

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MITOCHONDRIA-TOXIC AGENTS The antipsoriatic anthralin and the dicarboxylic acids used on topical applications in melanoma, i.e., azaleic acid and sebacic acid, are among the mitochondria-toxic agents that have been investigated. They lead to mitochondrial swelling, fragmentation, and disintegration. The process starts within 30 min of incubation in the presence of the cytotoxic agent, particularly with azaleic acid. After 30 min exposure to the agent, even if the cells are transferred to agent-free medium for a period of 3 to 7 days, no recovery is observed, as there is persistent damage. When mitochondria are labeled with dimethylaminostyrylpyridniummethyl iodine (DASPMI), following structural damage of the organelle, the probe is retrieved in lysosomes.

REFERENCES Kohen, E., Kohen, C., Morlière, P., Santus, R., Reyftmann, J.P., Dubertret, L., Hirschberg, J.G., and Coulomb, B., A microspectroßuorometric study of the effect of anthralin, an antipsoriatic drug, on cellular structures and metabolism, Cell. Biochem. Funct., 4, 157–168, 1986. Kohen, E., Kohen, C., Schachtschabel, D.O., Hirschberg, J.G., Shapiro, B.L., and Mcheileh, A., Microspectroßuorometry of human melanoma cells and Þbroblasts treated with azelaic acid. In: Photobiology, Riklis, E., Ed., 1991, pp. 315–318, Plenum Press, NY. Shapiro, B.L., Lam, F.H., and Fegal, R.J., Mitochondrial NADH dehydrogenase in cystic Þbrosis, Am. J. Human Genet., 34, 846–852, 1982.

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FIGURE 33 Control Þbroblast. A confrontation of DASPMI-stained mitochondria in a normal Þbroblast vs. the same in a cell treated by a mitochondria-toxic agent, azaleic acid, reveals considerable dislocation of the mitochondrial network, with fragmentation and granulation of these organelles (see Kohen, E. et al., 1991, previous page). While patterns obtained with different mitochondria-toxic agents or antibodies against mitochondrial enzymes are more or less similar, individual characteristic variations in the damage pattern are to be expected with different toxic agents. These chemically produced mitochondrial lesions are to be compared with localized or generalized photodamage due to UVA or visible irradiation with or without photosensitizers that produce very reactive oxygen radicals, similar to those produced by the respiratory chain in addition to other speciÞc activated oxygen species such, as singlet oxygen. Bessis in Paris and Gamaleya in Kiev (personal communication) have found that cell viability is incompatible with more than 30% damage in the mitochondrial population of a single cell. So far, methods of producing photodamage have been with ruby laser in Janus green-stained mitochondria, and with argon laser exciting the cytochrome band in unstained cells. Another challenging method of microirradiation may be the introduction within the living cell of molecular microirradiation sources. Wei Hong Han has introduced a single molecular light source. The most stable single carbocyanine dye C18(DIL) molecule emits about 200 million photons and has a lifetime of approximately 270 h before photobleaching. Work with such intracellular light emitters is still in its infancy, but it certainly points to future possibilities for intracellular microirradiation and localized photodamage. SP.

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A

B

FIGURE 34 Human Þbroblast grown for a week in the presence of 30 mM azaleic acid, a nine-carbon dicarboxylic acid used in the topical treatment of melanoma. (A) Phase. (B) (See color Þgures following page 42.) With rhodamine 123 used as a vital mitochondrial probe instead of DASPMI, ßuorescent fragments of damaged mitochondria are now loaded into the lysosomes. We do not know if such lysosomes persist in the cells as residual bodies (tertiary lysosomes), phagolysosomes, or if they could potentially be extruded from the cell. In view of such mitochondrial–lysosomal interaction, is it conceivable that there will also be a cytoskeletal activation in connection with the extrusion of damaged or debris-loaded organelles? As a far-fetched idea, could mitochondria–lysosome–cytoskeleton interactions have relevance to the study and therapy of lysosomal storage diseases through the accompanying intracellular upheavals? In the case of lysosomal storage disease, the cell is nonfunctional with immobilized super-loaded lysosomes. Could the intracellular mobilization produced by mitochondriatoxic agents, perhaps together with metabolic substrates of extramitochondrial pathways, result in some kind of cellular renewal? The problem we face is a situation in which the cell has practically ceased to be functional, as in the case of the glycoceramidase-deÞcient Gaucher cell, where the remaining cytoplasmic organelles are crushed by the storage microbodies. Can metabolic activation somehow help in getting rid of its lysosomal storage? SP.

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FIGURE 35A Normal Þbroblast treated with azaleic acid and stained with DASPMI. Staining with the vital probe is seen in both mitochondria and nucleus. There is certain mitochondrial damage, but for up to a week these organelles are better preserved than in similarly treated cells with the cystic Þbrosis trait. SP.

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FIGURE 35B Post-azaleic acid damage visualized with rhodamine-123 staining. It shows again coexistence of fragmented Þlamentous (i.e., damaged mitochondria) and ßuorescent globoid (i.e., debris-loaded lysosomes) structures. SP.

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FIGURE 35C Mitochondria in a cystic Þbrosis Þbroblast. The cell has remained for 4 days in azaleic acidcontaining medium as a drug fragilization test. Control Þbroblasts have been observed to remain for longer periods — up to 7 days in the presence of azaleic acid with partial permanence of mitochondrial integrity. However, the Þbroblasts with cystic Þbrosis trait do not last longer than 4 days under the same conditions and their mitochondria are totally disrupted. The rhodamine probe is then retrieved in the lysosomes as the result of mitochondrial autophagy. Micrograph shows extensive disruption of mitochondrial network. Prior to treatment with this mitochondria-toxic agent, the cell had a very well organized network, which could make one suspect that it corresponds to a single highly branched mitochondrion. Following incubation with azaleic acid, remnants of Þlamentous mitochondria are mixed with vesicle-like structures, which in the DASPMI-stained preparation may represent rolled over Þlamentous segments. Azaleic acid is used in the topical treatment of melanoma. Are cells submitted to such a treatment able to recover from the extensive mitochondrial damage? If they should recover, it could be interesting to research residual alterations in the progeny of such cells. Also there is no information about accompanying alterations in the other cytoplasmic organelles. There is some evidence that post azaleic damage, extramitochondrial cytoplasmic and nuclear glycolytic and malate pathways are activated. Could such activation or the use of other metabolic substrates, including 6-phosphogluconate, be effective in the cell’s recovery? The status of mtDNA after azaleic acid treatment is unknown. SP. (See color Þgures following page 42.)

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FIGURE 35D Same treatment as Figure 35C.

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FIGURE 35E Same treatment as Figure 35C.

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FIGURE 36 Same as Figure 35C with maximum autophagy of mitochondria resulting in heavy rhodamine loading of lysosomes. SP. (See color Þgures following page 42.)

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FIGURE 37 Extensive lysosomal autophagy in Þbroblast treated with azaleic acid. The DASPMI used for mitochondrial visualization is retrieved in lysosomes, most of which appear as giant phagolysosomes. SP.

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FIGURE 38 Melanoma 8255 maintained in azaleic acid for three days. The cell exhibits abnormal mitochondria. It would be expected that the mitochondria would be Þlamentous and branched as seen in other Þgures; however, they appear granular and coalescent, suggesting possible fusion. Such cells would require long-term viability tests and postpassage survival. It is not known whether discontinuation of azaleic acid treatment would result in the restoration of the original Þlamentous appearance. SP.

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FIGURE 39A Fibroblast maintained three days in the presence of 30 mM azaleic acid in Eagle’s medium with 10% calf serum. While several of the mitochondria are still Þlamentous, there is evidence of structural damage — apparent fragmentation and clumping, as well as bead-like forms, as described by Goldstein and Korczak for human Þbroblasts treated with cyanide (J. Cell Biol., 91, 392, 1981).)

FIGURE 39B Pseudocolor scale indicated. (See color Þgures following page 42.)

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FIGURE 40 Azaleic acid (10 mM), 30 min shock treatment. The cells are then moved to an azaleic acidfree medium and imaged a week later.

FIGURE 41 Azaleic acid. Pseudocolor scale indicated. (See color Þgures following page 42.)

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FIGURE 42 Azaleic acid, 30 min shock treatment, then imaged seven days after being kept in azaleic acidfree medium. (See color Þgures following page 42.)

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FIGURE 43 Azaleic acid, 30 min shock treatment, then imaged seven days after being kept in azaleic acid-free medium. Pseudocolor images.

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FIGURE 44 DASPMI-stained Þbroblast after anthralin treatment (23 µM, image representative of cells incubated from 15 to 30 min in the presence of this antipsoriasis drug). Control ßuorescence micrograph shows a network of Þlamentous mitochondria. In anthralin-treated cells, this Þlamentous organization is entirely disrupted and is replaced by ßuorescent globoid bodies. The change from Þlamentous to globoid strikingly starts from 30 to 60 min after addition of anthralin and continues in cells grown up to a week in its presence. Possible posttreatment recovery has not been investigated. These images seem to show more extensive damage than post-azaleic acid images. In the case of azaleic acid, we see the coexistence of partially broken Þlamentous structures with globoid bodies. Scanned microphotograph.

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MITOCHONDRIAL INHIBITORS With the uncoupler dinitrophenol, mitochondrial fragmentation is observed within 30 min. Treatment with the membrane potential suppressor results in extreme mitochondrial swelling and a beaded appearance of the organelle.

REFERENCES Juretic, O.D., Chen, H–C., Brown, J.H., Morell, J.L. and Westerhoff, H.V., Magainin-2 amide and analogues. Antimicrobial activity, membrane depolarization and susceptibility to proteolysis, FEBS Lett., 249, 219–223, 1989. Juretic, D., Hendler, R.W., Kamp, F., Caughey, W.S., Zasloff, M., and Westerhoff, H.V., Magainin oligomers reversibly dissipate membrane potential in cytochrome oxidase liposomes, Biochemistry, 33, 4562–4570, 1994. Westerhoff, H.V., Zasloff, M., Rosner, J.L., Hendler, R.W., De Wall, A., Gomez, A.V., Jongsma, A.P., Riethorst, A., and Juretic, D., Functional synergism of the magainins PCLa and magainin-2 in Escherichia coli, tumor cells and liposomes, Eur. J. Biochem., 228, 257–264, 1995.

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FIGURE 45 Dinitrophenol (DNP, 100 µM)-treated Þbroblast stained with DASPMI. After about 30 min of acute exposure to this uncoupler of oxidative phosphorylation, the mitochondria are completely disrupted, with persistence only of globoid fragments. These images resemble those obtained with other toxic agents destroying mitochondrial structure, such as anthralin and azaleic acid. In all these conditions it is expected that there will be severe disturbances in the mitochondrial regulation of cell metabolism, with release from control of extramitochondrial pathways, ie., glycolysis and hexose monophosphate shunt. One would therefore expect and may actually observe a volcano-like activation of these nonrespiratory pathways gone out of control. However, the activation or nonactivation of these pathways is not always predictable, and their timing cannot always be determined. It may also happen that cell metabolism will be completely inhibited. In other instances, glycolysis and hexose monophosphate shunt may survive the organelle damage and show the above-described intensiÞcation. Treatment with dinitrophenol results in anthralin-like images with total replacement of Þlamentous structures by globoid bodies. Posttreatment recovery is unknown. SP.

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FIGURE 46 NMuLi hepatocytes treated with rotenone, a compound used as a pesticide. The ßuorescence images are taken under conditions of NAD(P)H oxidation (aerobic, no substrate added). However, an intense ßuorescence in the blue region of the spectrum is noticed in granular bodies. The ßuorescence is at such a level of intensity that it could hardly be attributable to NAD(P)H per se. It is known that in the same way as the barbiturate amytal, rotenone blocks the NADH-cytochrome b reductase. This should result in NADH accumulation within the mitochondria. However, the ßuorescence observed is above what can be expected from the quantum yield of mitochondrial NADH. The observed ßuorescent bodies are more likely to be lysosomes. This opens a new possibility that there may be a so far undiscovered lysosomal (or other) pathway of energy metabolism. Alternatively, could it be that rotenone itself is ßuorescent under some conditions and the observed ßuorescence corresponds to its localization into the lysosomes, as a step toward its extrusion from the cell? SP. (See color Þgures following page 42.)

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FIGURE 47 Fibroblasts treated with the mitochondria-depolarizing peptide magainin. The original Þlamentous mitochondria have completely disappeared. They are replaced by globular-vesicular bodies, which may indicate swelling and rounding-up of these organelles following loss of membrane potential. This should be followed by ßuorescence probing of mitochondrial membrane potential and eventually ßuorescence probings of mitochondrial ions. In addition, it will be necessary to determine any possible loss of mitochondrial metabolic control and consequent repercussions in extramitochondrial (cytoplasmic and nuclear) glycolysis, phosphogluconate hexose monophosphate shunt, and malate pathways. NAD(P)H and succinate-induced ßavin ßuorescence changes must be studied. Cultivation of the cells after washing off the magainin medium and replacement in standard medium with glucose is recommended for evaluation of post-magainin recovery comparatively in normal and transformed cells, as well as in wild-type and mtDNA-deÞcient mutants. So far, such studies are possible in osteosarcoma and neuroblastoma cells. However, it is hoped that these could be extended to various lines of melanoma and hepatoma. SP. (See color Þgures following page 42.)

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PHOTOSENSITIZERS HEMATOPORPHYRIN, TPPQ Red-ßuorescing porphyrins accumulate in the cytoplasm. Upon continuous irradiation with UV (365 nm) they lead to accumulation of blue-ßuorescing Schiff bases. One must be careful not to confuse the ßuorescence of Schiff bases with that of equally blue-ßuorescing NAD(P)H and the methylumbelliferyl label of ßuorogenic substrates used in the evaluation of lysosomal hydrolases. The Schiff base emission spectrum is to the right of the NAD(P)H spectrum. If fragilization of lysosomal membranes by porphyrin irradiation is used to facilitate penetration of umbelliferyl substrates, it may be difÞcult to distinguish between the ßuorescence of released umbelliferyl label and that of accumulated Schiff bases. The latter accumulate diffusely in the cytoplasm, while the umbelliferyl label is largely conÞned to the lysosomes. Occasionally porphyrins also accumulate within lysosomes, leading to formation of phagolysosomes. Treatment with porphyrins results in post-UV irradiation swelling, fragmentation, and disintegration of mitochondria. In DASPMIstained preparation the ßuorochrome is retrieved within the lysosomes.

PRODUCTION

OF

SCHIFF BASES

BY

CONTINUOUS UV IRRADIATION

Accumulation of Schiff bases proceeds rapidly in the presence of photosensitizers, and at a slower pace in their absence. The reaction sequence is formation of lipid peroxides, followed by malonaldehyde, and ultimately Schiff bases, i.e., iminopropenes. The intermediate malonaldehyde is carcinogenic. The role of Schiff bases and lysosomal membrane permeabilization in carcinogenesis should be investigated.

REFERENCES Kohen, E., Reyftman, J.P., Morlière, P., and Santus, R., Microspectroßuorometry of ßuorescent photoproducts in photosensitized cells. Relationship to cellular senescence and quiescence in culture, Biochim. Biophys. Acta, 805, 332–336, 1984. Morlière, P., Kohen, E., Reyftmann, J.P., Santus, R., Kohen, C., Mazière, J.C., Goldstein, S., Mangel, W.F., and Dubertret, L., Photosensitization by porphyrins delivered to L cell Þbroblasts by human serum low density lipoproteins. A microspectroßuorometric study, Photochem. Photobiol., 46, 183–191, 1987. Morlière, P., Santus, R., Mazière, G.C., Gèze, M., Bazin, M., and Kohen, Photosensitization in living cells studied by microspectroßuorometry, J. Cell Pharmacol., 3, 29–37, 1992. Gèze, M., Morlière, P., Mazière, J.C., Smith, K.M., and Santus, R., Lysosomes, a key target of hydrophobic photosensitizers proposed for photochemotherapeutic applications, J. Photochem. Photobiol., 20, 23–35, 1993. Reyftmann, J.P., Contribution à l'étude de l'action photodynamique des porphyrines sur la membrane cellulaire. Intérêt de la microspectroßuorométrie cellulaire dans la détermination de mécamismes photobiologiques in vivo. THÈSS, Museum National d'Histoire Naturelle et Université Pierre et Marie Curie Paris VI, 1986.

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FIGURE 48A Hematoporphyrin (HP) localization in an L cell Þbroblast. Photosensitization damage in Þbroblasts results from treatment with 0.01% HP, a tetrapyrolic derivative which is an excellent photosensitizer capable of producing activated oxygen species such as singlet oxygen — a major cytotoxin — by photodynamic reactions. See HP ßuorescence in cell membrane and cytoplasm, with nuclear and perinuclear regions practically free of HP. Lysosomal membrane fragilization and mitochondrial fragmentation is expected. For mitochondrial observations one is referred to HP-treated DASPMI- or rhodamine-stained preparations. SP.

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FIGURE 48B Same with hematoporphyrin, also suggesting strong mitochondrial damage consistent with incorporation of the photosensitizer in mitochondria. Here rhodamine 123 is used as a mitochondrial probe. Autophagy of damaged mitochondria is seen.

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FIGURE 49A T4 hepatocyte (NMuLi) transformed at the fourth passage. The cells have been treated with Photofrin 2. The photosensitizer emitting red ßuorescence is found localized within lysosomes. Gigantic phagolysosomes are seen. Such cells can survive in culture after Photofrin 2 loading. Generally, there are heavy disturbances of cell metabolism following photosensitizer loading. Eventually, if the cells can be followed long enough the ultimate fate of phagolysosomes should be researched. Do they disintegrate or does the cell have the capability to extrude them?

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FIGURE 49B NMuLi cells, Þrst generation before transformation after Photofrin 2 treatment as in Figure 49A. The photosensitizer is retrieved in lysosomes. A cell with giant vacuoles is observed but there is little evidence of lysosomal gigantism.

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FIGURE 49C NMuLI transformed hepatocyte grown for 24 h in the presence of Photofrin 2. The cell is stained with DASPMI. There is severe fragmentation of mitochondria.

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FIGURE 50A Mitochondria in DASPMI-stained Þbroblast treated with Photofrin 2, a porphyrin derivative for use in early-stage or inoperable cancers of hollow organs by the so-called Photodynamic Therapy.

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FIGURE 50B As in Figure 50A, Þbroblast treated with Photofrin 2.

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FIGURE 50C Cells grown for 24 h in the presence of Photofrin 2 and stained with DASPMI. In cells where Photofrin ßuorescence is looked at with no DASPMI treatment, the photosensitizer ßuorescence is seen only in cytoplasmic organelles, and the nucleus remains completely unstained. However, when DASPMI is added large regions of the nucleus are stained and in the cytoplasm Photofrin ßuorescence is masked by the more intense DASPMI ßuorescence. In this preparation, the cells are swollen and the cytoplasm is entirely stained by DASPMI with no distinct imaging of organelles. Similar images have been recorded with global cytoplasmic staining in anthralin-treated Þbroblasts that have been stained with DASPMI.

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FIGURE 50D Fibroblast stained overnight with the porphyrin tetraphenylporphine quinoline (TTPQ), which preferentially localizes in lysosomes because of the quinoline group — a weak Lewis base — linked to the porphyrin. The cells are stained with DASPMI. An unusual image is obtained of spindle-like thickenings along the mitochondria. These could be evidence of localized damage or swelling. Followup beyond the initial 24 h is indicated; the ultimate fate of such cells and their mitochondria is currently unknown.

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FIGURE 50E Fibroblast maintained overnight in the presence of 4 µg/ml TTPQ. The cells were then treated with 10 µg/ml rhodamine 123 at 37oC, washed and incubated in basal Eagle’s medium with 20% calf serum for 1 h. There is disruption of the Þlamentous mitochondria characteristic for the cell with emergence of short rodlike or granular corpuscles. Mitochondrial morphology shows some analogy with changes seen in Figure 39 (which were obtained with another cytotoxic agent, i.e., azaleic acid). It is unknown whether restoration of original mitochondrial morphology is possible following discontinuation of the photosensitizer.

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FIGURE 50F Fibroblast treated as in Figure 50E. Similar changes in mitochondrial morphology.

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FIGURE 51A, B, C, D, E Photo-induced formation of Schiff bases — lipofuscin — in Cloudman melanoma cell by the UVA photo-oxidative stress (irradiation with 365 nm light). Lipid peroxidation endproducts, i.e., aldehydes and keto-alcohols, resulting from polyunsaturated fatty acid oxidation by reactive oxygen species react with free amino or sulphydryl (SH) groups in the cell. Photosensitized reactions in normal cells are produced by natural photosensitizers such as ßavins, NAD(P)H, and metal proteins.

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FIGURE 51C, D, E

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CARCINOGENS AND CANCER CHEMOTHERAPEUTIC DRUGS: AGENTS STIMULATING THE PROLIFERATION OF THE ENDOPLASMIC RETICULUM AND GOLGI TOGETHER WITH THE LOADING OF LYSOSOMES The above-named processes result in the formation of a characteristic multiorganelle complex, often paranuclear in localization. This is accompanied by Golgi gigantism. Agents so active are carcinogens, such as benzo(a)pyrene, and cancer chemotherapeutic agents, such as adriamycin. In the case of adriamycin, the drug Þrst accumulates in the nucleus and is then ejected into the cytoplasm, with subsequent accumulation within lysosomes. With benzo(a)pyrene and quinacrine mustard, there is also the appearance of ßuorescent channels running across the cytoplasm, which may represent dilated channels of the endoplasmic reticulum. Occasionally one or more nucleolar channels are identiÞed running from the nucleolus toward the nuclear membrane. All these structural changes may be related to the activation of detoxiÞcation processes such as ßavin-dependent mixed oxidase for metabolization, and possibly multidrug resistance (MDR) for eventual drug extrusion. Studies of concurrent responses to glycolytic substrate challenges, such as glucose-6-phosphate microinjections, reveal considerable activation of NAD(P)H associated pathways. Due to the multiplicity of benzo(a)pyrene metabolites, the analysis of the blue shifts obtained in the emission spectra presents considerable complexity. In principle, such spectra may be recorded pixel-by-pixel throughout the cell. Some of the most toxic metabolites (epoxides and epoxide DNA-adducts) have blue-shifted emission, while some of the extruded metabolites (detected in pericellular medium) seem red-shifted. The methods of deconvolution include: •

•

•

Similarity mapping — comparing recorded complex spectra to a library of known spectra of benzo(a)pyrene metabolites (see Rothman Malik reference in Chapter 3, Nuclear Probes) Matrix method — ßuorescence spectra are recorded in a matrix, the rows representing intensities at various emission wavelengths, each row under a different condition, i.e., excitation wavelength or experimental condition, each column corresponding to emission intensities at a speciÞc wavelength (the object of a doctoral thesis; see Fried, 1987 and Kohen et al., 1983) Fluorescence quenchers — to remove the contribution of a selected metabolite

Above studies of benzo(a)pyrene spectral imaging have been carried in different cell lines: •

• •

Cell cultures not selectively used in chemical carcinogenesis but on which spectroßuorometric data referent to carcinogen penetration, localization, and possible metabolization are already available, e.g., L Þbroblasts BRL (Buffalo rat liver) cells which are inducible for benzo(a)pyrene metabolization by 18 h preincubation in benzanthracene, comparing induced and noninduced cells CCL 228 (C3H/10T1/2C18) clone especially used in studies used in chemical carcinogenesis

REFERENCES Daudel, P., Croisy-Delcey, M., Jacquignon, P., and Vigny, P., Sur la structure d’un complexe résultant de l’action d’un epoxide aromatique polycyclique sur un acide deoxyribonucléique. C.R.. Acad.. Sci., Paris, 277, 2437–2439, 1973.

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Daudel, P., Croisy-Delcey, M., Alonso-Verduras, C., Duquesne, M., Jacquignon, P., Markovits, P., and Vigny, P., Étude par ßuorescence d’acides nucléiques extraits de cellules en culture traitées par le méthyl benzo(a)anthracène, C.R.. Acad.. Sci., Paris, 278, 2249–2252, 1974. Deumié, M., Kohen, E., Viallet, P., and Kohen, C., Rapid microspectroßuorometric studies in EL2 cells following intranuclear accumulation of dibenzocarbazoles, Histochemistry, 48, 17–27, 1976. Fried, M.R., Statistical methods for the determination of the number of independent ßuorescent emitters in living cells, May, 1987, Department of Physics, University of Miami. Gelboin, H.V., Benzo(a)pyrene metabolism, activation and carcinogenesis: role and regulation of mixedfunction oxidases and related enzymes, Physiol. Rev., 60, 1107–1166, 1980. Gelboin, H.V., Kinoshita, N., and Wiebel, F.J., Microsomal hydrolases: induction and role in polycyclic hydrocarbon carcinogenesis and toxicity, Fed. Proc., 31, 1298–1302, 1972. GrafÞ, A., Intracelluläre Benzprenspeicherung in lebenden Normal- und Tumorzellen (Mitteilung), Z. Krebsforschung, 50, 196–219, 1940. Kohen, E., Kohen, C., and Hirschberg, J.G., Microspectro-ßuorometery of carcinogens in living cells, Histochemistry, 79, 31–52, 1983. Salmon, J.M., Kohen, E., Kohen, C., and Bengtsson, G., Microspectroßuorometric approach for the study of benzo(a)pyrene and dibenzo(a,h)anthracene metabolization in single living cells, Histochemistry, 42, 61–74, 1974.

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FIGURE 52A Human skin Þbroblast (line 61 obtained from Dr. B.L. Shapiro, Department of Oral Biology, University of Minnesota, 1991) from a culture maintained continuously for 1 month in the presence of 15 µM carcinogenic agent, i.e., benzo(a)pyrene. This compound is found to accumulate in a perinuclear network formed by the Golgi apparatus, the ER, and the lysosomes. The constitution of such a multiorganelle complex can be investigated from three standpoints: spectroscopic, physiopathologic, and therapeutic (connected to cell detoxiÞcation activity). A scan of the multiorganelle complex by ßuorescence excitation spectroscopy brought to its highest accuracy by Fourier interferometry may help to detect various metabolites of the original agent. A set of spectral images characteristic of the various metabolites being stored in a computer memory can be used for the deconvolution of the spectral images over various locations of the observed multiorganelle complex. From the point of physiopathology, one could attempt to relate the formation of the multiorganelle complex to cell energy metabolism, using metabolic substrates, modiÞers — activators and inhibitors — and mitochondria-toxic agents. Both preventive and therapeutic applications are conceivable if we can mobilize and organize the formation of the multiorganelle network toward the elimination of the cytotoxic compounds. One possibility is that despite the lack of evidence for accumulation within the nucleus, minute amounts of very active metabolites are actually reaching genetic sites. The question has been asked if one single molecule of carcinogen metabolite can be localized at a genetic site by ßuorescence. Monomolecular localization of ßuorescent molecules has been discussed and it is possible (see Hirschfeld in Figure 23A legend). One should therefore try to expand the limits of ßuorescence detection to such a level. Whether single molecular localization is possible or not, every cell presents its own pattern of multiorganelle detoxiÞcation network production. It may become possible to relate the formation and control of such networks to cell diagnostics and therapeutics. The carcinogen is localized in lysosome-like vesicles and a large paranuclear network. The latter is reminiscent of the Golgi apparatus stained for thiamine pyrophosphatase. Fluorescence spectra can be recorded from such structure and compared to spectra from other cytoplasmic and nuclear regions. Within the living cell, benzo(a)pyrene is converted to over 40 metabolites. For this reason, the deconvolution of ßuorescence emission spectra recorded pixel-by-pixel presents enormous complexities. When the 406 nm/460 nm ratio of the intracellular emission spectrum is measured in L cell Þbroblasts, there is generally a nuclear blue shift as compared to benzo(a)pyrene in solution. The blue shift is randomized over the nuclear and cytoplasmic regions as the cultured cells are maintained for days in the presence of the carcinogen. Comparatively, the surrounding medium is red-shifted, which could indicate extrusion of red-shifted metabolite conjugates such as glucuronides or sulfates, or the metabolite 3-hydroxybenzo(a)pyrene. (See color Þgures following page 42.)

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FIGURE 52B Benzo(a)pyrene (BP) emission spectrum in solution and cytoplasm of L Þbroblast gown in the presence of BP.

FIGURE 52C Time evolution of benzo(a)pyrene (BP) spectrum.

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FIGURE 53 Fibroblast maintained for 24 h in the presence of the carcinogen benzo(a)pyrene (1 µM). The benzo(a)pyrene ßuorescence persists up to a week after removal of the carcinogen from the medium. The distribution of the carcinogen is somewhat different from that in Figure 62, except for the absence of the agent within the nucleus. The carcinogen is primarily localized in the lysosomes within a perinuclear cloud, which may correspond to the Golgi apparatus. These cells survive long-term culture in the presence of benzo(a)pyrene. Thus, it would be interesting to use the most reÞned Fourier excitation–emission spectral imaging to follow the possible evolution of metabolites. The recognition of individual metabolites will require spectral deconvolution using a bank of metabolic images with characteristic spectral properties. A challenging question is: Can the evolution of metabolite(s) spectra be related to energy metabolism? This can be studied by adding intermediates or modiÞers of the main energy pathways through cell perfusion or microinjection. Cells already malignant, such as osteosarcoma or neuroblastoma, may be submitted to a similar treatment and followed through culture up to several weeks. It remains to be determined whether target sites in the process of carcinogenesis are the primary sites of localization observed in Figure 52 through Figure 54. We do not even know at this time whether the almost impossible localization at genetic sites as hypothesized by many researchers is indeed crucial. Resolution of that aspect rests upon the ability to detect single ßuorescent molecules or a few such molecules at speciÞc sites within the nucleus. Originally GrafÞ thought that the Þrst localization and effect of ßuorescent carcinogens was in the mitochondria. At that time, in the 1940s, the separate identity of lysosomes was unknown. Even now, despite current knowledge of cytoplasmic organelles by cell fractionation methods and ultrastructural analysis, there is justiÞcation to probe for a certain overlap of organelle function within the intact cell. Some of the blue ßuorescence attributed to mitochondrial NAD(P)H may actually be lysosomal, particularly in aging cells, and itself due to metabolism or storage and formation of lipofuscin-like pigments. (See color Þgures following page 42.)

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FIGURE 54A Benzo(a)pyrene-treated Þbroblasts. The image is strikingly similar to some of the images obtained in cells grown with the Golgi probe NBD-ceramide (see Figure 10). This is simply because what we are observing is ßuorochrome accumulation in the Golgi apparatus; in one case the ßuorochrome is a Golgi probe, in the other case it is the carcinogen itself. The variety of benzo(a)pyrene localization sites revealed here and in other Þgures is most likely a consequence of the lipophilic character of this carcinogenic agent; its distribution depends on experimental conditions such as concentration, incubation time, and cellular aging, i.e., number of passages.

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FIGURE 54B Fibroblast treated with benzo(a)pyrene. The cell was then treated with sorbitol and subsequently submitted to hypotonic shock. The treatment resulted in formation of swollen vesicles throughout the cytoplasm. Some of the benzo(a)pyrene is retrieved in unswollen, small lysosome-like corpuscles. There is also formation of what appears to be a nucleolar channel that extends from within the nucleolar toward the nuclear membrane.

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FIGURE 54C Beno(a)pyrene-grown Þbroblasts. The ßuorescent carcinogen is seen to accumulate in lysosomal bodies and a rather small paranuclear network consistent with the Golgi apparatus. An interesting Þnding is the presence of three nucleolar channels that seem to be conßuent toward a paranuclear structure which may be a part of the Golgi-ER complex. One may wonder if these channels are not carrying benzo(a)pyrene or its metabolites toward extranuclear organelles in what may be conceived as a pathway to protect the cell's genetic apparatus from “xenobiotic attack.” Modern cameras with point spread function (PSF) correction or confocal imaging could be helpful in the study of the relationship between nuclear and paranuclear structures. It is noteworthy that in this Þgure the lysosomes are quite small, and there is no evidence of phagolysosomes as seen in other cases of treatment with cytotoxic agents.

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FIGURE 54D Other Þbroblast treated with benzo(a)pyrene. The distribution of the cytotoxic agent is quite different from in Figure 54C. There is a massive accumulation into lysosomes, which form a perinuclear cloud. Nucleolar channels are not visible. The nucleus shows complete absence of the toxic ßuorochrome. Whatever ßuorescence is seen in the central nuclear region can be interpreted as coming from overlying or underlying lysosomes. This image seems in conßict with the mutation theory of carcinogenesis. Of course, this is only in appearance, because it is impossible to exclude the possibility that a few benzo(a)pyrene metabolite molecules are Þnding their way to the genetic apparatus. Some of the Þbroblasts show enlarged ßuorescent lysosomal bodies that may be actual phagolysosomes.

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FIGURE 54E Another feature of benzo(a)pyrene treatment. In this case, the cytotoxic agent is seen to accumulate within Golgi-like channels and lysosomes. (See color Þgures following page 42.)

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FIGURE 55A Cloudman melanoma cells incubated in the presence of adriamycin. The nuclei are hyperloaded with the ßuorescent cancer chemotherapeutic, which is also a carcinogen.

FIGURE 55B Human Þbroblast incubated for 6 h in the presence of adriamycin and then stained with the Golgi probe NBD-ceramide. Note the presence of adriamycin-loaded ßuorescent channels (? Golgi ER cisterns). (See color Þgures following page 42.)

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FIGURE 55C Adriamycin-treated Þbroblast. The agent is both a cancer chemotherapeutic and a carcinogen. The ßuorochrome accumulates in the nucleus (see Figure 55A) and is then ejected toward cytoplasmic organelles, such as lysosomes, as part of a detoxiÞcation process.

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FIGURE 55D Adriamycin-treated Þbroblast. Very large and compact Golgi apparatus.

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4 Genetic Diseases The probing of lysosomes has been accomplished with fluorogenic umbelliferone substrates such as methyl umbelliferyl glucoside (UMBG) and UG9 4-nonylumbelliferyl-b-D-glucoside. The penetration of fluorogenic substrates is facilitated by: • • •

Fragilization of lysosomal membrane by photosensitizers of the porphyrin group Use of detergents such as N-dodecyl imidazole Incubation with apolipoprotein to facilitate penetration of fluorogenic substrate through the cholesterol pathway

If the fluorogenic substrate is not channeled through the cholesterol substrate, it is treated by the cell as a xenobiotic and accumulates within a multiorganelle perinuclear complex often constituted by the Golgi and the ER with participation of lysosomes. The fluorescent probes used by Gatt (1993) are lissaminerhodaminedodecanoyl-glucocerobroside (LR12GC) and pyrenedodecanoyl trihexosyl ceramide (P12THC). They work in the opposite way of fluorogenic probes. In the latter case, fluorescence of the fluorophore moeity is released if the probe is cleaved by glucoceramidase. However, when a fluorophore is cleaved from a fluorescent probe, then the glucoside moeity is digested, the cleaved fluorophore cannot be retained; it is extruded, which leads to a loss of intracellular fluorescence. Conversely, fluorogenic probes are designed for postcleavage intracellular retention of the fluorescence-masking glucoside moeity. Particularly, the nonyl group has been entered in the design of UG9 to facilitate retention due to the larger size of the liberated fluorophore. In this chapter, applications are shown in the study of storage diseases associated with hydrolase deficiencies leading to accumulation of glucoceramide (Gaucher) and sphingolipids (Niemann-Pick).

REFERENCES Gaucher, P.C.E., De l’épitheliome primitif de la rate, hypertrophie idiopathique de la rate sans leucémie, Thèse de Doctorat en Médecine, 28 Janvier 1882. Octave Doin, Éd., Paris. Kohen, E., Kohen, C., Hirschberg, J.G., Santus, R., Grabowski, G., Mangel, W., Gatt, S., and Prince, J., An in situ study of beta-glucosidase activity in normal and Gaucher fibroblasts with fluorogenic probes, Cell Biochem. Funct., 11, 167–177, 1993. Mahley, R.W., Apolipoprotein E: cholesterol transport. Protein with expanding role in biology, Science, 140, 622–629, 1988. Marks, D.L. and Pagano, R.E., Clathrin-dependent and -independent internalization of plasma membrane sphingolipids intitiates two Golgi targeting pathways, J. Cell Biol., 154, 535–547, 2001. Pagano, R.E., Puri, V., Dominguez, M., and Marks, B.L., Membrane traffic in sphingolipid storage disease, Traffic, 1, 807–815, 2000. Puri, V., Watanabe, R., Singh, R.D., Dominguez, M., Brown, J.C., Wheatley, C.L., Puri, V., Watanabe, R., Sun, X., Wheatley, C.L., Marks, D.L., and Pagano, R.E., Nature Cell Biol., 1, 386–388, 1999. Scriver, C.R., Beaudet, A.L., Sly, W.S., and Valle, D., The Metabolic and Molecular Basis of Inherited Disease, Vols. I–III, McGraw Hill, 7th ed., 1995. Sun, X., Marks, D.L., Park, W.D., Wheatley, C.L., Puri, V., O'Brien, J.F., Kraft, D.L., Lundquist, P.A., Patterson, M.C., Pagano, R.E., and Snow, K., Niemann-Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1, Am. J. Hum. Genet., 68, 1361–1372, 2001.

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FIGURE 56 Normal and Gaucher fibroblasts are treated with a lysosomal fluorogenic probe — UG9 (4-nonylum-

belliferyl-b-D-glucoside), 13 mg/ml. There are recorded instances of cells remaining in the presence of UG9 for no longer than 1 h. The fluorogenic substrate becomes fluorescent after digestion by glucoceramidase. The enzyme has a lysosomal and a cytoplasmic variety. Fibroblasts from a Gaucher patient were submitted to a treatment similar to that of the normal fibroblasts. In examples of both cases, fluorochrome accumulation in lysosome-like bodies was observed. One would have expected less fluorescence in the Gaucher fibroblasts due to partial or total deficiency of the hydrolase, i.e., glucoceramidase. Fluorescence spectra recorded from normal and Gaucher fibroblasts have generally shown weaker, or much weaker, fluorescence in the cells with the Gaucher trait. However, spectral studies occasionally reveal the existence of Gaucher cells in which the fluorogenic reaction of the glucoceramidase probe is quite intense. In the face of this ambiguity, is it still possible to develop a functional identification and characterization of Gaucher cells with UG9 or similar probes? It seems necessary to establish a scale of graded levels in the activity or deficiency of glucoceramidase within the lysosomes as well as the cytoplasm. On this basis, different degrees of enzyme activity or deficiency may be identified. Once such scaling is established, different methods of cell therapy may be investigated. Not only gene therapy should be considered, but also co-culture of enzyme-defective and normal cells. Another challenging approach may be to research, in these Gaucher cells obviously stuffed with defective lysosomes, the triggering of energy-metabolism-dependent and cytoskeleton-mediated extrusion of these disabled bodies. For the particular Gaucher cells showing fluorochrome emission, it is conceivable that the fluorogenic substrate cleavage by ceramidase is rather nonspecific and induces the liberation of nonylumbelliferyl which is then treated as a toxic foreign material. This has indeed been observed in overnight incubation with fluorogenic agents without apolipoprotein (ApoE). In such cases, a response of cytoplasmic organelles may be expected with formation of a multiorganelle aggregate including involvement of Golgi and ER. Fibroblasts were grown in the presence of the fluorogenic probe UG9 without addition of ApoE, a mediator of the lysosomal pathway for cholesterol or like substrate incorporation. The probe had not entered the cell through the apolipoprotein-guided pathway and was not recognized as a lysosomal substrate. Consequently, it was simply recognized as a toxic foreign compound, a xenobiotic, and it was incorporated into the fibroblast’s multiorganelle detoxification complex comprising Golgi, ER, and lysosomes. In such instances, the large fluorescent bodies seen could be interpreted as being either phagolysosomes or components of a dispersed Golgi apparatus. Thus, for the added UG9 probe two possible fates are to be expected: 1) incorporation through the ApoE-mediated cholesterol pathway with targeting to lysosomes and in situ glucoceramidase activity leading to release of the umbelliferyl fluorochrome, or 2) nonspecific processing of the fluorogenic substrate as a xenobiotic and accumulation of fluorochrome in Golgi, ER, and lysosomes as a xenobiotic awaiting extrusion or metabolization. This figure is an example of the second possibility, observed in MKE fibroblasts obtained from Dr. G. Grabowski, Human Genetics, Mount Sinai, 1993. Figures 57A, B, C, and D are examples of the second possibility.

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FIGURE 57A Targeting of the fluorogenic probe UG9, 13 mg ml–1, to the lysosomes, as seen in this image, is more satisfactorily accomplished in the presence of ApoE, 1.2 mg ml–1. The cells were incubated in the ApoE, UG9 medium for 3 h. Postcleavage accumulation of the fluorochrome moiety of UG9 is seen in large lysosome-like bodies, suggesting phagolysosomes. The observed bodies are too large to be accounted for by single lysosomes. It seems therefore that the process is accompanied by coalescence and fusion of lysosomelike bodies. This image represents a first step in the use of fluorogenic-substrate-added fibroblasts for the diagnosis of Gaucher disease (glucoceramidase, i.e., beta-glucosidase deficiency) and eventually the evaluation of gene or other therapy. A suggestion that a diagnostic test may result from these studies comes from the incubation and growth of normal and Gaucher fibroblasts up to several days in the presence of the glucoceramidase inhibitor conduritol B epoxide (20 mM). The fluorogenic response was considerably more suppressed in the Gaucher cells as compared to normal fibroblasts.

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FIGURE 57B UG9 in the presence of ApoE.

FIGURE 57C Similar to Figures 57A and B.

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FIGURE 57D Similar to Figures 57A, B, and C. (See color figures following page 42.)

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FIGURE 57E An alternative to ApoE-mediated internalization of the probe is facilitation of probe penetration by the use of a detergent, such as N-dodecylimidazole, 100 mg ml.-1. Instead of UG9, another probe synthesized by W. Mangel (1993) is fluoresceinyl(bis)-b-D-glucopyranoside, 50 mg ml-1. The fluorescence rise was monitored from 45 sec to 1 h 15 min. There was a relatively high background fluorescence after addition of the probe, but this did not prevent the observation of the fluorogenic reaction. An increase of fluorescence over background, most likely due to cleavage of the probe, started at about 3 min with continuous rise until 1 h 15 min. The maximum of the emission spectrum recorded from the rising fluorescence was at about 544 nm, which is consistent with the fluorogenic group fluoresceinyl.

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FIGURE 58A Fluorescence micrograph of human fibroblast obtained from Dr. G. Grabowski (Division of Medical Genetics, Dept. of Pediatrics, Mount Sinai School of Medicine, NY, 1993) and treated with the lysosomotropic detergent N-dodecyl imidazole to achieve permeabilization of the lysosomal membrane. Using the fluorogenic probe fluoresceinyl(bis)-b-glucopyranoside, intragranular, or intralysosomal, localization of the cleaved probe is observed. This is probably the first record of intralysosomal in situ fluorogenic reaction. Intralysosomal localization of fluorescence-labeled glucosylceramide has reportedly been obtained by Dr. S. Gatt, Jerusalem (personal communication to Dr. Grabowski, 1997).

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FIGURE 58B Phase photomicrograph of the same lysosomotropic detergent-treated cells showing extensive granularity and swelling of the cytoplasm.

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A

B

FIGURE 59 (A) Colocalization of Rh-CtxB and Dil-LDL with caveolin-1-GFP in human skin fibroblasts. Cells were

transfected with a plasmid expressing caveolin-1-GFP (COOH–terminal) and subsequently incubated with 0.2 mM Rh-CtxB, fluorescent endocytic marker, or 0.5 mg/Dil-LDL, fluorescent LDL, for 30 min at 37oC. The samples were then observed by confocal microscopy. Note the extensive colocalization of Rh-CtxB-red fluorescence — (but not Dil-LDL) with caveolin-1GFP-green fluorescence. No crossover between red and green fluorescence channels could be detected in control cells labeled with marker alone, i.e., Rh-CtxB or Dil-LDL, or in caveolin-1-GFP (green fluorescence)-labeled cells containing no marker. The results suggest that in skin fibroblasts CtxB is a valid marker for caveolae, as defined by the presence of caveolin-1GFP. (Puri, V., Watanabe, R., Singh, R.D., Dominguez, M., Brown, J.C., Wheatley, C.L., and Pagano, R.E., Figure 4, J. Cell Biol., 154, 535–547, 2001. Courtesy of the Rockefeller University Press.) (B) Colocalization of endosomes labeled by fluorescent LacCer or SM with endocytic markers. Cells were incubated with 0.5 mM BODIPY-LacCer and 0.2 mM Rh-CtxB or 0.5 mg/ml Dil-LDL for 30 min at 10oC, washed and warmed for 30 sec at 37oC in the absence of inhibitors before fluorescence microscopy. Note the extensive colocalization of BODIPY-LacCer — green fluorescence — and Rh-CtxB — red fluorescence — visible as yellow–orange fluorescence, whereas little colocalization of BODIPY-LacCer was seen with Dil-LDL 9S (see overlay). (Puri, V., Watanabe, R., Singh, R.D., Dominguez, M., Brown, J.C., Wheatley, C.L., and Pagano, R.E., Figure 5, J. Cell Biol., 154, 535–547, 2001. Courtesy of the Rockefeller University Press.)

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FIGURE 60 BODIPY-LacCer targeting in normal, variant, and classical NPC cells. Living NPC fibroblasts on glass cover slips were pulse-labeled with BODIPY-LacCer for 45 min at 37oC and were observed by fluorescence microscopy. Normal and NPC fibroblasts were obtained from the Coriell Institute for Medical Research. Other NPC fibroblasts were from the NPC Cell Repository of the Division of Laboratory Genetics at the Mayo Clinic in Rochester, MN. Images are representative of >80% of cells observed for each experiment. Note that BODIPY-LacCer was targeted to the Golgi apparatus in both normal and NPC variant cells but was concentrated in punctate structures in classical NPC cells. Bar = 10 mm. G = Golgi complex. (Sun, X., Marks, D.L., Park, W.D., Wheatley, C.L., Puri, V., O'Brien, J.F., Craft, D.L., Lundquist, P.A., Patterson, M.C., Pagano, R.E., and Snow, K., Figure 2, Am. J. Hum. Genet., 68, 1361–1372, 2001. Courtesy of American Journal of Genetics.)

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FIGURE 61 Effect of cholesterol on sphingolipid targeting in normal and sphingolipid storage disease (SLSD) fibroblasts. Cholesterol depletion restores normal LacCer (lactosylceramide) trafficking in SLSD fibroblasts. Cells were grown in complete medium containing fetal bovine serum (FBS) (controls) or in medium containing lipoprotein-deficient serum (LPDS) to deplete cellular cholesterol, pulse-labeled with BODIPYLacCer, and observed under the fluorescence microscope. In normal cells, cholesterol depletion enhanced the labeling of the Golgi complex. In Niemann-Pick type A (NPA) and GM1 gangliosidosis cells, labeling of the endosomes/lysosomes was greatly reduced and labeling of the Golgi complex increased after cholesterol depletion.

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FIGURE 62 Excess cholesterol in normal fibroblasts confers an SLSD phenotype for LacCer transport. Cells were grown in complete medium containing FBS (controls) or in LPDS containing 125 mg ml-1 LDL for 24 h to raise cellular cholesterol levels to an excess. The cells were then pulse-labeled with BODIPY LacCer and observed under the fluorescence microscope, or fixed and stained with antibody to Niemann-Pick type C1 (NPC1). Note the absence of Golgi labeling by BODIPY-LacCer in cells incubated with an excess of LDL, compared with the Golgi labeling seen in normal fibroblasts grown in FBS. Scale bars represent 10 mm. (Puri, V., Watanabe, R., Dominguez, M., Sun, X., Wheatley, C.L., Marks, D.L., and Pagano, R.E., Figure 2, Nature Cell Biology, 1, 386–388, 1999. Courtesy of Nature Cell Biology.)

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FIGURE 63 Top: Normal human fibroblasts and GM1 fibroblasts were grown in complete medium with FBS (controls) and LPDS (lipoprotein-deficient serum). The cells were then pulse-labeled with BODIPY-LacCer and observed under the fluorescence microscope. Bottom: Normal human skin fibroblasts were grown with 50 mg ml-1 LDL (control) or 125 mg ml-1 (excess chol) for 24 h. Note the absence of Golgi labeling by the LacCer analog in cells incubated with an excess of LDL relative to that seen for control fibroblasts grown in FBS. Bars, 10 ìm. (Pagano, R.E., Puri, V., Dominguez, M., and Marks, D.L., Figure 3B, Traffic, 1, 807–815, 2000. Courtesy of Munksgaard.)

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Differentiation and 5 Cell Cell Pathology Hepatocytes. Nude mouse liver cells (NMuLi) hepatocyte clones have been followed in the course of spontaneous transformation over several generations. The nontransformed early clones have an epitheloid morphology and globoid mitochondria. The subsequent transformed clones exhibit a fibroblastoid morphology, and the mitochondria change from globoid to filamentous. Golgi gigantism is observed in the transformed clones. Myoblast. In fusion-competent mouse myoblasts the mitochondria are globoid and the Golgi is prominent. In fusion-deficient myoblasts the mitochondria are filamentous and the Golgi is fragmented, dispersed. Keratinocytes. As keratinocytes undergo the process of spontaneous transformation in culture, two mitochondrial patterns are observed: globoid and filamentous. Wild-type osteosarcoma and mitochondrial DNA-deficient mutants. Mostly globoid, large mitochondria are present. Characteristically, such large globoid mitochondria are also present in the many tentacle-like extensions of these cells. Human colon cancer cells. Short rod-like mitochondria have been labeled with dimethylaminostyrylpyridniummethyl iodine (DASPMI), tetramethylrhodamine-methylester (TMRM), and the mitochondrial oxygen probe PRE4. In quinacrine-treated colon cancer cells, occasional phagolysosome formation is noticed.

REFERENCES Engel, A.G. and Banker, B.Q., Myology, Vol. 1–2, McGraw-Hill, NY, 1986. Kohen, E., Kohen, C., Prince, J., Pinon, R., Hirschberg, J.G., Santus, R., Morlière, P., Schachtschabel, D.O., and Gatt, S., Microspectrofluorometry of organelle interactions in hepatocytes treated with cytotoxic agents, J. Cell Pharmacol., 3, 8–21, 1992. Hirschberg, J.G., Kohen, E., and Kohen, C., Microspectrofluorometry: its application to the study of living cell structure and function, Trends in Optical Engineering, Trivandrum, Kerala, India, 1993. Kohen, E., Hirschberg, J.G., Kohen, C., and Monti, M., NAD(P)H and Schiff base fluorescence spectroscopy, imaging and maximum sensitivity micrographs at the convergence of cellular detoxification, senescence and differentiation. In: Proc. Adv. in Fluorescence Sensing Technol., IV, Jan. 24–27, 1999, San Diego, CA, Vol. 3602, pp. 172–183, SPIE, Bellingham, WA. Kohen, E., Hirschberg, J.G., Kohen, C., Schachtschabel, D.O., Monti, M., and Stanikunaite, R., Fluorescence spectral imaging of organelle interactions. In: Optical Diagnostics of Living Cells, Vol. 3, Progress in Biomedical Optics and Imaging, Jan. 24–25, 2000, San Jose, CA, Vol. 3921, 218–231, SPIE, Bellingham, WA. Kohen, E., Gatt, S., Schachtschabel, A., Schachtschabel, D.O., Kohen, C., Agmon, V., Hirschberg, J.G., Monti, M., and Roisen, F., Multiprobe fluorescence imaging and microspectrofluorometry of cell transformation and differentiation: implications in terms of biochemistry and biotechnology, Biotechnol. Appl. Biochem., 29, 191–205, 1999. Kohen, E., Gatt, S., Schachtschabel, A., Schachtschabel, D.O., Kohen, E., Agmon, V., Hirschberg, J.G., and Monti, M., Microspectrofluorometry and fluorescence imaging in the study of human cytopathology, Microscopy Research and Technique, 51, 469–480, 2000.

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Hirschberg, J.G., Kohen, E., Kohen, C., Monti, M., Stanikunaite, R., Özkütük, N., and Schachtschabel, D.O., The potential and application of Fourier Interferometry to cell cytometry towards predictive oncology, 5th Int. Symp. Predictive Oncology and Therapy, Geneva, Switzerland, Oct. 28–31, 2000, Cancer Detect. Prev., 24/Suppl., 2000. Hirschberg, J.G., Kohen, E., Örnek, C., Monti, M., and Berry, J.P., Fluorescence imaging of mitochondrial localization in malignant cells, Biomedical Optical Spectroscopy and Diagnostics, OSA Biomedical Topical Meetings, April 7–10, 2002, Fontainebleau Resort and Towers, Miami Beach, FL, Abstract TuD22. Kohen, E., Hirschberg, J.G., Örnek, C., Monti, M., and Berry, J.P., Fluorescence imaging of mitochondrial responses to glucose challenge and probes in mitochondrial DNA-deficient osteosarcoma cells, Cancer Detec. Prev., 2002 Symp. Vol., 6th Int. Symp. Predictive Oncology and Intervention Strategies, Pasteur Institute, Paris, Feb. 9-12, 2002. Programs and Abstracts, p. 382, Abstr. 382. Kohen, E., Hirschberg, J.G., Örnek, C., Monti, M., Berry, J., and Leblanc, R., Fluorescence imaging of mitochondrial responses to glucose in osteosarcoma, Microscopy and Microanalysis 2002, Expo Issue Vol. 8, Suppl. 1, 2002, Quebec City, Canada, Aug. 5–8. Kohen, E., Hirschberg, J.G., Örnek, C., Berry, J., and Monti, M., Fluorescence spectral imaging of wild type and mitochondrial DNA-deficient malignant cells in culture, Proc. Abstr., 7th World Congress on Advances in Oncology, 5th Int. Symp. Molecular Medicine, Oct. 10–12, 2002, Hersenissos, Crete, Greece, Abstr. 138. Kohen, E., Gatt, S., Schachtschabel, A., Schachtschabel, D.O., Kohen, C., Agmon, V., Hirschberg, J.G., Monti, M., and Roisen, F., Multiprobe fluorescence imaging and microspectrofluorometry of cell transformation and differentiation: implications in terms of applied biochemistry and biotechnology, Biotechnol. Appl. Biochem., 29, 191–205, 1999. Kohen, E., Gatt, S., Schachtschabel, A., Schachtschabel, D.O., Kohen, C., Agmon, V., Hirschberg, J.G., and Monti, M., Microscopy Res. Tech., 51, 469–480, 2000. Kohen, E., Hirschberg, J.G., Örnek, C., Monti, M., and Berry, J.P., Fluorescence imaging of mitochondrial localization of metabolism in malignant cells, OSA Biomedical Topical Meetings: Biomedical Optical Spectroscopy and Diagnostics, BOSD, pp. 562–564, Fontainebleau Hilton Resort and Towers, Miami Beach, FL, April 7–10, 2002. Kohen, E., Özkütük, N., Hirschberg, J.G., Pantazis, P., Balan, K., Monti, M., and Haroon, S., New fluorescence imaging studies on colon and breast cancer cells, Proc. Abstr., 8th World Congress on Advances in Oncology and 6th Int. Symp. Molecular Medicine, Oct. 2003, Hersenissos, Crete, Greece.

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FIGURE 63A Nude mouse hepatocyte (NMuLi cell), normal clone. Mitochondria are rather globoid and consistent with the mitochondrial image obtained in histological preparations of normal liver cells.

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FIGURE 63B Nude mouse hepatocyte (NMuLi cell). Clone having undergone malignant transformation. The originally polygonal epithelial cells have undergone a change to elongated, spindle-shaped fibroblastoid type. The mitochondria are now filamentous. The processes of these cellular and mitochondrial morphological changes are now under investigation. They may be viewed under the general category of epithelial cells undergoing mesenchymal drift in the course of malignant transformation. Whether such cells have lost their functional specialization, such as bile production, is unknown at this stage.

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FIGURE 64 Hepatocyte treated with the Golgi probe NBD-ceramide. A paranuclear gigantic Golgi apparatus is seen. Research up to the present indicates that Golgi gigantism may be related to cellular transformation. The NMuLi cells seen here are known to spontaneously transform in culture after a few passages. Such clones are useful to follow for mitochondrial and Golgi alterations in the course of transformation and increasing malignancy and could be of interest to study the apoptotic process during the progression of malignancy.

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FIGURE 65A NMuLi. Fura 2.

FIGURE 65B NMuLi. Fura 2.

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FIGURE 66A Osteosarcoma. Mitochondria stained with MitoTracker Green.

FIGURE 66B Osteosarcoma. Mitochondria stained with MitoTracker Green.

FIGURE 66C Osteosarcoma. Mitochondria stained with MitoTracker Green.

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FIGURE 67 Human colon cancer mitochondria. DASPMI-stained human colon cancer. Image obtained with CCD camera using integrater.

FIGURE 68A Human colon cancer lysososomes (atebrine-stained). Image obtained with CCD camera using integrater.

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FIGURE 68B Human colon cancer lysososomes (atebrine-stained). Image obtained with CCD camera using integrater.

FIGURE 68C Human colon cancer lysososomes (atebrine-stained). Image obtained with CCD camera using integrater.

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FIGURE 68D Comparison with normal fibroblast lysosomes (quinacrine treatment). See also nucleolar channel.

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FIGURE 69 Human melanoma culture cell stained with the mitochondrial probe rhodamine 123. It is generally possible, by looking at the localization of the fluorescence, to discriminate between rhodamine 123 and DASPMI, even though both fluorochromes are in comparable spectral regions in terms of both excitation and emission. In the case of DASPMI, within the nucleus there are nucleolar regions intensely stained with the probe while the nucleus is clear when rhodamine 123 is used. The mitochondria of melanoma are globoid as in many epithelial cells. However, it is possible that a certain mitochondrial swelling occurs with rhodamine 123. According to some views, melanocytes, the cells from which melanoma originates, are considered of epithelial origin, and they are located near the basal layer of the epidermis. Other views hold that melanocytes are close to cells of neural origin.

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FIGURE 70 Fusion-competent myoblast treated with NBD-ceramide. A compact, unipolar, large Golgi apparatus is seen. (See color figures following page 42).

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FIGURE 71 Fusion-competent myoblast treated with the mitochondrial probe DASPMI. The mitochondria are largely fusiform, and they produce a beaded appearance with local regions of slightly elongated globoid thickening. This again raises the question of mitochondrial compartmentation and requires further studies with potentiometric drugs such as TMRM, as done in 1989 by Loew on neuroblastoma. (See color figures following page 42.)

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FIGURE 72 Fusion-deficient myoblasts stained with NBD ceramide. The Golgi apparatus is fragmented and smaller than in the fusion-competent myoblasts. This difference remains unexplained so far, and we do not know how it may affect the myoblast fusion function to construct a functional myocyte. (See color figures following page 42.)

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FIGURE 73A Electron microscopic image of mitochondria in fusion-competent myoblast. The organelles are filamentous, confirming the fluorescence image of similar cells in Figure 71. (See color figures following page 42.)

FIGURE 73B Electron microscopic image of mitochondria in fusion-deficient myoblast, consistent with Figure 72.

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6 Cell-to-Cell Communication Two aspects have been investigated primarily in reconstituted pancreatic islet cells and insulinoma cells: 1. The cell-to-cell spread of fluorescent dyes, i.e., fluorescein, carboxyfluorescein, lucifer yellow. In monolayer cultures microcommunicating territories of two to six cells are observed. The studied cluster is circled with a diamond-tip objective and submitted for electronmicroscopy to identify intercommunicating cells. Thus it is found that insulinsecreting B cells are connected to glucagon-secreting A cells and somatostatin-secreting D cells. A special optical system that includes a gold-plated beam splitter was designed to follow visually the movement of dyes while a recording of fluorescence is made simultaneously. It was then possible to follow the dye as it moved to remote sites within a cluster through long intercellular processes. The modulation of cell-to-cell communication by glucose metabolism, cyclic-AMP analogs, and first-generation antidiabetic drugs such as tolbutamide and glibenclamide was investigated. Intercellular transit times in the order of 0.1 sec were found. Compared to normal pancreatic islet cells, intercellular transit times appear slowed down in insulinoma, to 1 sec or more. Similar differences in transit times are found between glia and glioma cells. 2. Metabolic cooperation. Glycolytic substrate, such as glucose-6-phosphate (G6P), is injected into an islet cell within a cluster. The injected intermediate is not fluorescent by itself; however, its processing along the glycolytic chain leads to NADH formation, and the latter fluoresces in blue. Therefore, microinjection of G6P results in blue fluorescence within the injected cell and, if there is cell-to-cell communication, within intercommunicating cells. Intercellular transit times in the order of 0.1 sec are found.

REFERENCES Kohen, E., Kohen, C., Thorell, B., Mintz, D.H., and Rabinovitch, A., Intercellular communication in pancreatic islet monolayer cultures; a microfluorometric study, Science, 204, 862–865, 1979. Kohen, E., Kohen, C., and Rabinovitch, A., Cell-to-cell communication in rat pancreatic islet monolayer is modulated by agents affecting islet secretory activity, Diabetes, 32, 95–98, 1983. Meda, P., Probing of conexin channels. Evidence for involvement of junctional coupling in pancreatic secretion. In: Analytical Use of Fluorescent Probes in Oncology, NATO ASI Series, Series A; Life Sciences, Vol. 286, Kohen, E. and Hirschberg, J.G., Eds., 1966, pp. 149–156, Plenum Press, NY.

Patterns of communication in reconstituted pancreatic islet cell aggregates. Combined phase and fluorescence images. Only the intercommunicating cells within reconstituted aggregate are fluorescent; the remainder of the cells are not fluorescent.

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FIGURE 74 Combined phase-fluorescence image. Reconstituted cluster of pancreatic islet cells; about four cells communicant.

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FIGURE 75 Reconstituted aggregate. Two clusters of communication each corresponding to a separate microelectrophoretic injection of fluorochrome; two cells intercommunicant, four cells intercommunicant.

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FIGURE 76 Two clusters; two and four cells intercommunicant.

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FIGURE 77 Apparently no communication, only injected cell communicant, with possibly some pericellular diffusion of fluorochrome.

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FIGURE 78 Two to three cells communicant.

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FIGURE 79 Three cells communicant.

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FIGURE 80 Two clusters of communication; two and three cells.

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FIGURE 81 Two clusters of communication; two and three cells.

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FIGURE 82 Three cells communicant.

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FIGURE 83 At least four cells communicant.

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7 The Study of Microecosystems As revealed by studies on intercellular communication, within a cluster of cells there are groups forming integrating units. Interestingly, such units are not only formed between adjacent cells. Dyes such as fluorescein and carboxyfluorescein, when injected into a cell æ occasionally bypassing immediate neighbors æ are seen to move through long intercellular processes into remotely located cells. This is especially noticed within clusters of reconstituted pancreatic islet cells. The functional significance may be in terms of secretory activity synchronization. Scanning through organelles, one can discern how their activities are synchronized or coordinated within the cells, which are part of an integrating unit. The studies of Meda (1995) in cocultures of pancreatic islet cells and neurons open the possibility of pacemaker nerve cells regulating the activity of endocrine cells. While the investigation of microecosystems is quite important in the area of cellular pathophysiology, it exhibits even greater significance and promise in the field of cellular immunology. Marcel Bessis was one of the pioneers to initiate the idea of cancer cell ecology in terms of the interactions between killer cells and malignant cells. The discovery of perforin (see Laskarin et al., 1999) has brought microecology to the molecular level and made possible its exploration by fluorescence imaging of cell organelles. A third area of interest is the study of carcinogenesis in cocultures of hepatocytes, liver endothelial cells, and reticuloendothelial cells with carcinogen metabolites moving from one cell type into another. Thus it has been determined that the carcinogenicity of vinyl chloride is enhanced by preliminary metabolization in endothelial cells and subsequent transfer into hepatocytes. When Bessis originated the idea of microecology, he was thinking of its therapeutic applications in hematology, particularly in the treatment of leukemias. Therefore, tremendous applications and possibilities exist in cellular immunology. A challenging study is on the immunological synapse of cytotoxic C lymphocytes (CTL) with target cells. Activated CD8 cytotoxic T lymphocytes (CTL) play an important role in destroying tumorigenic and virally infected cells. The cell-kill effect is highly efficient and involves only transient interaction between CTL and target cells. The fact that it is a transient interaction makes it possible for CTL to be serial killers, because following interaction with a first target cell they can move onto a second cell, then a third, and so on. Movies of living cells have shown that CTL are capable of destroying targets within times as short as 2 to 10 min and then killing further targets only 6 min later. Upon target cell recognition, CTL rapidly polarize their microtubule organizing center (MTOC), Golgi complex, and lytic granules toward the target cell. These granules, which contain the protein capable of destroying the target cell, fuse with the CTL plasma membrane at the place of contact and release their content toward the target. The study of microecosystems may also prove beneficial for in vitro studies of plant physiology, with possible implications for agriculture and biotechnology. A natural microecosystem is encountered in cultures with lichen cells with coculture of fungal and algal cells. In terms of the hydrogenproducing alga Chlamydomonas reinhardtii, possible genetic engineering of algal effectiveness may be explored by gene transfers in cocultures of the alga with other organisms. Another interesting model is provided by the individual unsynchronized or synchronized activities of yeast cells. It is conceivable that fluorescence imaging of organelle and enzyme activity in multicellular microecosystems opens a whole domain of opportunities in both biomedicine and biotechnology, leading to the microengineering of new therapies and improved energy sources. The methods are starting to become available, and progress depends on the increasing ability to resolve intra- and intercellular functions at the levels of micro- and nanocompartments.

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REFERENCES Laskarin, G., Strbo, N., Sotosek, V., Rukavina, D., Faust, Z., Szekires, Barthog, J., and Podack, E.R., Progesterone directly and indirectly affects perforin expression in cytolytic cells, Am. J. Reprod. Immunol., 42, 312–320, 1999. Meda, P., Junctional coupling of pancreatic b-cells. In: Pacemaker Activity and Intercellular Communication, Huizinga, J.D., Ed., 1995, pp. 275–290, CRC Press, Boca Raton, FL, cited in Meda, P., Analytical Use of Fluorescent Probes in Oncology, Kohen, E. and Hirschberg, J.G., Eds., 1996, pp. 149–156, Plenum Press, NY. Stinchcombe, J.C., Rossi, G., Booth, S., and Griffiths, G.M., The immunological synapse of CTL contains a secretory domain and membrane bridges, Immunity, 15, 751–761, 2001.

FIGURE 84 Morphological events occurring in the cytotoxic T lymphocyte (CTL) upon target cell recognition. Confocal image shows CTL mixed with P815 target cell stained with antibodies against cathepsin (blue), tubulin (green), and talin (red). The scale bar represents 4 mm. An immunological synapse rapidly forms in CTL, with a ring of adhesion proteins surrounding an inner signaling domain. Lytic granule secretion occurs in a separate domain within the adhesion ring, maintaining signaling protein organization during exocytosis. Target plasma markers are transferred to the CTL as the cells separate. Electron microscopy reveals continuities forming membrane bridges between the CTL and target cell membranes, suggesting a possible mechanism for this transfer. CTL are serial killers capable of destroying target cells within times as short as 2 to 10 min and then killing further target cells only 6 min later. Upon target cell recognition, CTL cells rapidly polarize their microtubule organizing center (MTOC), Golgi complex, and lytic granules toward the target cell. These lytic granules, which contain the proteins capable of destroying the target cell, fuse with the CTL plasma membrane at the site of contact and release their contents toward the target. (Stinchcombe, J.C., Rossi, G., Booth, S., and Griffiths, G.M., Sir William Dunn School of Pathology, Oxford, U.K., Figure 2G, in The immunological synapse of CTL contains a secretory domain and membrane bridges, Immunity, 15, 751–761, 2001. Courtesy of Immunity.) (See color figures following page 42.)

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A

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FIGURE 85A AND B Secretory lysosomes insert between the signaling molecule patch and the adhesion ring. CTL-P815 conjugates stained with antibodies against Lck (red), CD11a (green), cathepsin (blue) in frame B. Antibodies against granzyme A (green) and actin (blue) in frame F. B and F are projected confocal sections taken 0.4 mM apart through the sample. The lytic granules insert to the side of the signaling protein marker within the adhesion ring and clearly show that the signaling and secretory molecules are distributed into two distinct domains within the adhesion ring. Different stages of granule secretion and polarization are happening simultaneously with some granules already inserted between the signaling patch and adhesion ring while others are still polarizing. (Stinchcombe, J.C., Rossi, G., Booth, S., and Griffiths, G.M., Figures 4B and 4F, Immunity, 15, 751–761, 2001. Courtesy of Immunity.) (See color figures following page 42.)

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FIGURE 86 Cell-to-cell communication between two kinds of cell in culture, presumably pancreatic islet cell and fibroblast. Such communication may be critical for structural and functional regulation. Seemingly, communication may also be established between endocrine cells and neurons, which may be connected to pacemaker activity.

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8 Biotechnology Experiments aimed at unraveling changes due to drug effects in organelle structures and interactions are modeled in diploid and hexaploid yeast cells. Studies include globoid mitochondrial visualization by dimethylaminostyrylpyridniummethyl iodine (DASPMI), MitoTracker Green, and tetramethylrhodamine-ethylester (TMRE). A major application of work modeled in yeast is the study of metabolism in hydrogen-producing algae, Chlamydomonas reinhardtii, as they react to various treatments, e.g., UV irradiation, perfusion of substrate, metabolic modifiers, and xenobiotics. To reach the organelles and nanocompartments of chlamydomonas, fluorescence imaging spectroscopy using Fourier interferometry, with options for confocal microscopy and scanning near-field optics, (currently in development) would provide a much improved method. Imaging Fourier interferometry on the excitation side holds a greater promise than on the emission side in terms of signal-to-noise ratio. An excitation–emission design is also being contemplated. For these studies, the development of a fluorogenic substrate for the algae’s hydrogenase would be desirable. Theoretically it would seem that better understanding of the algal functioning at the micro- or even nanostructural level will be one way to improve the biotechnological performance of Chlamydomonas rheinhardtii. For fluorescence imaging cell immobilization is required; however, the flagellate organism chlamydomonas is highly mobile. One way of bypassing this difficulty has been to select flagelladeficient, i.e., bald, 2+ and 2- CC478 Chlamydomonas reinhardtii, or membrane-deficient algae such as CW15+ and -CC-277, TR261.OO. In these cells, the highly fluorescent large chloroplast was an obstacle to the observation of other cellular structures. For the purpose at hand, supposedly chlorophyll-free yellow strains were selected: CC-1168 Y1AMT+, a stable yellow in the dark mutant; CC-2359 LTS1-30MT, a carotinoid-deficient small chloroplast membrane structure, very light-sensitive; and CC-2769 Yellow 29-MT, apparently blocked in chlorophyll synthesis. Upon long-term cultivation in darkness, some of the yellow mutant colonies exhibited green revertants. In preliminary observations using the black and white CCD, even the yellow mutants were found to exhibit strong fluorescence associated with chloloroplast-like bodies. Recently it has been possible to immobilize chlamydomonas in polylysine-coated microchambers, which considerably facilitates the fluorescence imaging of these organisms. Imaging studies show that under various conditions of excitation (365, 436, 546 nm), a highly fluorescent crescentor oval-shaped large chloroplast is identified. In MitoTracker Green-treated preparations, occasionally globoid structures suggestive of mitochondria are facing the chloroplast. Using a tricolor CCD, no chloroplast fluorescence could be observed in the yellow mutants; however, chloroplast fluorescence was visualized among the green revertants of green colonies. The discrepancy between no observation of chloroplast fluorescence in the yellow colonies using the tricolor CCD and its observation in black-and-white CCD is easy to explain. The tricolor CCD allows only live imaging; the black-and-white CCD has an integrater which allows frame accumulation and therefore observation of fluorescences undetectable in the tricolor system. Every nanocompartment of the alga, i.e., any space defined by a pixel, is a relatively large volume compared to molecular size. Thus, the spectrum derived from such a volume contains contributions from a number of different molecules, i.e., chlorophylls and other pigments, each with its own spectrum and modified by interactions between them. Similarity mapping is a useful method whereby the sample is composed of a number of spatially separated components, each characterized by a known and unique spectrum, and the task is to detect and map all components (see Rothman, Malik, Bar-Am, and Cabib reference in Chapter 1). The steps of this algorithm are: 1) the known set of spectra of the components are stored in a spectral library, 2) for every pixel a comparison is made between its measured spectrum

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and all spectra in the library, 3) each pixel is identified with the component whose spectrum is most similar to the pixel’s spectrum, and 4) each pixel is displayed in a previously established code identifying the specific component, forming a so-called “classified image.” This image highlights and enhances small differences that are difficult to distinguish in a conventional image. Similarity mapping based on fluorescence spectrum has been used for a nondestructive analysis and comparison of pigmentation in different regions of the red alga Porphyra linearis. Thus, by using Fourier transform multipixel spectroscopy, the subcellular localization of each pigment, i.e., chlorophyll, phycoerythrin, and phycocyanin, was revealed. The same methodology offers promising possibilities to characterize the pigmentation of different regions in the chlamydomonas, and to follow their alterations and interactions during the activity of the hydrogen-producing alga. These preliminary investigations are to be pursued, specifically with various organelle and enzyme probes. It is not completely unrealistic that the studies can be extended to the nanocompartments of the hydrogen-producing organism through scanning at the submicron level. From the standpoint of biotechnology, the more we learn about the metabolic regulation of the hydrogenproducing algae, the more we may enhance our tools for future applications. It is also predictable that the efficiency of the alga will be improved by genetic engineering.

REFERENCES Govindjee, Y.L., Structure of the red fluorescence band in chloroplasts, J. Gen. Phys., 49, 763–780, 1966. Harris, E.H., The Chlamydomonas Source Book, A Comprehensive Guide to Biology and Laboratory Use, Academic Press, San Diego, CA, 1989. Kohen, E., Hirschberg, J.G., Schachtschabel, D.O., Monti, M., and Leblanc, R., An investigation of hydrogenproducing algae by fluorescence imaging, 1st Int. Energy, Exergy and Environment Symp. IEES-1, Hotel Princess, Izmir, Turkey, July 2003. Kohen, E., Hirschberg, J.G., Özkütük, N., Leblanc, R., Veziroglu, T.N., and Monti, M., Fluorescence imaging and photoacoustic imaging of hydrogen-producing algae, ICAST 2003, 3rd Int. Conf. Adv. Strategic Technologies, Knowledge-Based Technologies for Sustainable Development, Penang, Malaysia, Aug. 12–14, 2003. Kohen, E., Hirschberg, J.G., and Leblanc, R., Novel imaging methods for the study of organelle structure and function in hydrogen-producing algae, European Hydrogen Energy Conference, Sept. 2–5, 2003, Grenoble France, EHA, European Energy Association. Kohen, E., Hirschberg, J.G., and Özkütük, N., Hypothesis V. Hydrogen Power Theoretical and Engineering Solutions Int. Symp., Sept. 7–10, 2003, Porto Conte, Sardinia, Italy. Veziroglu, T.N., Hydrogen-production by biological processes: a survey of the literature, Int. J. Hydrogen Energy, 26, 13–28, 2001.

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FIGURE 87 Yeast, Saccharomyces cerevidae stained with DASPMI. The mitochondria are globoid and peripherally located.

FIGURE 88 Yeast stained with MitoTracker Green, Molecular Probes, Eugene, OR. Globoid mitochondria are seen, but compared to Figure 87 they are considerably more rare and less bright. Fluorescences obtained with MitoTracker Green can equal the intensity obtained with DASPMI. The lower fluorescence intensity in this particular instance may be due to the fact that saccharomyces may have been obtained from another source or that the preparation may have aged.

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FIGURE 89A, B, C, D, E, AND F Hydrogen-producing Chlamydomonas reinhardtii. The large fluorescent bodies are chloroplasts. The natural red fluorescence of chlorophyll in this organelle largely masks the fluorescence of other pigments or organelles. An attempt was made to visualize the mitochondria with DASPMI. The smaller fluorescent bodies opposite to the chloroplast may be mitochondria.

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FIGURE 89C

FIGURE 89D

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FIGURE 89E

FIGURE 89F

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9 Instrumentation Fluorescence spectroscopy in the microscope is usually far superior to ordinary absorption spectroscopy because, if practiced under the best conditions, there is a virtual absence of parasitic light. In absorption spectroscopy of single living cells, their thinness, usually between 0.5 and 5 mm, causes the absorption to be very small, often less than 1%. This means that observations are made in the presence of background light many times more intense than the absorption signal containing the information. If, for example 1% of the light is absorbed, which is often the case for the nearly transparent living cell, the observation must be made in the presence of background light 100 times as strong as the signal itself. Living cells are often very sensitive to radiation, in which case it is of paramount importance to keep the intensity of the exciting light to a minimum. This has led to the greatest care being exercised to gather as much information as possible with the least amount of excitation irradiation. In addition, many of the phenomena exhibited in living cells change rapidly in time, often in milliseconds, so that gathering the data from the whole spectrum at the same time is desirable. It is possible to obtain quasi-simultaneous fluorescence spectra by using a linear optical multichannel analyzer (OMA) with as many as 525 channels. Cooling the detector to solid carbon dioxide temperature tends to reduce parasitic noise and so improve signal-to-noise (S/N). In choosing a dispersing element, the modern blazed diffraction grating has usually been found superior to the prism because of its superior dispersion, which allows a broader entrance aperture for a given spectral resolution. By a similar argument, Fourier and Fabry–Perot interferometers have been shown to have an analogous advantage over diffraction grating. However, they have not yet been widely applied to the measurement of fluorescence spectra in living cells, and such designs (see Hirschberg references in Chapter 10) are currently in the development stage. The remarkable expansion of fluorescent probes and the rise of one-, two-, and three-dimensional microspectrofluorometry of cell metabolism, organelle interactions, and microarchitecture, have revived the morphological approach aimed at unraveling structure and function relationships in intact living cells. The highly sensitive techniques recently introduced in microscopy range from multiple-parameter spectral imaging to fluorescence photobleaching recovery, fluorescence resonance energy transfer, fluorescence polarization microscopy, polarized fluorescence photobleaching recovery, pixel-by-pixel deconvolution in living cells of multiexponential fluorescence lifetimes, and combined fluorescence and x-ray microscopy. The ultimate rewards include an enhanced perspective of in situ intracellular processes and a revolution in our understanding of cellular biochemistry, pathology, and pharmacology. There is also the realistic hope, crucial from the standpoint of biotechnology, of comprehending the control mechanisms of biochemical processes within living cells at both qualitative and quantitative levels.

REFERENCES Casperson, T., Die Eiwweissverteilung in den Strukturen des Zellkernen, Chromosoma, 1, 562–619, 1940. Casperson, T. and Lomakka, G., Recent progress in quantitative cytochemistry: instrumentation and results. In: Introduction to Quantitative Cytochemistry II, Wied, G.L. and Bahr, G.F., Eds., 1970, Academic Press, NY. Hirschberg, J.G., Kohen, E., and Kohen, C., Microspectrofluorometry, in Advances in Optical and Electron Microscopy, Vol. 14, pp. 121–211, Academic Press, San Diego, 1994.

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Jackson, H. and Moore, H., Encyclopaedia Britannica, Vol. 15, 14th ed., p. 446, Encyclopedia Britannica, NY, 1932. Kohen, E., Microfluorometric studies of the metabolism in the single living cell, Akademiska Avhandling, doctoral thesis, Dept. of Pathology, Karolinska Institute, Stockholm, Sweden, Sept. 5, 1973. Kohen, E. and Hirschberg, J.G., Cell Structure and Function by Microspectrofluorometry, Academic Press, San Diego, 1990. Kohen, E., Kohen, C., Hirschberg, J.G., Santus, R., Morlière, P., Kasten, F.H., and Ghadially, F.N., Optical Methods in Cell Biology. In Encyclopedia of Human Biology, Vol. 5, pp. 561–585, Academic Press, NY, 1991. Kohen, E. and Hirschberg, J.G., Applications of Optical Engineering to the Study of Cellular Pathology, Vols. 1 and 2, Research Signpost, Trivandrum, Kerala, India. Stevens, J.K., Mills, L.R., and Trogadis, J.E., Three-Dimensional Confocal Microscopy, Academic Press, San Diego. Thorell, B., Studies on the formation of cellular substances during blood cell formation, Diss. med Stockholm Karrolinska Institutet, Oct. 21, 1947, doctoral thesis, Henry Kimpton, London.

FIGURE 90 Schematics of microspectrofluorometer.

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FIGURE 91 Schematics of microspectrofluorometer.

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Spherical Mirror Tungsten Lamp Rg2 Schott Filter Fast Aspheric Lens Beam Splitter

Annular Aperture

Very Fast combination of Fresnel Lenses

Object Short Pass Filters

High Power Phase Objetive Phase Ring

Long Pass Filter

Shutter

Ploemopal Iluminator

Fused Silica Lens Spherical Mirror Mercury Arc

Relay Lens Eyepiece

Beam Splitter One or Two Dimensional Scan

Adjustable Aperture Filter Mounted in a Sector

SIT

Reley Lens Small Incandescent Lamp

Beam Splitter Red Filter

Zoom Lens

Diffraction Grating Plane Mirror

FIGURE 92 Schematics of microspectrofluorometer.

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FIGURE 93A Photograph of microspectrofluorometer setup with helium–cadmium laser for excitation in the long UV.

FIGURE 93B Micromanipulating setup around the cell incubation chamber for microspectrofluorometry. Substrates and metabolites are introduced into the cells grown in the chamber by microelectrophoretic injection. The micromanipulators are pneumatic-type Cailloux. The original Cailloux manipulators were hydraulic; later they were converted to pneumatic.

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FIGURE 93C Photograph of microspectrofluorometry setup with high-pressure mercury arc replacing the helium–cadmium laser as a source of excitation. With the appropriate excitation filters and the classical Ploem blocks equipped with excitation, dichroic, and emission filters, fluorescence excitation can be carried out from 360 to 800 nm (long ultraviolet to red). The dichroic filters are for separation of excitation and emission wavelengths. The excitation–emission arrangement used in microspectrofluorometry is also used for fluorescence imaging with a CCD camera.

FIGURE 93D A view of the microscope stage with the microelectrophoresis–microinjection setup.

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FIGURE 93E Microinjection setup.

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FIGURE 93F Microinjection setup.

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Methods and 10 Novel Instrumental Designs TWO-PHOTON EXCITATION MICROSCOPY The two-photon excitation method (TPEM) has a potential advantage over conventional wide-field digital deconvolution (DDM) or laser scanning confocal microscopy (LSCM) because of its intrinsic three-dimensional resolution and the absence of background fluorescence. In two-photon excitation imaging, the photobleaching and autofluorescence are considerably reduced, since the infrared pulse laser light illumination occurs only on the focal plane. In DDM or LSCM, one-photon UV or visible light illuminates the whole field of view and considerable photobleaching occurs above and below the focal plane. Two-photon excitation imaging with infrared excitation is a superior imaging system for UV-absorbing fluorophores.

REFERENCE Lakowicz, J., Recent developments in fluorescence spectroscopy, fluorescence lifetime imaging, microscopy, long lifetime metal ligand probes, multiphoton excitation, and light quenching. In: Applications of Optical Engineering to the Study of Cellular Pathology, Vol. 1, Kohen, E. and Hirschberg, J., Eds., Research Signpost, Trivandrum, India, pp. 33–46. Masters, B., Three-dimensional optical imaging of tissue with two-photon excitation laser scanning microscopy. In: Applications of Optical Engineering to the Study of Cellular Pathology, Vol. 1, Kohen, E. and Hirschberg, J., Eds., Research Signpost, Trivandrum, India, pp. 173–182. Periamasi, A., Sköglund, P., Noakes, C., and Keller, R., An evaluation of two-photon excitation versus confocal and digital deconvolution microscopy imaging in Xenopus morphogenesis, Microscopy Research and Technique, 47, 172–181, 1999. Samarinski, H., Gryczyaski, L., and Lakowicz, J., Calcium-dependent fluorescence lifetimes of Indo-1 for one- and two-photon excitation of fluorescence, Phytochem. Photobiol., 58, 341–345, 1993.

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FIGURE 94 Schematic illustration of laser scanning confocal microscopy (LSCM) and digital deconvolution microscopy (DDM). Emission filter wheel (EMFW); filter wheel controller unit (FW-CU); excitation filter wheel (EXFW); CCD camera control unit (CCD-CU); confocal control unit (C-CU). (See Periasamy, Sköglund.)

FIGURE 95 Illustration of light illumination and detector configuration, used in one- and two-photon imaging systems. Wide-field digital deconvolution microscopy (DDM); laser scanning confocal microscopy (LSCM); two-photon excitation microscopy (TPEM); dichroic mirror (DM). (See Periasamy, Sköglund.)

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FIGURE 96 Demonstration of excitation power vs. signal-to-noise of two-photon images in xenopus gastrula and neurula tissues. Two-photon images were acquired at 870 nm and about 22 nm deep. The signal-to-noise ratio was better than 22 mW compared to powers of excitation. The acquired two-photon images were deconvoluted using DeltaVision software. The deconvolution option helps to improve TPEM images by removing the background noise in deep tissue imaging. (Periasamy, A., Sköglund, P., Noakes, C., and Keller, R., V.M. Keck Center for Cellular Imaging, Gilmer Hall, University of Virginia, Charlottesville, Dept. of Biology, Figures 4 and 5, Microscopy Research and Technique 47, 172–181, 1999. Courtesy of Microscopy Research and Technique.)

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FIGURE 97A AND B Advantage of two-photon fluorescence microscopy over one-photon fluorescence microscopy of thick specimen. Chondrocytes in rat cartilage head cap were stained with Oregon Greenphalloidine F-actin probe and with the Hoechst nucleus probe. (A) Fluorescence micrograph of chondrocytes by one-photon absorption of the 488 nm light of an argon laser by the actin probe penetration depth: 64 mm. (B) Fluorescence micrograph of chondrocytes by two-photon absorption of the 780 nm light of a femtosecond MIRA 900 laser by the Hoechst probe. Penetration depth: 219 mm. This figure illustrates the benefits of multiphoton excitation fluorescence microscopy: deeper spatial resolution, improved signal-to-noise ratio, clear examination of thick sample, Z-axis scanning, negligible phototoxicity, reduced photobleaching of the probes, and reduced blurring of the image by light scattering. (Courtesy of Dumas, D., Grossin, L., Cauchoix, G., Gentils, M., Santus, R., and Stoltz, J.F., Comparison of wide-field deconvolution and confocal microscopy in bioengineering. Interest of multiphoton microscopy in the study of articular cartilage, Biorheology, 40, 253–259, 2003.)

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FOURIER INTERFEROMETRY FOR EXCITATION–EMISSION FLUORESCENCE SPECTRAL IMAGING This apparatus, in development, aims at the achievement of imaging Fourier interferometry both on the excitation and emission sides, as shown in Figure 98 through Figure 100 (see Hirschberg references). Compared to the black-and-white or color fluorescence imaging microscope–TV systems described above, this apparatus holds greater potential for the structural and functional analysis of chlamydomonas. Such Fourier interferometry for fluorescence imaging can be achieved by a two-beam interferometer of the Michelson type, the Sagnac type, or the recently constructed Pentaferometer type (Figure 99). The instrument may be operated with a Pentaferometer on the excitation side only or the emission side only, as well with two Pentaferometers, one on the excitation side and the other on the emission side (Figure 100). So far, Gemperlein in Munich has used Fourier interferometry on the excitation side for the analysis of visual pigments in the butterfly. The Applied Spectral Imaging Group (Carlsbad, CA, and Migdal Ha’Emek, Israel) and Malik in Israel have used Fourier interferometry on the emission side for cytopathological studies in mammalian cells (see also Hirschberg et al., 1998). Imaging Fourier interferometry on the excitation side often holds greater advantages than on the emission side. The shape of the excitation spectrum is dependent on more highly excited energy levels of the fluorescent molecules than is that of the emission spectrum, and is therefore more sensitive to the environment of the fluorochrome. Moreover, when damage or alteration of the living cell under study limits the intensity of the excitation radiation that can be utilized, the position of the spectral selection element is crucial. The light throughput of this element is far less than 100%. Therefore, the placing of the spectral selection element before the object, such as required for the excitation spectrum, is advantageous. For the emission spectrum, the spectral selection element has to be placed after the object, a situation in which loss in light intensity is hardly affordable. It is easy to conclude that for the same fluorescence yield, significantly less illumination will fall on the cell in the case of excitation spectrum than in the case of emission spectrum. Consequently, the living cell is better protected from irradiation damage when the excitation rather than the emission spectrum is recorded. As described in the pioneering work of Weber, in the case where it is necessary to distinguish between the fluorescence of several fluorophores in a mixture, knowledge of the excitation as well as emission spectrum may be much more informative than when the emission spectrum only is measured (15). Also, complementation of fluorescence imaging by photoacoustic imaging will yield information about the component of the excitation energy that goes into internal conversion rather than fluorescence (Figure 101, Weber, 1961). Three organelles are selected for fluorescence imaging of normal and pathological cells. The mitochondrial probes are dimethylaminostyrylpyridniummethyl iodine (DASPMI), tetramethylrhodamine-methylester (TMRM), tetramethylrhodamine-ethylester (TMRE), and MitoTracker Green (Molecular Probes, Eugene, OR). Recently a probe for evaluation of mitochondrial oxygen has been introduced, i.e., (1¢¢-pyrenebutyl)-2-rhodamine ester (PRE4), cf. Ribou, Vigo, and Salmon, University of Perpignan, France, 2002. The probe enables dual fluorescence excitation, i.e., UV for the oxygensensitive pyrene butyl and blue for the mitochondrial membrane potential-sensitive rhodamine. With these probes mitochondrial morphology, globoid vs. filamentous, is identified, as well as drug-induced organelle swelling and fragmentation. Lysosomal probes are lysosomotropic compounds, e.g., atebrine, or fluorogenic substrates of lysosomal hydrolases. Mitochondrial damage is accompanied by retrieval of mitochondrial probes within lysosomes as a result of autophagocytosis. Lysosome probe overloading leads to formation of phagolysosomes. With the Golgi probe NBD-ceramide, organelle gigantism is observed in malignant hepatocytes and organelle dispersion in genetically deficient myoblasts. To such multiparameter fluorescence imaging, nuclear pattern recognition may be added eventually with fluorescence in situ hybridization (FISH) techniques. While one- or two-wavelength excitation has been used up to this time, designs are in progress for fluorescence excitation–emission imaging by Fourier interferometry. It is also planned to recuperate the part of the excitation energy

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that does not go to fluorescence by a complementary design of photoacoustic microscopy. Studies initiated on normal and malignant hepatocytes, keratinocytes, osteosarcomas, and mastocytomas are now extended to HCT116 human colon cancer cells and MCF-7 human breast adenocarcinoma cells. The usefulness of the method is being investigated in 9-nitrocamptothecin-grown cells.

REFERENCES Gemperlein, R., What can you do with a complex common stimulus. In: Applications of Optical Engineering to the Study of Cellular Pathology, Vol. 2, Kohen, E. and Hirschberg, G., 1999, pp. 99–119. Hirschberg, J.G. and Kohen, E., Instrumentation design for study of metabolic control in living cells, in Analytical Use of Fluorescent Probes in Oncology, NATO ASI Series, Series A; Life Sciences, Vol. 286, pp. 293–298, Plenum Press, NY, 1996. Hirschberg, J.G., Vereb, G., Meyer, C.K., Kirsch, A.K., Kohen, E.K., and Jovin, T.M., Interferometric measurement of fluorescence excitation spectra, Applied Optics, 37, 1953–1957, 1998. Hirschberg, J.G. and Kohen, E., Pentaferometer: a solid Sagnac interferometer, Applied Optics, 38, 136–138, 1999. Hirschberg, J.G., Kohen, E., Kohen, C., Örnek, C., and Berry, J.P., Fourier interferometery with spectral fluorescence imaging for the study of mitochondrial distribution in living normal/malignant cells, Hybrid and Novel Imaging and New Optical Instrumentation for Biomedical Applications. Progress in Biomedical Optics and Imaging, Vol. 2, No. 34. European Conferences on Biomedical Optics. Proc. SPIE, Vol. 4434 Boccara, A.-C. and Oraevsky, A.A., Eds., June 20–21, 2001, 18, Munich, Germany. Hirschberg, J.G., Kohen, E., Örnek, C., Monti, M., and Berry, J.P., Towards fluorescence excitation imaging by a Fourier interferometer probing of molecular order or chaos, Proc. Abstr. 7th World Congress on Advances in Oncology, and 5th Int. Symp. on Molecular Medicine, Oct. 10–12, 2002, Hersenissos, Crete, Greece, Int. J. Molecular Medicine, 10, Suppl. 1, 2000, Abstr. 139. Kohen, E., Hirschberg, J.G., Kohen, C., Schachtschabel, D.O., Stanikunaite, R., and Monti, M., Fourier interferometry as applied in microspectrofluorometry and fluorescence imaging of living cells, Fiber and Integrated Optics, 20, 411–425, 2001. Kohen, E., Hirschberg, J.G., Örnek, C., and Berry, J.P., Fourier interferometry/spectral imaging of response to metabolic challenge in perinuclear mitochondria of malignant cells, 23rd Ann. Int. Conf. IEEE Engineering in Medicine and Biology (EMBC 2001), Oct. 25–28, 2001, Istanbul, Turkey. Rothman, C., Malik, Z., Bar-Am, I., and Cabib, O., In: Applications of Optical Engineering to the Study of Cellular Pathology, Vol. 2, Kohen, E. and Hirschberg, G., 1999, pp. 121–132. Weber, G., Enumeration of components in complex systems by fluorescence spectroscopy, Nature, 190, 27–29, 1961.

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FIGURE 98 Field-widened Michelson interferometer. The advantages are that it is much more luminous than a classical Michelson and that it has no moving parts. From the entrance port the light passes through chamber 1, which is filled with a gas with a low index of refraction, such as helium. Chamber 2 is filled with a gas of high refractive index, i.e., freon. The beam splitter produces two optical paths that are geometrically identical, one passing through chamber 1 and the other through chamber 2. The entrance port is imaged at the exit port by concave mirrors. Thus, the whole breadths of the entrance and exit apertures interfere together, in position as well as in angle. This provides both field and aperture widening. The optical path difference between the beams is changed by varying the pressure of the gas in the two chambers, keeping the pressure equal in each chamber to eliminate mechanical stress. Since the refractive indices of the two gases are different, the interfering optical paths vary, producing the Fourier transform of the spectrum.

FIGURE 99 The pentaferometer. A standard “penta” prism was cut down the center as shown, and the long cut faces were polished. A beam splitter was produced by evaporation on one of the long faces in one of the prism halves. The opposite sides were silvered to form mirrors. The two halves were finally cemented together. In use, the entering light is separated into two beams by the beam splitter; they exit together and focus on a slit, forming the interference fringes. The interferometer is rotated to sweep the fringes past the slit, producing the Fourier transform of the incident light.

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FIGURE 100 Two interferometers are coupled to a tissue-culture microscope, a fluorescence excitation source, and a computer. The excitation is modulated according to wavelengths by the first interferometer and sent to the microscope, where it excites the sample. The resulting fluorescence is in turn modulated by the second interferometer. The modulation frequency is markedly different in the two interferometers, to distinguish the excitation and emission spectra. The signal is then sent to a computer, where the two spectra are recovered, using a Fast Fourier Transform (FFT) program.

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FIGURE 101 Combined photoacoustic and phase microscope. When a molecule is excited, it can return to the ground state in two ways, either by emitting a photon in fluorescence or without a photon generating heat. In the latter case, if the excitation is periodic, sound may be produced and detected. A tissue culture inverted microscope with an object immersed in liquid is shown, designed to measure the photoacoustic effect, along with phase-contrast viewing. The excitation is passed into the LCA, which modulates the light with different frequencies, depending on position. The array is imaged in the object plane of the microscope. A small microphone is placed in the liquid surrounding the object to detect the sound. The resulting signal is amplified and sent to a computer, which sends the sound from each pixel in the object to its proper position in a display, so that an acoustic image is produced. For phase contrast the visible light source is used, and the image is captured by the CCD. A similar setup can be used with the fluorescence excitation source to produce a fluorescence image, and thus to obtain both the fluorescence and photoacoustic images.

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EXCITATION–EMISSION FLUORESCENCE IMAGING COMBINED WITH PHOTOACOUSTIC MICROSCOPY. A COMBINED FLUORESCENCE IMAGING AND PHOTOACOUSTIC MICROSCOPY DESIGN FOR STUDIES IN CANCER CELLS The fluorescence excitation energy is essentially retrieved in fluorescence emission, internal conversion, and intersystem crossing. Due to the lower probability of intersystem crossing, except for photosensitizers, most of the energy is recuperated in fluorescence and internal conversion. In terms of revealing cell structure and function, the former corresponds to fluorescence imaging and the latter to photoacoustic imaging. Thus a design combining both will be a powerful tool in the recognition of physiopathological processes within the organelles of the living cell. For combined fluorescence and photoacoustic imaging, a liquid crystal array (LCA) is placed in the excitation train conjugate to the object. The LCA is modulated so that each pixel in turn is transparent for a very short period, about 10 microsec, remaining opaque the rest of the time. Since the LCA is imaged on the object, the result is that each pixel is illuminated in turn and fluorescence is viewed through the same LCA. The nanosecond fluorescence process is much faster than the time each pixel of the LCA remains open. The fluorescence image is obtained on a CCD camera. For acoustic microscopy (AM) a miniature microphone is placed in the liquid containing the object to be studied, i.e., the living cell. The pulses of light give rise to acoustic pulses that are transmitted to the computer, which displays the magnitude of the pulses on an oscilloscope screen, positioning them in correspondence to the successively transparent regions in the LCA. Thus an image of the acoustic response to the excitation is presented directly without any need for scanning. An important advantage is that fluorescence microscopy and acoustic microscopy can be incorporated in the same instrument and can operate simultaneously. The method can be applied to the organelles of living cancer cells, specifically on the organelles currently used for multiparameter characterization of these cells. Broad applications are contemplated in terms of organelle alterations in the course of cell transformation, as well as cell diagnostics and therapeutic trials. It is hoped that it will be possible to carry such fluorescence studies at the level of nanocompartments in organelles of living cells (Figure 102).

REFERENCES Kohen, E., Hirschberg, J.G., Berry, J., Özkütük, N., Örnek, C., Monti, M., Leblanc, R., Schachtschabel, D.O., and Haroon, S., Towards Fourier interferometry fluorescence excitation/emission imaging of malignant cells combined with photoacoustic imaging, Hybrid Imaging Techniques and Other Novel Imaging Methods, Eur. Conf. Biomedical Optics 2003, June 20–23, Munich, Germany. Kohen, E., Hirschberg, J.G., and Leblanc, R., Fluorescence imaging and photoacoustic microscopy design for studies in cancer cells, Proc. Abstr. 8th World Congress on Advances in Oncology and 6th Int. Symp. Molecular Medicine, Oct. 2003, Hersenissos, Crete, Greece, Int. J. Mol. Med., Athens, Greece.

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169

B

C

FIGURE 102 A model of a multiparameter (multidimensional) system for tumor cell diagnostics, prognostics, and drug trials. Three organelle fluorescence parameters are used: (A) mitochondria, (B) Golgi, and (C) lysosomes. It is highly desirable to add a nuclear DNA parameter, specifically based on fluorescence in situ hybridization (FISH). In the case of osteosarcoma with mitochondrial DNA-deficient mutations, imaging of the mitochondrial DNA would be a welcome development. (See color figures following page 42.)

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COMBINED CONFOCAL FLUORESCENCE AND ACOUSTIC MICROSCOPY (CCOFAM) WITHOUT NECESSITY FOR SCANNING In ordinary confocal fluorescence microscopy (CFM), the full image is obtained by mechanically scanning over the object. This is accomplished either by two-dimensionally translating the stage carrying the object or by using movable mirrors to effect the same result. Such a process is inherently slow and thus suffers from the objection that if living cells are being studied, changes in the object may occur during the scanning period. Here we take advantage of the great rapidity of the fluorescence process, about 10-9 sec or 0.001 msec. Instead of using either a pinhole aperture or a slit as in the classical method, the whole object is illuminated and no scanning is necessary. A two-dimensional LCA is placed in the excitation optical train conjugate to the object. The LCA is modulated so that each pixel in turn is transparent for a short time, remaining opaque the rest of the time. The process is similar to that in a flat television or computer monitor. The exciting light is passed through the LCA on the way to the object. The result is that each pixel of the object is illuminated in turn with the short pulses of the exciting light. The resulting fluorescence is viewed through a second LCA, modulated in the same way as the excitation unit. The response time of the liquid crystals and the resolution required will determine the frequency of the images which can be obtained. For example, if the liquid crystal can be cycled at 2 mHz, the transparent periods can be of the order of 1/2 msec duration, each separated by 1/2 msec. Then, for a field of 1000 ¥ 1000 or 106 total pixels, a frequency of one image per second can be obtained. Thus a gain of at least an order of magnitude can be obtained over classical CFM. It is also probable that liquid crystals can be modulated as much as 20 times faster. If this is true, flickerless real-time CFM can, for the first time, be a reality. Since each pixel is being illuminated separately, and the fluorescence from the same pixel location is also being observed separately, the result is the same as that which is obtained with classical CFM. Planes in thick specimens can be studied without interference from above and below. The advantage is that the images are obtained without scanning; the whole two-dimensional region in the object can be seen practically at once. In ordinary AM, as in CFM, the image must be obtained by a scanning process over the object. Since this is a long and tedious process, we describe here a method to obtain the whole acoustic image simultaneously. We also take advantage of the rapidity of the acoustic process which, like fluorescence, is of the order of 10-9 sec. The LCA is used in the same way for excitation as in the confocal fluorescence described above. For AM, a small microphone is placed in the liquid containing the object to be studied, i.e., a living cell. The pulses of exciting light give rise to acoustic pulses, which are transmitted by the microphone to the computer, which displays the magnitude of the pulses on an oscilloscope screen, positioning them in correspondence to the transparent region in the LCA. Thus an image of the acoustic response to the excitation, AM, is presented directly without any need for scanning. An important advantage here is that AM and CFM can be incorporated in the same instrument, CCOFAM, and can operate simultaneously.

REFERENCES Kohen, E., Hirschberg, J.G., Berry, J., Özkütük, N., Örnek, C., Monti, M., Leblanc, R.M., Schachschabel, D.O., and Haroon, S., Towards interferometry fluorescence excitation/emission imaging of malignant cells combined with photoacoustic microscopy, Hybrid Imaging Techniques and Other Novel Imaging Methods, Eur. Conf. Biomedical Optics 2003, June 22–25, Munich, Germany. Puppels, G.J., de Mul, F.F.M., Otto, C., Greve, J., Robert-Nicoud, M., and Arndt-Jovin, D.J., Studying living cells and chromosomes by confocal Raman spectroscopy, Nature, 347, 301–303, 1990.

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Lemasters, J.J., Laser scanning confocal microscopy of cell function, in Analytical Use of Fluorescent Probes in Oncology, Kohen, E. and Hirschberg, J.G., Eds.,1996. Tam, A.C., Applications of photoacoustic scanning techniques, Reviews of Modern Physics, 58, 381–431, 1986.

IS THE STUDY OF NANOCOMPARTMENTS IN LIVING CELLS FEASIBLE? The ultimate milestone in low-level monitoring is single molecule detection within intracellular nanocompartments. So far this has remained at the level of demonstration, not application. Potential applications include sequencing DNA, probing nanometer scale environments, monitoring individual molecule reactivities, studying the variability of molecular conformations, detecting disease at an early stage, and devising nanostructures and molecular scale imaging probes for single molecule microscopy. The most stable carbocyanine dye C18 (Dil) emits about 200 million photons. Recent advances in optical detection, such as the avalanche diode and ICCD, have enabled us to detect single photons. With 200 photons/sec for adequate S/N ratio, a single Dil molecule will have a lifetime of approximately 278 h before photobleaching. This single molecule light source has a low rate of photobleaching and a high quantum yield, valuable indicators of the feasibility of a single molecule light source.

REFERENCE Tan, W., Lou, J.H., and Kopelman, R., Intracellular sensing and optical imaging beyond the diffraction limit. In: Applications of Optical Imaging to the Study of Cellular Pathology, Vol. 2, Kohen, E. and Hirschberg, J.G., Eds., 1999, Research Signpost, Trivandrum, Kerala, India.

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11 Conclusion This atlas illustrates both the power and the limitations of the present microscopic techniques of fluorescence for the study of structure and function in normal and pathological cells. The factors illustrating the strengths of the techniques are: 1. The availability of specific fluorescence probes for the structures of cell organelles which are capable of converting in fluorescence emission most of the energy accumulated by a single molecule of the probe which has absorbed one or more photons at the appropriate wavelength. The specificity, the luminescence of the probe, and the high sensitivity of the currently available detection systems, including the intensified charged couple device (ICCD), allow numerous biomedical and biotechnological applications. A further advantage is the superiority of these nondestructive methods, which permit high resolution cellular pharmacology studies at the subcellular level on populations of intact living cells without the pollution and global destruction associated with radioactivity techniques. 2. Opposite to all other techniques of cellular biochemistry, microfluorometry, combining topographic resolution with spectral analysis, makes it possible to establish in real time the precise mapping and kinetics of the structural modifications and molecular interactions in the intact living cell. This is performed simply by following the fate and properties of specifically appropriate, in some instances even tailored, fluorescent probes. This feature is particularly significant for the study of cell metabolism and catabolism, i.e., respiration cellulaire, intercellular signaling, intra- or intercellular communication, and the current genetic perturbations, such as gene transfer and “knockout” techniques. 3. The development of femtosecond lasers highly powerful in the infrared region with the emergence of microfluorometers for two- and three-photon absorption allows expansion of classical single-photon studies either in steady-state or fluorescence lifetime studies. The advantage of multiphoton fluorescence studies are higher resolution, amelioration of signal-to-noise ratio, facilitated examination of thick samples, reduced photobleaching of the probe, and reduced blurring of the image by the diffusion of light. Currently, one weakness of the techniques is the obvious link of the microfluorometer's performance to the limitations of the detection systems, i.e., CCD camera sensitivity or image processing system. It is in fact the level of perfection attainable in the detection system which on one hand allows improvement of the quality and the resolution of the topographic image, and on the other hand pushing back of the actual limits of the notion of real-time measurements by reducing the image accumulation time required to seize the intra- or intercellular processes with a satisfactory signal-to-noise ratio. Much is to be expected from image acquisition and treatment technologies being developed for space studies and astronomy. All progress attained on image acquisition in astronomy rapidly reaches the domain of image acquisition in microscopy. Thus can one hope that the contemplation of this atlas will convince the researcher in biology, neophyte or well established, as well as the experts in opto-electronics, that the applications of microfluorometry and fluorescence imaging in biomedical and biotechnological sciences will affect a broad domain in research and development to unravel the secrets of life through the microecologic and microethologic aspects.

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Index A A-cells, pancreatic islet, 28, 29, 129 Acoustic techniques combined with fluorescence imaging combined confocal fluorescence and acoustic microscopy (CCOFAM), 170–171 fluorescence spectral imaging with photoacoustic microscopy, 168–169 Adeno(Cre), 30 Adhesion ring, lymphocyte, 143 Adriamycin, 8, 40, 86, 96, 97, 98 Algae, 145, 146 a-cells, pancreatic islet, 28, 29, 129 Amsacrine, 18, 20 Anthralin, 52, 67 Apolipoprotein E, 13, 99, 100, 102, 103 Apoptosis, 117 Atabrine, see Quinacrine ATP hydrolysis, GSH-stimulated, 32 Autofluorescence cell-to-cell communication, 129 CV1 cells, 31 imaging techniques and, 159 mitochondria, 7 NAD(P)H, 28, 29, 30 pancreatic islet cells, 28, 29, 30 Autophagocytosis, 1, 13; see also Phagolysosomes autofluorescence, 31 azaleic acid-treated cells, 60, 61 hematoporphyrin-treated cells, 74 Avalanche diode, 171 Azaleic acid, 52 autophagocytosis, 60. 61 fibroblasts, cystic fibrosis, 57, 58, 59, 60 fibroblasts treated with, 54, 55, 63, 64, 65, 66 melanoma cell, 62 NADH transients, 27

B B-cells, pancreas, 28, 29, 30, 129 Benzo-(a)-pyrene, 8 emission spectra, 86 emission spectra and time evolution, 89 fibroblasts treated with, 90, 91, 92, 93, 94 human skin fibroblast, 88 b-cells, pancreatic islet, 28, 29, 30, 129 b-glucosidase, UG9 and, 17 BIODIPY-LacCer, 107, 108 GM1 fibroblasts, 111 SLSD phenotype induction in normal fibroblasts, 110 Biotechnology, 145–150

BRL (Buffalo rat liver), 86

C C18(DIL), 53 Cailloux manipulators, 155 Calcein, 5 Calcium probe, Fura-2, 19, 118 Cancer cell ecology, 141 Cancer chemotherapeutic drugs, 8, 27, 86–98 Carbocyanine dye, 53, 171 Carboxyfluorescein cell-to-cell communication, 129 microecosystems, 141 Carcinogenesis/transformation, 113–127 Carcinogens, 8, 86–98 cell lines for studying, 86 microecosystems/cell ecology, 141 Caveolin-1-GFP, 107 CCD cameras, xv, 25, 173 combined excitation/emission–photoacoustic system, 168 combined phase–photoacoustic system, 167 Hoechst dye-stained nuclei, 21 with integrater, 120, 121 microspectrofluorimetry, 156 one- and two-photon imaging systems, 160 tricolor, 145 CCL 228 cells, 86 CCOFAM (combined confocal fluorescence and acoustic microscopy), 170–171 CD8 lymphocytes, 141, 142, 143 Cell differentiation and cell pathology, 113–127 Cell membrane, hematoporphyrin fluorescence in, 73 Cell-to-cell communication, 129–139 cytotoxic T lymphocytes–P815 conjugates, 142, 143 pancreatic islet cells and fibroblasts, 144 Chlamydomonas reinhardtii, 141, 145, 146, 148, 149, 150 Chlorophyll, 145, 146, 148, 149, 150 Chloroplasts, 147 Cholesterol fibroblast, SLSD phenotype induction, 110 lipid storage disease cells, 109 Cholesterol pathway, 13, 99, 100 Chromatin, 32 Chromosomes, 18 Classified image, 146 Cloudman melanoma cells, 84, 85, 96 Cocultures T lymphocytes and tumor cells, 141, 143, 144 pancreatic islet cells, 141, 144 Coenzyme reduction

175

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176 NADH, see NADH metabolic probes, 27 Colocalization of Rh-CtxB and Dil-LDL with caveolin-1-GFP, 107 Colon cancer cells, human, 164 cell differentiation and cell pathology, 113 mitochondria and lysosomes, 120 Combined fluorescence and acoustic techniques combined confocal fluorescence and acoustic microscopy (CCOFAM), 170–171 fluorescence spectral imaging with photoacoustic microscopy, 168–169 Combined fluorescence and x-ray microscopy, 151 Confocal fluorescence microscopy, 5, 145, 170–171 Cre recombinase, 30 CV1 cells, 7, 31 Cystic fibrosis fibroblasts, 57, 58, 59, 60 Cytogenetics, 18 Cytoplasm autofluorescence, see Autofluorescence hematoporphyrin fluorescence in, 73 Cytoskeleton, 54 extrusion of disabled organelles, 100 fibroblasts, DASPMI-stained, 3 lysosome processing, 34 Cytotoxic drugs, 33–98, 42 carcinogens and cancer chemotherapeutic drugs, 86–98 Golgi, 51 lysosomotropic, 13, 33–51 mitochondria, 1 inhibitors, 68–71 toxic agents, 52–67 photosensitizers, 72–85 Cytotoxic T lymphocytes, 141, 142, 143

D DAPI, 7 DASPMI (dimethylaminostyrylpyridiniummethyl iodine), 1, 52, 72, 113, 145 anthralin treated fibroblasts, 67 autophagocytosis, 61 azaleic acid-treated cells, 55 Chlamydomonas reinhardtii, 148, 149, 150 Fourier interferometry, 163 human colon cancer cells, 120 melanoma cells, 123 mitochondria, 2, 3, 4, 6 Photofrin 2-treated fibroblasts, 78, 79, 80 Photofrin 2-treated hepatocytes, 77 quinacrine-treated cells, 49, 50 TTPQ-treated fibroblasts, 81 yeast, 147 D-cells, pancreas, 129 Deconvolution methods, 86, 90, 151, 161 Delta cells, pancreatic islet, 129 DeltaVision software, 161 Detergents, 13, 99, 104, 105, 106 Detoxification, xv, 13; see also Multiorganelle paranuclear detoxification complex

Differentiation, cell, 113–127 hepatocyte transformation, 115, 116, 117, 118 myoblast, 124, 125, 126, 127 Digital deconvolution microscopy (DDM), 159, 160, 161 Dil, 171 Dil-LDL and RhCTXB colocalization with caveolin-1GFP, 107 Dimethylaminostyrylpyridiniummethyl iodine, see DASPMI Dinitrophenol, 68, 69 DNA, 7 fluorescent probes, 20 multiparameter imaging model, 169 quinacrine and, 38 DNA adducts, 86 DNA typing, 18 N-Dodecyl imidazole, 13, 99, 104, 105, 106

E Electron microscopy myelinosomes, 36 myoblasts, 127 quinacrine-treated rat liver cell, 35 ultrastructural map of L cell fibroblast, xvi Emission filter wheel (EMFW), 159 Endoplasmic reticulum benzo-(a)-pyrene-treated cells, 88 carcinogens and cancer chemotherapeutic agents, 86–98 multiorganelle paranuclear complex, 13, 38; see also Multiorganelle paranuclear detoxification complex myelinosomes, 36 Endothelial cells, 141 Epoxides, 86 Erroneous detoxification, xv Excitation–emission fluorescence spectral imaging, see Fluorescence spectral imaging

F Fabry-Perot interferometry, 151 Fast Fourier Transform (FFT) program, 166 Fibroblasts, xvi autophagocytosis, 60, 61 cell–cell communication with pancreatic islet cell, 144 colocalization of Rh-CtxB and Dil-LDL with caveolin-1-GFP, 107 cytotoxic drugs adriamycin, 96, 97, 98 anthralin, 67 azaleic acid, 54, 55, 56, 57, 58, 59, 60, 61, 63, 64, 65, 66 benzo-(a)-pyrene, 88, 89, 90, 91, 92, 93, 94 dinitrophenol, 69 phenobarbital, 51 quinacrine, 34, 35, 38, 39, 40, 41, 42 DASPMI stained, 53

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177

fluorescence probes, vital, xv Golgi, 8, 9, 10, 11, 12 mitochondrial, 2, 3, 5 nuclear, 19 hereditary metabolic disorders cystic fibrosis, 57, 58, 59, 60 Gaucher disease, 100, 101, 102, 103 SLSD phenotype induction, 110 sphingolipid storage disease (SLSD), 109 metabolic probes, 27 NBD-ceramide, 8, 9, 10, 11, 12 normal, 122 photosensitizers hematoporphyrin-treated, 73, 74 Photofrin 2-treated, 78, 79, 80 TTPQ-treated, 81, 82, 83 Filters, microspectrofluorimetry, 156 Flavins, 84 Fluorescein, 129 Fluoresceinyl(bis)-a-glucopyranoside, 105 Fluoresceinyl(bis)-b-D-glucopyranoside, 104 Fluorescence in situ hybridization (FISH), 18 Fourier interferometry, 163 multiparameter imaging model, 169 Fluorescence photobleaching recovery, 151 Fluorescence polarization microscopy, 151 Fluorescence probes, vital, xv, 1–21 Golgi, 8–12 lysosomal, 13–17 mitochondrial, 1–7 nuclear, 18–21 Fluorescence quenchers, deconvolution methods, 86 Fluorescence spectral imaging, 88, 145, 151 combined with photoacoustic microscopy, 168–169 Fourier interferometry for, 163–167 Fourier interferometry, 88, 145, 151, 163–167 Fourier transform multipixel spectroscopy, 146 Fura-2, 19, 118

G Gaucher disease, 99, 100, 101, 102, 103 GDH-produced thiol (RSH) groups, 23 Genetic diseases, 99–111 Giant lysosomes, 13; see also Phagolysosomes Glibenclamide, 129 Glucagon-secreting cells, 28, 129 Glucoceramidase, 17, 99, 100, 101 Glucoceramides (Gaucher disease), 99 Glucose-6-phosphate, 129 Glucose-8-phosphate, 26 Glucose kinase-inactivated cells, 30 Glucose response/glycolytic substrate addition, 23, 24, 26 mitochondria, 25 NADH transients, 26, 27 pancreatic islet cells, 29 Glutathione, nuclear compartmentation, 32 Glutathione dehydrogenase (GDH), 23, 32 Glycolysis cell-to-cell communication, 129

dinitrophenol and, 69 glucose response/glycolytic substrate addition, 23, 24, 25, 26, 27, 29 magainin and, 71 GM1 gangliosidosis, 109, 111 Golgi complex, 142 BIODIPY-LacCer, 108 cell differentiation and cell pathology, 113 cytotoxic drugs and, 51 adriamycin, 96, 98 benzo-(a)-pyrene, 88, 91, 93, 95 carcinogens and cancer chemotherapeutic agents, 86–98 quinacrine, 41 fluorescence probes, vital, 8–12 Fourier interferometry, 163 lipid storage disease cells, 109 GM1 fibroblasts, 111 SLSD phenotype induction in normal fibroblasts, 110 lysosome complexation, 41 multiorganelle paranuclear complex, 13, 38; see also Multiorganelle paranuclear detoxification complex myoblasts, 124, 126, 127 NBD-ceramide, 51, 117 NMuLi cells, 117 NBD-ceramide treated, 117 transformation, 113 Golgi–endoplasmic reticulum complex adriamycin-treated cells, 96 benzo-(a)-pyrene-treated cells, 93 Golgi gigantism, 86 Fourier interferometry, 163 NBD-ceramide treatment of NMuLi cells, 117

H Hematoporphyrin, 72, 73 Hepatocytes, 8, 32, 113, 164 benzo-(a)-pyrene-treated, 89 cell differentiation and cell pathology, 113 cocultured with reticuloendothelial and endothelial cells, 141 Fourier interferometry, 163 NMuLi, see NMuLi cells Photofrin 2-treated, 75, 76, 77 quinacrine-treated, 35 Hereditary metabolic disorders, 34 cystic fibrosis, 57, 58, 59, 60 Gaucher disease, 99, 100, 101, 102, 103 SLSD phenotype induction, 109, 110 sphingolipid storage disease (SLSD), 99, 109 Hoechst dyes, 18, 21, 162 Human breast adenocarcinoma cells, 164 Human colon cancer cells, 113, 120, 164 Human melanoma cells, 27; see also Melanoma cells Human skin fibroblast, 88, 107 Hydrogen-producing algae, 146, 148, 149, 150 Hydrolases, lysosomal, 13, 72

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178 Fourier interferometry, 163 UG9 and, 17

I Image intensifier, 6 Immunofluorescence, pancreatic islet cells, 28 Immunological synapse, 141, 142, 143 In situ fluorigenic reaction, lysosome, 105 Instrumentation, 151–158 Insulin, 28 Insulinoma, 129 Insulin-secreting B cells, 28, 29, 30, 129 Integrater, 120, 121 Intensified charge coupled device (ICCD), 25, 171, 173 Interferometry, Fourier, 163–166 Intranuclear matrix, 32 InvestiGater, xv

J Janus green, 53

K Karyotyping, spectral (SKY), 18 Keratinocytes, 113, 164 Krebs cycle, 23

L Lactosylceramide (LacCer) BIODIPY, 107, 108 cholesterol depletion and, 109 SLSD phenotype induction in normal fibroblasts, 110 Lasers, photodamage, 53 Laser scanning confocal microscopy (LSCM), 159, 160 L cell fibroblasts, xvi benzo-(a)-pyrene-treated, 89 hematoporphyrin-treated, 73, 74 metabolic probes, 27 Lipid peroxides, 72 Lipofuchsin, 84, 85 Lipofuchsin-like pigment, autofluorescence, 31 Liquid crystal array, 168 Lissaminerhodaminedodecanoyl-glucocerebroside (LR12GC), 99 Liver cells NMuLi hepatocytes, see NMuLi cells quinacrine, 35 LR12GC, 99 Lucifer yellow, cell-to-cell communication, 129 Lymphocytes, 141, 142, 143 Lysosomal hydrolases, 13, 17, 72, 163 Lysosomes autofluorescence, 31 autophagocytosis, 1, 60, 61; see also Autophagocytosis; Phagolysosomes

cytotoxic drugs, 33–51 azaleic acid-treated cells, 60, 61 benzo-(a)-pyrene-treated cells, 88, 92, 93, 94, 95 carcinogens and cancer chemotherapeutic agents, 86–98 quinacrine plus nitramine treated NMuLi cells, 44, 45, 46 quinacrine-treated cells, 40, 47, 48, 49, 50 rotenone and, 70 cytotoxic T lymphocytes, 143 DASPMI and, 52 fluorescence probes, vital, 13–17 Fourier interferometry, 163 genetic diseases, 34, 54, 99–111 human colon cancer cells, 120, 121 membrane permeabilization, 13, 99, 104, 105, 106 mitochondrial interactions, 54, 55, 56 multiorganelle paranuclear complex, 13; see also Multiorganelle paranuclear detoxification complex multiparameter imaging model, 169 normal fibroblasts, 122 photosensitizing agents Photofrin 2-treated fibroblasts, 78, 79, 80 Photofrin 2-treated hepatocytes, 75, 76, 77 TTPQ-treated fibroblasts, 81 Schiff bases and, 72

M Magainin, 71 Mastocytomas, 164 Matrix method, deconvolution, 86 MCF-7, 164 Melanoma cells, 123 adriamycin, 96 azaleic acid-treated, 62 metabolic probes, 27 photo-oxidative stress, 84, 85 Membrane permeabilization, lysosomes, 13, 99, 104, 105, 106 Metabolic activation, 57, 86 Metabolic disorders, hereditary, see Hereditary metabolic disorders Metabolic inhibitors, xv, 68–71 Metabolic map, xix Metabolic probes, 23–32 Metabolism, xv, xvi; see also NADH; NAD(P)H cell-to-cell communication, 129 dinitrophenol and, 69 glucose response/glycolytic substrate addition, 23, 24, 25, 26, 27, 29 fluorigenic substrates, 13 magainin and, 71 recovery from azaleic acid treatment, 57 N-Methyl-N-2,4,6-tetraanilinetetranitrobenzamine (nitramine), 43, 44, 45, 46 Methylumbelliferyl glucoside (UMBG), 99 Michelson interferometer, field-widened, 165 Microcompartmentation, mitochondrial, 2

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Index Microecosystems, 141–144 Microelectrophoresis, 156 Microinjection setup, 156, 157, 158 Microirradiation, 53 Microspectrofluorometry, 151, 152, 153, 154, 155, 156 Microtrabecular network, xix Microtubule organizing center (MTOC), 141, 142 Mitochondria autophagocytosis, 60, 61; see also Autophagocytosis; Phagolysosomes cell differentiation and cell pathology, 113 Chlamydomonas reinhardtii, 148 cystic fibrosis fibroblasts, 57, 58, 59, 60 cytotoxic drug-treated cells anthralin, 67 azaleic acid-treated fibroblasts, 54, 55, 57, 58, 59, 60, 63 azaleic acid-treated melanoma cells, 62 inhibitors, 68–71 magainin, 71 quinacrine, 49, 50 rotenone, 70 toxic agents, 52–67 DASPMI staining, 49, 50 fluorescence probes, vital, 1–7 Fourier interferometry, 163 human colon cancer cells, 120 melanoma cells, 123 metabolic probes, 23, 25, 27 multiparameter imaging model, 169 myoblasts, 125, 127 NADH fluorescence, 31 NMuLi transformation, 115, 116, 117 osteosarcoma, MitoTracker Green, 119 photosensitizers, 13 hematoporphyrin, 74 Photofrin 2-treated fibroblasts, 78, 79, 80 Photofrin 2-treated hepatocytes, 77 TTPQ, 81, 82, 83 rhodamine-123, 54 yeast, 147 Mitochondrial DNA, 7 Mitochondrial fragmentation, 1 Mitochondria–lysosome interaction, 54, 55, 56 MitoTracker Green, 1, 145 Fourier interferometry, 163 osteosarcoma, 119 yeast, 147 Monochlorobimane (BmCl), 23, 32 Morphometry, xvi Multichannel analyzer, fluorescence spectroscopy, 151 Multidrug resistance (MDR), 40, 86 Multiexponential fluorescence lifetimes, 151 Multiorganelle paranuclear detoxification complex, 13, 38, 99, 100 benzo-(a)-pyrene, 88 carcinogens and cancer chemotherapeutic drugs, 86–98 Multiple-parameter spectral imaging, 151 Myelinosomes, 13, 35, 36, 37 Myoblasts cell differentiation and cell pathology, 113

179 DASPMI staining, 125 NBD-ceramide, 124, 126 ultrastructure, 127

N NADH, 25, 27, 31 cell-to-cell communication, 129 metabolic probes, 27 rotenone and, 70 NAD(P), 23 NAD(P)H, 23, 86 autofluorescence, 28, 29, 30 CV1 cells, 31 magainin and, 71 photosensitizers, 84 rotenone and, 70 Schiff bases and, 72 Nanocompartment study feasibility of, 171 optical sensing techniques, 18 NBD-ceramide, 8, 9, 10, 11, 12 adriamycin-treated cells, 96 Fourier interferometry, 163 myoblasts, 124, 126 NMuLi treatment, 117 phenobarbital-treated fibroblast, 51 N-dodecyl imidazole, 13, 99, 104, 105, 106 Near-field optical probes (NFOs), 18 Near-field scanning optical microscopy (SNOM), 18 Neoplastic cells cell-to-cell communication, 129 transformation, see Transformation Nerve cells, pacemaker, 141 Niemann-Pick disease cells, 99, 108, 109, 110 Nitramine, NMuLi, 43, 44, 45, 46 9-Nitrocamptothecin, 164 NMuLi (nude mouse liver hepatocyte) cells, 43, 44, 45, 46, 113 cell differentiation and cell pathology, 115, 116, 117 NBD-ceramide treatment, 117 Photofrin 2-treated, 75, 76, 77 rotenone treated, 70 transformation, 115, 116, 117 Nonylmethylumbelliferylglucoside, see UG9 Novel methods and instrumental designs, 159–171 combined confocal fluorescence and acoustic microscopy (CCOFAM), 170–171 excitation–emission fluorescence spectral imaging combined with photoacoustic microscopy, 168–169 Fourier interferometry for, 163–167 nanocompartment study, feasibility of, 171 two-photon excitation microscopy, 159–162 Nuclear matrix, 32 Nuclear membrane, paranuclear microchannels, 39, 40 Nuclear pump, 40 Nucleolar channel, 122 benzo-(a)-pyrene-treated cells, 92 quinacrine-treated cells, 38 Nucleolus

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180 DASPMI recovery in, 1 melanoma cells, 123 Nucleus adriamycin-treated cells, 96, 97 azaleic acid-treated cells, 55 benzo-(a)-pyrene-treated cells, 94 DASPMI recovery in, 1 DASPMI staining, 49, 50 fluorescence probes, vital, 18–21 Fourier interferometry, 163 melanoma cells, 123 monochlorobimane, 32 multiparameter imaging model, 169 paranuclear microchannels, 39 quinacrine-treated cells, 38, 49, 50 Nude mouse liver hepatocyte, see NMuLi cells

O One-dimensional scans, xvi One-photon fluorescence microscopy, 162 Optical multi-channel analyzer, 151 Oregon Green, 162 Organelle morphometry, xvi Osteosarcoma, 164 cell differentiation and cell pathology, 113 glucose response, 24 mitochondria, 119 multiparameter imaging model, 169 Oxidative damage, see Reactive oxygen species Oxygen, mitochondria, 1, 53, 113

P Pacemaker nerve cells, 141 Pancreatic islet cells, 28, 29, 30 cell-to-cell communication, 129–139 coculture with neurons, 141 Paranuclear complex, see also Multiorganelle paranuclear detoxification complex benzo-(a)-pyrene-treated cells, 88, 93, 94 NBD-ceramide treatment of NMuLi cells, 117 phenobarbital-treated fibroblast, 51 Paranuclear microchannels, 39, 40 Pentaferometer, 165 Perforin, 141 Phagolysosomes, 13, 41; see also Autophagocytosis azaleic acid-treated cells, 60, 61 benzo-(a)-pyrene-treated cells, 94 colon cancer cells, 113 Fourier interferometry, 163 Photofrin 2-treated hepatocytes, 75 quinacrine-treated cells, 34, 35, 40 Phase microscopy, combined with photoacoustic microscopy, 167 Phenobarbital, 51 Photoacoustic microscopy combined with fluorescence spectral imaging, 168–169 combined with phase microscope, 167

Fourier interferometry with, 164 Photobleaching fluorescence recovery, 151 imaging techniques and, 159 instrumentation for imaging, 151 one- versus two-photon fluorescence microscopy, 162 single-molecule light sources and, 171 Photodamage, 53 Photofrin 2 fibroblasts treated with, 78, 79, 80 hepatocytes (NMuLi) treated with, 75, 76, 77 Photo-oxidation, 84, 85 Photosensitizers, 13, 72–85, 99 Phycocyanin, 146 Phycoerythrin, 146 Picrylnitromethamine (nitramine), 43, 44, 45, 46 Pigment localization, algae, 146 Pixel-by-pixel recording, xvi, 151 Polarization of cells, cytotoxic T lymphocytes-P815 conjugates, 143 Polarized fluorescence photobleaching, 151 Porphyra linearis, 146 Porphyrins, 72, 99; see also Photofrin 2 PRE4, 113, 163 P12THC, 99 Pyrenebutylrhodamine (PRE), 1 Pyrenedodecanoyl trihexosyl ceramide (P12THC), 99

Q Quasi-simultaneous fluorescence spectra, 151 Quinacrine (Atabrine), 8, 13, 14, 15, 16, 33, 37, 38, 42, 47, 48, 49, 50 colon cancer cells, 113 DASPMI staining with, 49, 50 fibroblasts, 35, 40 Fourier interferometry, 163 human colon cancer cells, 120, 121 NMuLi, 43, 44, 45, 46 normal fibroblasts, 122 Quinacrine mustard, 8, 86

R Reactive oxygen species, 53 mitochondrial damage, 7 photodamage and, 73 Redox complexes, 23 Redox state, 32 Reduced glutathione (GSH), 32 Reticuloendothelial cells, 141 Rh-CtxB and Dil-LDL, colocalization with caveolin-1-GFP, 107 Rhodamine-123, 1 azaleic acid-treated cells, 54, 56 fibroblast, cystic fibrosis, 57, 58, 59, 60 Fourier interferometry, 163 hematoporphyrin-treated cells, 74 melanoma cells, 123

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181

mitochondria, 5 TTPQ-treated fibroblasts, 82, 83 Rotenone, 70 RSH groups, 23

S Saccharomyces cerevisiae, 21, 145, 147 Scanning near-field optical microscopy (SNOM), 18, 145 Schiff bases, 72, 84, 85 Sebacic acid, 27, 52 Similarity mapping, 86 Single-molecule probes, 18, 171 Skin fibroblasts, 88, 107 Somatostatin-secreting D cells, 129 Sorbitol, 92, 93 Spectral deconvolution, 86, 90 Spectral imaging, 151 benzo-(a)-pyrene, 86 Fourier interferometry for, 163–166 Spectral karyotyping (SKY), 18 Sphingolipids (Niemann-Pick disease), 99 Sphingolipid storage disease (SLSD) fibroblasts, 109, 110 Sulfhydryl groups, 84

colon cancer cells, 120, 121 hepatocytes, 115, 116, 117, 118 melanoma cells, 123 microecosystems/cell ecology, 141 Tricolor CCD, 145 Tumor cells cytotoxic T cells and, 141, 142, 143 metabolic probes, 24 multiparameter imaging model, 169 Two-dimensional LCA, 170, 171 Two-dimensional scans, xvi Two-photon excitation microscopy (TPEM), 29, 159–162

U UG9 (nonylmethylumbelliferylglucoside), 17, 99, 100, 101, 102, 103 Ultrastructural map, xvi Ultraviolet-absorbing fluorophores, 159 Ultraviolet photodamage, 53, 72, 84, 85, 145 Umbelliferones, 99; see also UG9 Umbelliferyl group, UG9, 17 UMBG (methylumbelliferyl glucoside), 99

V

T Tetralyte, tetryl (nitramine), 43, 44, 45, 46 Tetramethylrhodamine-ethylester, see TMRE Tetramethylrhodaminemethylester, see TMRM Tetraphenylporphine quinoline (TTPQ), 81, 82, 83 T4 heptatocytes, Photofrin 2-treated, 75, 76, 77 Thiol probes, 32 Three-dimensional scans, xvi T lymphocytes, 141, 142, 143 TMRE (tetramethylrhodamine-ethylester), 1, 145, 163 TMRM (tetramethylrhodaminemethylester), 1, 5, 163 Tolbutamide, 129 Topoisomerase inhibitors, 18, 20 TPPQ, 72 Transformation, 113 cell-to-cell communication, 129

Video images, xv Vital fluorescence probes, see Fluorescence probes, vital Voxel-by-voxel recording, xvi

X Xenobiotics, xv, 13, 42 X-ray microscopy, 151

Y Yeasts, 21, 145, 147

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COLOR FIGURE 1.2

COLOR FIGURE 1.3b

COLOR FIGURE 1.4

COLOR FIGURE 1.5a,b

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COLOR FIGURE 1.10

COLOR FIGURE 1.8

a.

b.

c. COLOR FIGURE 1.11

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COLOR FIGURE 1.12

COLOR FIGURE 1.14

COLOR FIGURE 1.17a

COLOR FIGURE 1.17b

COLOR FIGURE 2.18b

COLOR FIGURE 2.18c

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COLOR FIGURE 2.19a

COLOR FIGURE 3.22a

COLOR FIGURE 3.23a

COLOR FIGURE 3.23c

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COLOR FIGURE 3.30

COLOR FIGURE 3.31

COLOR FIGURE 3.34b

COLOR FIGURE 3.35c

COLOR FIGURE 3.36

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COLOR FIGURE 3.39b

COLOR FIGURE 3.42

COLOR FIGURE 3.52a

COLOR FIGURE 3.41

COLOR FIGURE 3.46

COLOR FIGURE 3.47

COLOR FIGURE 3.53

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COLOR FIGURE 3.54e

COLOR FIGURE 5.70

COLOR FIGURE 3.55b

COLOR FIGURE 5.71

COLOR FIGURE 5.73a

COLOR FIGURE 4.57d

COLOR FIGURE 5.72

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COLOR FIGURE 7.84

a

COLOR FIGURE 7.85a,b

b COLOR FIGURE 10.102

c