Quick Photoshop for Research: A Guide to Digital Imaging for Photoshop 4x, 5x, 6x, 7x

Quick Photoshop for Research A Guide to Digital Imaging for Photoshop 4x, 5x, 6x, 7x

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Quick Photoshop for Research A Guide to Digital Imaging for Photoshop 4x, 5x, 6x, 7x Jerry Sedgewick Department of Neuroscience University of Minnesota Minneapolis, Minnesota

KLUWER ACADEMIC PUBLISHERS NEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

eBook ISBN: Print ISBN:

0-306-47938-9 0-306-47375-5

©2002 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow Print ©2002 Kluwer Academic/Plenum Publishers New York All rights reserved No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: and Kluwer's eBookstore at:

http://kluweronline.com http://ebooks.kluweronline.com

Preface Quick Photoshop for Research: A Guide to Digital Imaging contains essential information for the use of Photoshop specific to researchers. This step-by-step guide is the only book published for users whose needs are not for the purpose of graphic or web design: instead, this book only addresses the tools and functions necessary for the ethical enhancement of scientific images, and subsequent layout of these images into figures or plates. The aim is to provide information about digital imaging in an easy-to-follow guide from the beginning of the imaging process to its end. Additional information about scanning and acquiring images via a digital camera or laser/PMT system is also covered, as well as information about printers and PowerPoint. This book is intended for occasional users, as well as beginning and intermediate users primarily in the life sciences, though it can be applied to forensics, astronomy and engineering. It can also be distributed from core imaging facilities from within universities and corporations for those who use microscopes, scanners, confocal systems, and other imaging devices. Actions can be downloaded from quickphotoshop.com to automate functions covered in this book. Part 1 of the book covers what users should know about imaging, ethics, computer requirements, differences between vector and imaging programs, setting up preferences in Photoshop, and calibrating computer screens before using Photoshop. Part 2 explains Photoshop functions for imaging: open, duplicate, rotate, flip, undo, history, zoom, crop, mode; along with corrections for dust and scratches, color and contrast, and for adding color. Part 3 addresses what functions are used when working with several images to make a plate. You'll learn about layering, cropping several images to the same dimensions, combining images, adding white space for lettering, using the text tool, and changing the orientation of text. Part 4 covers the lay-out of multi-image figures or plates; the addition of symbols such as boxes, arrows, and circles; how to fill with patterns; the construction of scale bars; what file formats need to be saved; how to set resolution for output; the steps for converting RGB files to CMYK maintaining color saturation and contrast; ways to solve hue shifts in grayscale photographs; methods for organizing and archiving files; and specific ways to integrate Photoshop with PowerPoint. This book finally addresses what every researcher needs to know about when working with images with the most popular and widely-used imaging program. It speaks to what researchers know intrinsically in a world that has become far more visual: images often convey more than words; and, like it or not, images leave an impression in recipients' minds, often making the difference between a grant that receives more points rather than less, or between a paper that is accepted in a prestigious journal versus one that is less desirous. Like it or not, images can make or break careers.

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Acknowledgments Thanks to the following individuals Layout and design: Jerry Sedgewick, Laurel Connett, and Linda Peterson Cover design: Nam Pham Editing: Laurel Connett, Reena Maheshwari, Aria Williams, and Jenny White Templates: Jill Plumb-Smith Cover photos: Paul Letourneau, Ph.D. (fluorescent growth cone), Stan Erlandsen, Ph.D. (EM of mosquito) Inside photo credits: Martin Wessendorf, Ph.D. (fluorescent neurites); Stan Erlandsen, Ph.D. (EM photos) Special thanks to my wife, Jan Shafer, and to my colleagues, John Oja and Jim McCabe, without whom this book would never have been completed.

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Contents Part 1 Before Getting Started In Photoshop Flatbed Scanners

2

Typical Choices When Scanning Image Type Resolution/Output Size (or Scaling) Resolution: Setting by File Size Filter Options Contrast, Gamma, Brightness

Film Scanners Scanning Tips Digital Camera Systems

6 7 9

5 Steps to Follow When Acquiring Digital Images White Balancing (Color Images Only: Brightfield) Correct for Uneven Illumination Avoid Over-Saturation Image or Frame Average: Darkfield (Fluorescence) Complementary Filtering (primarily for grayscale cameras) Other Useful Solutions When Acquiring Images

Part 2

15 17 19 21 22

Calibrating Your Monitor

Setting Memory for Photoshop on a Macintosh Visual Definitions Overview of Toolbar Setting up The Photoshop Workspace Troubleshooting Load Actions in Photoshop

25 26 27 28 29 30

Mode

39

Quick Photoshop: Steps for Single Images

Marquee Tool and Guidelines Open Image/Duplicate

32 33

Open Image File -- Standard Image Files When File Does Not Appear Once your File is Open Duplicate

Rotate/Flip

35

For Small Degrees of Rotation For Interactive and High Degree of Rotation (useful for rotating parts of an image)

Undo & History Zooming In & Out

36 37

Keyboard Commands For Zoom (Preferred) Moving Image While Zoomed In (Navigating)

Crop

Making Non-pixelated, High Resolution Images from Graphing, Drawing & Word Processing Programs Differences Between a Drawing Program (Vector Graphics) and a Paint Program (Image Files) Ethics and Photoshop Computer Requirements for Running Photoshop Computer Screens (Monitors)

38

Changing Mode To RGB (color) Changing Mode to Grayscale (or 16-bit to 8-bit)

Dust and Scratches Sharpening Contrast & Color: A Primer Contrast/Color Auto Actions

40 41 42 43

Correcting Color and Contrast for Typical Images (generally from scanned negatives, slides, or digital cameras) If Auto-Balance Doesn't Auto-determine Hues Correctly and White Areas Exist in Image File

Contrast/Color Manual Adjustments Manual Adjustment of Contrast Manual Adjustment of Color Correcting Color Shift in Whites & Selected Areas

44

Part 2 Quick Photoshop: Steps for Single Images Contrast/Color Darkfield & Drawings or Graphs

47

Adding Color 48

Contrast For Images with Gray or Neutral Backgrounds (such as gels or electron microscopy images) Reducing Graininess of Films

Manual Color Controls/Subtle colors

50

Hue/Saturation Changing the Color Blue

Contrast for Darkfield (bright objects on black background) Contrast for Images Only Containing Pure Black and White (called "bitmap" in Photoshop)

Contrast: Measuring Pixels & Matching Background Gray Levels

Color: Changing Certain Colors

49

51

Adding Color to Grayscale Images (Darkfield) Adding Pseudocolor to Grayscale images (for visualization purposes) Minimizing Grayscale/Color Values (for visualization purposes)

Subsequent Steps for Single Image Final Resolution and Dimensions for Single Images

52 53

Adding White Space For Lettering in Photoshop Manual Lettering of Images (4x, 5x) Lettering in Photoshop (6x, 7x) Enhancements to Lettering

63 64 65 66

Variations (for subtle modifications of color and contrast)

Part 3 Combining More Than One Image and Adding Lettering Layers Layers: Actions & Flattening Cropping Several Images to the Same Dimensions

55 56 57

Cropping Image Files that are at the Same Resolution and Size Cropping Images That are the Same Resolution, but Different Sizes Cropping Images of Different Sizes and Resolutions to a Specified Size

Merging Images Combining >1 Image Combining 2 (or More) Images, or Parts of Images, Into 1

Outlining Black Lettering in White, or White Lettering in Black (for lettering against "noisy" backgrounds) Placing a Box Behind Text

Alignment and Orientation of Type 60 61

Rows or Columns of Lettering at 45 degrees Rotate and Align Text

67

Part 4

Finalizing Figures

Auto, Single & Multi-Image Figures (or Plates)

70

Manual Layout of Multiple Images to Make a Figure or Plate

Working With Color CMYK Files (for Publication and Pre-Press Printers) 75

Templates Based Upon Images at Non-fixed Resolutions Templates Based Upon Fixed-resolution Images (e.g., from digital camera systems) Manually Adding Images to a Page Deleting Extraneous Image Area

Symbols

78

Symbols from Symbol.psd file

Adding Symbols Manually

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Arrows and Dashes Boxes and Circles Keyboard Symbols Photoshop 6.0 Symbols

Filling Image Areas with Patterns Magnification and Scale Bars Saving Files Prints and 35mm Slides From Digital Files Overview of Printers

Setting Resolution and Dimensions For Output

86

For Pre-press printers, and Photographic Printers Laser Printers and Ink Jets For Publication Resolution Values from Digital Systems and Scanned Films

From Image Files at Various Sizes From Image Files at Fixed Resolutions Auto-Layout Corrections For Lettering Re-positioning Both Images and Lettering Contact Sheet II

81 82 83 84

88

Check To See How Much Color Shifts Method 1. Color and Contrast Restoration on CMYK Images with Small Color Shifts Method 2. Color and Contrast Restoration on RGB Images with Large or Problematic Color Shifts CMYK Correction for Blue Steps to Follow When Color Matching is Near-Impossible Reviving Contrast in the CMYK file

Working with PowerPoint

97

To make JPEG images for Importing into PowerPoint To Correct for Darkened Images in PowerPoint Setting Dimensions of Images to Match PowerPoint PowerPoint Files to Photoshop Making White-on-Blue or Yellow-on-Blue Slides to Match PowerPoint

Color Shifts with Grayscale Photographs Organizing & Archiving Files Summary of Steps Index

100 101 102 104

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Part 1

Before Getting Started In Photoshop While an extraordinary number of image problems can be solved by using Photoshop, the frustration of correcting these problems can be far more timeconsuming than avoiding the problems in the first place. This is even more true when considering that only a handful of Photoshop experts exist to do the job right in any work community, and when considering the ethics of changing “original” image data (see more on page 19).

Thus, it is of paramount importance to be more careful and knowledgeable about acquiring images than you are about enhancing those images in Photoshop. This section should be as carefully read as any of the Step-by-Step sections that follow.

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Flatbed Scanners Typical Choices When Scanning: • • • •

Image Type Resolution/Output Size Filtering Options Contrast, Gamma, Brightness

With cheaper scanners, you may not see subtle color or grayscale graduations, especially in pastel versus primary colors. Also, be aware that the advertised resolution of a scanner is normally twice the true optical resolution. The advertised resolution is interpolated (pixels are duplicated to double resolution).

Image Type Color materials. Set at True Color or RGB color (whether 16.8 million, 32-bit, 24bit, etc.) for scanning color materials. Photographic type materials (with a range of grays). Set at Grayscale. If a choice of “bit-depth” is shown (8-bit, 10-bit, 12-bit, and so on), use default setting or 8-bit unless the image density or intensity will be measured (while a higher bit-depth

“sees” a greater number of gray levels, it normally becomes 8-bit again once printed or made into a 35mm slide). Text or line drawings in black/white. Set at Bitmap, Black-and-White, or Binary. At this setting, only pure blacks and pure whites (black/white) will be scanned.

Indexed Color (256 colors) or CMYK. Do not use either of these settings. Indexed Color only records 256 colors and is intended only for the web. You should let Photoshop convert RGB to Indexed Color after scanning your document. CMYK records color intended only for printing presses and is used primarily by graphic designers. Your computer screen shows colors as a mixture of red, green, and blue, thus it makes more sense to scan as RGB. Convert the file later in Photoshop.

Some scanner software makes image type choices for you. To avoid having your choice changed by automation, either change the automatic image type option in a preferences or options menu that may have come with your software; or, after previewing and outlining your area of interest — and before your final scan — select the image type.

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Resolution/Output Size (or Scaling) “Resolution confusion” defines most situations in the digital world. In the photographic/microscopy fields, resolution is thought of as the ability to discriminate closely set details in space, and, in the past, films with different resolutions were chosen depending on how finely resolved the image needed to be. It means about the same thing in the digital world, but that ability depends on how many pixels comprise your image. The caveat would seem to be, "the more the better," but there are times when the number of pixels chosen EXCEEDS either the resolution of what is being digitized (when you scan a newspaper photo, for example), or the output device to which these images will eventually be printed. Publishers normally print images at resolutions no higher than 133 lines per inch (but 300 pixels per inch is required on the original image), where text and line drawings are printed at 1200 dots per inch (dpi). Thus, scans at resolutions higher than that are pointless. Even 35mm film is super-pixelated (or what is called over-sampled) when it is scanned at resolutions higher than 1500 pixels per inch for digital duplication.

inch (used synonymously with dots per inch). One usually need not consider dots or pixels per inch until printing to the output device, but resolution at the beginning of the process is thought of in terms of what comes at the end: outputting for a printing device. In the end, the real resolution of an image is not pixels or dots per inch, but simply a measurement of pixels across and down. When resolution is expressed as so many dots or pixels per inch, the reference is to OUTPUT resolution. For the sake of making resolution choices easier, the highest possible resolution becomes your starting point: the resolution required by a publisher. Resolutions for making 35mm slides, on-screen slide presentations, digital prints, web images, and images to be sent to reviewers all can either remain at the same resolution, or be made into images with less pixels across and down (the latter comprises typical choices for on-screen presentations and web images). Choose from the following tables when you set resolutions on a scanner.

What follows are some rules of thumb to aid in choosing the number of pixels per

Output Resolution and Output Size or Scaling (Required by Most Journals): Image Type

Output Resolution

*Output Size: 1 column/2 column

Color or Grayscale scans without text: Color/Grayscale with text EM negatives Text and Black/White Drawings (bitmap)

300 dots per inch (dpi) 600 dots per inch (dpi) 800-1200 dots per inch (dpi) 1200 dots per inch (dpi)

3.3 inches wide (8.5cm)/6.85 inches wide (17.4cm) 3.3 inches wide (8.5cm)/6.85 inches wide (17.4cm) 3.3 inches wide(8.5cm)/6.85incheswide(17.4cm) 3.3 inches wide (8.5cm)/6.85 inches wide (17.4cm)

*Dimensions indicated only apply to some journals and are shown here only as “ballpark” or “safe” settings.

Output size: If Output Size is not available on your scanner software, use the “file size” method on the following page. Also use the “file size” method for a less detailed way to estimate a starting point for resolution settings. Text & black/white images. Smooth edges do not exist when zooming in on text, graphs, and drawings, even at 1200 dpi. The problem is that we can zoom in far

more than at a “normal” viewing distance (a comfortable reading distance for 12 point type). Print out on an ink jet before re-scanning to make a judgement about quality, or scan at 600 dpi as grayscale. Whites can be whitened and blacks blackened using contrast adjustments in Photoshop (see Page 48), and the image will contain smooth edges.

3

Resolution: Setting by File Size The number of pixels that comprise an image (the more pixels, the higher the resolution) is tied proportionately to the size of the file, which is measured in bytes, kilobytes, or megabytes. Certain file sizes are sure to contain enough pixels for nearly any end, whether the material you scan is intended for an ink jet printer, PowerPoint, a 35mm slide, the web, or publication in a journal. Typically, a file size of > 1200 pixels is adequate for most scanned images without text. These files can

Setting Resolution According to the “File Size” Approach: Image Type

File Size

Color Scans without text Color Scans with text Grayscale without text Grayscale with text Binary

6-8 megabytes ~10 megabytes 2-3 megabytes 4-6 megabytes Ignore file size and set to 1200- 2400dpi (the smaller the text, the larger the dpi).

Be sure to save the original as a separate file in Photoshop. Files scanned at these resolutions can be made much larger in file size and dimensions without losing detail. If only a part of a scanned image is used, as in an EM negative, but the part has not yet been determined, scan at a higher file size.

4

be made significantly larger, but it is pointless to save at resolutions which exceed output resolution of high-end printing devices. In this instance, the number associated with output resolution settings using dots per inch are unregarded until just before printing or sending the file to a publisher.

Filter Options Often scanner software includes several options for filtering your image. The most common include descreen (often median filtering) and sharpening functions. Sharpen. Sharpen functions work far better in Photoshop, so it is best to turn off any sharpening in the scanning software. Descreen (or median). This filter reduces or nearly eliminates the dot pattern in pictures from magazines and journals. These dots are typically at a density of 133 dots per inch(dpi), also called lines per inch (1pi). Newspapers are less dense at 80 dpi. Scanner software can do a great job of nearly eliminating the dot pattern to solutions that end up simply blurring the scanned image using a median filter (median filters really don't work well; these are made to retain edges and blur everything else). The best descreen functions use a fourier transform in their process. More information about fourier space can be found in “The Image Processing Handbook” by John Russ, or in the software manual that accompanies the “Image Processing Toolkit,” (a software program that provides Photoshop plug-ins) by Chris Russ (members.aol.com/ImagProcTK/). However, most descreen functions use a median filter, so be aware that descreen may not always give you the results you want.

Contrast, Gamma, Brightness Most scans turn out looking like the original when contrast, brightness and gamma settings are set to automatic. However, in situations where contrast is too broad or uninterpretable by the software, you’ll have to set it manually. What’s most important is to make sure detail is visible in the blackest and whitest parts of your image. The best way to do that is through adjusting the gamma, which is a way to affect mostly the midtones, though these changes can also affect the blackest and whitest parts of the image. If gamma is not available, adjust white and black levels (also called brightness and contrast).

5

Film Scanners The same adjustments are made with film scanners as with flatbed scanners, except that film scanners allow for a far greater selection of image types, and the method for setting resolution is different. Image types. Film scanners allow for making a selection of image type on the basis of the kind of film used. For the most part, slide film is Ektachrome or Fujichrome and color negative film, as of this printing, is typically Kodak Gold. Generic settings that may be entitled “raw negative” and “raw slid e” exist if you are unaware of the film type.

Setting Film Scanner Resolution by File Size Color scans Grayscale Full resolution

6

6 - 8 megabytes 3-4 megabytes 15 megabytes for entire image

Resolution. The resolution of film is based upon pixels across and down. Use the “file size” approach.

Film which contains lettering will come out fuzzy. The image will need to be relettered, or you can sharpen in Photoshop (but don’t expect great results).

Scanning Tips Moire patterns. When scanning pictures from books and magazines, often you will see a moire pattern on the computer monitor, which looks a lot like two screens that are positioned at cross axis. This is NOT a resolution problem, it is simply the phenomenon of the dot pattern that makes up the computer screen crossed against the dot pattern on the magazine picture. Zoom in to make your evaluation. However, you may also notice a moire if you print out on a laserjet printer. This is because the laserjet also screens another dot pattern over your image to print. The easiest way to get around this is to print out on an inkjet. Disappearing lines. Also, when images are zoomed out, horizontal lines can disappear. Always zoom in to check to see that everything was scanned to your satisfaction. Pictures and text from books and journals. Whenever scanning from books and journals, you need to place a black piece of paper behind the page you are scanning to avoid any bleed-through from printed material on the other side of the page. Paper is usually thin enough that text bleeds through from the backside. You may want to Descreen to eliminate dot pattern if it is a picture (drawings usually don't get printed with a dot pattern). 3D objects. Do not hesitate to scan three-dimensional objects on a flatbed scanner. You'd be surprised at how focused the image can be, depending on the quality of the scanner. I have seen good results at up to 40mm depth through plexiglass. Microscope slides and like materials. Low power images can be taken with a flatbed scanner, useful up to about 2x - 4x. You will need to scan at ~1800ppi or better at true optical resolution. Certain slide scanners can be adapted to scan microscope slides. If you can adapt, these scanners give even more resolution, with the advantage of perfectly even illumination in background Pixelated lettering. Lettering that is zoomed up too far will look pixelated. Zoom out to a normal viewing distance to evaluate the quality of your scan; remember that horizontal lines may disappear. Print out results to evaluate on an ink jet printer, if you wish to be sure.

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Coomasie blue gels. These gels can be scanned dry or wet (as long as care is taken to keep water from running to the edge and seeping into the electronic equipment). Because the blue color of coomasie is so light, the gel either needs to lay on a sheet of darkened glass, or the contrast needs to be high while brightness is turned down. If the gel is wet, be sure to wet the glass with a squirt of water before transferring. Smooth out the bubbles that form underneath. Scan as quickly as possible to avoid the accumulation of more bubbles due to the heat from the scanner.

Scanning of Gels

Autorads. Autorads, or x-ray films, are fairly straightforward. Scanner software can read these easily with its internal auto settings. If the film is exceptionally grainy, however, the graininess may be removed by placing the film on a sheet of glass, and then raising that glass off the surface by using coins or consistently sized spacers. Gels can suffer from slight out-of-focus detail and still look good. Don't be timid about de-focusing bands to eliminate grain. Electron microscopy films. Beware of Moisture rings if placing on glass. Do not press on glass. I haven’t found a good way to prevent these rings from occuring when in contact with glass, except to raise the film slightly off the glass using strips of tape.

Arrangement of gel and glass on scanner

Moisture Rings

Nitrocellulose. Any material is easier to scan than nitrocellulose. The stain is so dim that every last wrinkle in the paper must be brought out to see bands. Place the nitrocellulose blot on a piece of darkened glass and raise the glass off the surface with coins or spacers and then raise contrast and lower brightness. Note that nitrocellulose is notorious for moisture rings (see above). Newer scanners typically do not use glass for large format films. Moisture ring on EM Film

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Digital Camera Systems 5 Steps to Follow When Acquiring Digital Images: • White Balance (Color Images Only: Brightfield) • Correct for Uneven Illumination in the Field • Avoid Over-Saturation • Image- or Frame-Average • Use "Opposite" Filters (For Grayscale Images Only)

Digital cameras for the consumer market typically do not contain all these protocols. However, white balancing can often be done manually, as well as avoiding over saturation (by manually setting shutter speed and aperture).

White Balancing (Color Images Only: Brightfield) When cameras are turned off and then turned on again, they have no reference to what is white, and images take on an overall cast of color. Thus, many digital and video systems provide—either in the camera (using an internal auto-white balance feature) or in the software—the white balancing option. Off-Microscope white balancing. Take an image of a white card before taking images of your samples. Be sure that the card is appropriately lit in a position in which the card reflects the “average” amount of light. How to white balance on microscope. When the camera is on a microscope, microscope slides need to be removed so that the camera can read off the light coming from the light source. Once these cameras are white balanced, they read colors correctly until the camera is turned off or the light level is turned up or down (thus changing the color temperature of the light). If you can't white balance, set color temperature. If your digital camera does not have the option for white balancing (or if you use film), then you need to set your light either to daylight or to tungsten lighting. (On a microscope, markings should exist to guide you regarding where to set the slider control for the light, typically a PHOTO indication). If no markings exist, then the light should be turned all the way up (what would be known as 3200 degrees kelvin on a color temperature scale, and Problems with DIC. If your microscope has the functions for DIC/ Nomarski, be sure the DIC prisms are not in the light path, or bands of colored light will exist in the background. The DIC prisms do what prisms do: separate light into its color components.

would also be the setting for TUNGSTEN film). You can also turn the light all the way up and flip a blue filter into place. In that instance, the light is considered daylight (5500 degrees kelvin on a color temperature scale, and also the setting for DAYLIGHT film). These two settings should make subsequent color balancing easier in Photoshop. Neutral Density filters, if available, can be used in the light path to reduce brightness of light.

White balancing is done with brightfield images (dark objects on a white background) NOT with darkfield, such as fluorescence (bright objects on a black background). With darkfield, not only do pure hues exist, making color interpretation unmistakeable; but the lamps and lasers do not provide color temperature references.

Color Temperature is a reference to light on the basis of whether the light is “warmer” or “cooler.” Measurement values are given for light in degrees kelvin. A warm light would be incandescent light: a yellow light. A cool light, on the other hand, would be sunlight on a blue sky day, which tends toward blue. If you think of an iron pot being heated by a fire, a cooler pot would be yellow, where a much hotter pot would be bluish. It’s precisely that conception which gave rise to this way of thinking about and referring to light.

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Correct for Uneven Illumination More than anything else, correcting for uneven illumination is critical for good imaging. It is especially important with low magnification imaging: the world in which uneven illumination can interfere with a good representation of your sample. In non-microscopic conditions, take a picture of a white card in the same manner described on previous page. When using a microscope, take a picture of the light source with microscope slide removed from the image area (be sure sample under coverslip was in focus before doing so). Take a picture of that, making sure no part of the image is oversaturated (too white). If your camera adjusts brightness automatically based upon the brightest point, oversaturation witll not occur. If, however, the digital system is not automatic, manual controls will have to be set so that a slight gray can be observed at the brightest area. Many systems allow for a LUT overlay to show oversaturated pixels (see page 12 for more info). Auto-correction for uneven illumination. With many digital systems, the option of taking what is called a "blank field," a "flatfield," or a "shading" image can be introduced into an automated process so that uneven illumination is auto-corrected while acquiring subsequent images: look for this possibilty. In acquisition software, the flatfield image can be incorporated into an automated imaging operation in which the flatfield image is subtracted from subsequent images. More ways to correct for uneven illumination. Software solutions can only solve minimal problems with uneven illumination. Frosted glass, clouded plexiglass, or a frosted cut out from transparent film can be put in the light path to even the light from edge to edge to avoid spotlight effects or any other illumination problems. If uneven illumination still exists, you can purchase a gradient-type filter available at professional camera stores. Gradients come in different styles. Choose the gradient that fits your lighting defect and place in the light path (effective only at a fixed magnification). Manual shading correction: take blank field image for subtraction later. If no option exists internally in the camera or in software, then take a picture of a flatfield image. Save the image to be used later in Photoshop, or even better, use Image J (NIH Image) to correct for uneven illumination. The method is outlined at the web site www.quickphotoshop.com.

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Microscopes. On a microscope, Köhler illumination should be set at higher magnifications; at lower magnifications (<10x), Köhler is not necessary, but the condensor lens should be flipped out of the way or removed. For fluorescence, microscope manuals come with your scope. Use these to align the bulb and mirror of your fluorescent source. No consistent method exists for obtaining shading from fluorescent sources, though fluorescent plastic may work as a “white card.” These can be cut out from fluorescent clipboards at office supply stores. Focus slightly below the surface, since the surface itself can act like a mirror. A good explanation of Köhler can be found at http://micro.magnet.fsu.edu.

Avoid Over-Saturation Avoid saturating white- and black-end of images (especially important with confocal images). Avoid increasing brightness, or lowering black level so that image details are lost. Nothing is more unsightly than blobs of gleaming, flat, fluorescent-stained cells on a puddle of pure black.

Rule of Thumb: In Photoshop you can always INCREASE contrast (make blacks blacker and whites whiter), but you cannot create detail where it never existed (where pixels are over-saturated).

Pixels can be measured. By placing the cursor over representative pixels on your computer screen, pixel values often display at bottom of the screen in your acquisition sofware. In Photoshop, the values are indicated in the Info Box. In menu under Windows, select Show Info (see page 48) A pixel value of 0 indicates over-saturation in the blacks; a pixel value of 255 (8-bit), 4096 (12bit) and 65,536 (16-bit) indicates over-saturation on the white end. It may be opposite on a Macintosh.

Choose highest bit-depth available. Your camera system may include a higher bitdepth than the standard 8-bit-per-channel. Choose a higher bit-depth for greater resolution of gray values and somewhat more ability to make out details in the shadow and bright areas. Your software should also have the option of saving as 16-bit. Photoshop can only open 8- and 16-bit image files. Human eyesight doesn’t see all grays. More detail exists than we are able to detect on the monitor. Human eyesight is limited to the ~100 gray level. Detail is unseen, especially in the shadows or in the black region. Thus it's better to keep detail in blacks — in case you need it — than it is to throw it away by over- saturating.

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How to Set Levels When Acquiring Images to Avoid Oversaturating Pixels. Using LUT overlay. Confocal and CCD software often includes a pseudocolor overlay to show when blacks or whites are over-saturated. On Bio-Rad® software, for example, a LUT (Look Up Table: a graphic overlay that is not part of the image) named SetCol is used to show green wherever the image is over-saturated in the black region, and red where the image is over-saturated in the white region. By adjusting both the white level (alternately called brightness) and the black level (alternately called contrast), you can bring pixels “in range.” By providing this overlay, our limited vision, and the variances of how the computer screen is set in terms of brightness, no longer play a part in determining where to set the black and white levels (this is especially important when subsequent intensity or density measurements will be made).

Please note that density/intensity measurements cannot be taken when the gamma deviates from a linear slope of one. Any bending of the gamma results in a log scale, and makes subsequent measurements incorrect. Also note that background should be subtracted when subsequently measuring density/intensity to correct for dead pixels on the CCD chip. The background image is acquired when light does not illuminate sample. This image can be saved, then subtracted later, or included in automated protocol that may have come with acquisition software.

LUT Overlay

Look for pseudocolor overlays, often called LUTs, in your image acquisition program. Change gamma when whites/blacks are out of range. Some instances occur in which whites are so intense in parts of your image, and dark values so dark, that your only option for capturing both within a single image is to change the gamma value of the camera (not always possible on a CCD camera). Gamma settings “bend” the midtone grays, typically increasing the brightness of the darkest pixels in your image. If a gamma setting does not exist, take two separate pictures: one underexposed to capture the light part of the image, and the other overexposed to capture the dark parts. Recombine in Photoshop or Image J (see www.quickphotoshop.com).

Image or Frame Average: Darkfield (Fluorescence) The camera chip can become hot while acquiring, and it produces what is called dark noise. This is electronic noise, and it makes your images look grainy or snowy. The most ideal way to correct this problem is to take several frames and average them together. A frame average amount of 8 to 16 is a good bet. The effectiveness of frame averaging is exponential. A frame average of 2 increases signal to noise by a factor of 1. An average of 4 increases signal to noise by a factor of 2, 8 increases s/n by a factor of 3, 16 by a factor of 4, and so on. If you can't frame average. If you are using a system in which this is not possible, take four or so images of the same sample and average later in Photoshop (see www.quickphotoshop.com/imaging_corrections.pdf).

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Other causes for noise. Some cameras allow for the option of setting electronic gain. The higher the gain, the more introduction of graininess or snowiness, especially in the background. Lower gain, if possible, to reduce noise. Binning. Some camera systems allow for binning. In that instance, sensors on the CCD chip are grouped together so that, say, four sensors are used to collect one pixel (versus one sensor per pixel). In that manner, CCD chips become significantly more sensitive and less prone to noise, but the number of pixels across and down (resolution) is lowered.

Complementary Filtering (Primarily for Grayscale Cameras)

Other Useful Solutions When Acquiring Images

Hues can be darkened or lightened by choosing appropriate filters. These can be placed in the light path, or in front of a lens. The most useful way to acheive darkening of a color is to choose the filter that is its opposite, or complement. The degree of darkening depends the intensity of the hue. Thus, a blue filter will increase darkness and contrast in its complement (yellow or brown). Conversely, it will decrease contrast and lighten any color of blue. If you wish to make certain stains stand out to be measured, or if you wish to diminish non-specific hues, filters can often accomplish that end. These are best used with camera systems yielding grayscale images.

De-interlace (for video). Video frames occur at 30 frames per second. To acheive 30 frames most efficiently, essentially only 1/2 the image is collected per frame. Since video is read out in horizontal lines, each frame can include every other horizontal line and still present enough information to "fool" those who view it. In that manner, one frame includes even lines (2, 4, 6, 8...) and the next includes odd lines (1,3,5,7...). This is called an interlaced image. Some video software allows for de-interlacing options, to "fill in" the missing horizontal lines. If it isn't available, Photoshop can be used (under Filter, choose Video or Other, then De-Interlace).

Glare. Problems with glare can be solved three ways: • If possible, scan on a flatbed scanner. Try unlikely samples, including those that are 3-dimensional. • If possible, submerge in a bath of water. • Polarize the light. Purchase polarizing filters for BOTH the light sources AND a rotatable filter for your imaging system. Place filter in front of your light source on its own stand. If more than one light source exists, set polarizing filters at the same axis (axis should be marked). Then turn filter in front of camera lens while viewing through camera. Glare should disappear when turned to the point at which you obtain cross-polarization. Continued on next page >

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Functions For Brightening Dim Samples Accumulate (for dim signals). Accumulate can be chosen to brighten an exceptionally dim signal. Accumulate adds one frame to another when acquiring an image. Thus, brightness builds as images are acquired. Electronic noise also increases, but this can be used with frame averaging to improve signal to noise. Integration (a cheaper alternative to buying a cooled camera: cooling is to reduce dark current noise as a result of heated sensors on the camera's chip). Another way to increase signal from dim samples is to “integrate on the chip.” This is really no different than increasing shutter speed on a 35mm camera. Integrating allows the chip to collect photons over longer periods of time, but it, too, increases noise and requires frame averaging.

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Making Non-Pixelated, High Resolution Images from Graphing, Drawing, & Word Processing Programs Many drawing, graphing, UNIX, and word processing programs do not provide the option for making high resolution image or text & drawing files. With the exception of a few notable programs like CorelDraw, Canvas, Illustrator, and the newest version of PowerPoint, options for saving or exporting files to TIFF, BMP (bitmap: in this instance, a bitmap is simply an image file, NOT a pure black and pure white drawing), or PICT can only be done at 72 dpi. The problem with a resolution that spreads text and lines across only 72 dots or pixels per inch is twofold: • 72 dots per inch comprises too few pixels and text looks chunky and pixelated. • No provision is made for creating text with a ghost image behind it for confusing the eye (what is called anti-aliased lettering). Only programs that allow you to save or export files at higher than 72dpi, and also allow you to check "anti-aliased lettering," will produce high enough resolution. Images saved as JPEG can look good, but these are compressed files, and some image information is thrown away (judge for yourself).

Please note that the indication of an output resolution of 72 dpi does not always indicate low resolution of the image itself. If the source of the image is a digital camera or Photomultiplier Tube (PMT)/laser system, then Photoshop is guessing the output resolution incorrectly.

Setting Save As, or Export Options in Programs that Allow High Resolution Images and Text: 1. Set image size in height and width to desired size, or choose from below. Set at these resolutions, depending on image type: Image Type Resolution Output Size: 1 column/2 column Color or Grayscale images without text 300 dots per inch (dpi) 3.3 inches wide (8.5cm)/6.85 inches wide (17.4cm) Color/Grayscale with text 600 dots per inch (dpi) 3.3 inches wide (8.5cm)/6.85 inches wide (17.4cm) Text and Black/White Drawings (binary) 1200 dots per inch (dpi) 3.3 inches wide (8.5cm)/6.85 inches wide (17.4cm) 2. If option exists, check anti-aliasing.

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If you can only produce low resolution, aliased files from your drawing/graphing/word processing program, try one of the following methods:

Method One (Works 100%)

Method Two (Works 99%)

Print the File/Scan Result 1. Print the file to an ink jet or laser printer. 2. Scan resulting page on a flatbed scanner (see “Flatbed Scanners” for details). 3. Save to an image file (such as TIFF).

Save Big/Make Small in Photoshop. 1. Quadruple the size of your graph or drawing and its page size. 2. Export to a TIFF, PICT or BMP file at 72 dpi. 3. Open in Photoshop and scale down. Rough edges will disappear,

Making EPS (Encapsulated Postscript) File -- on a Mac

Choose postscript printer. Check Write or Print to file and EPS (if possible). Rename file with .eps or .ps (for postscript file).

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Method Three (Mostly works: postscript files work intermittently) Save as Illustrator File, EPS (Encapsulated PostScript), PostScript, or Adobe Acrobat 1. Look for some way to either Save, Export, or print the file to an Adobe Illustrator file, available on some software programs. Better yet, buy the full version of Adobe Acrobat* and print to an Acrobat file (PC), or drag to into the Acrobat printer icon (Macintosh). These files open in Photoshop. Be sure to set resolution/size accorting to chart (p. 18) if prompted in Photoshop. 2. If no option exists, then open your print dialogue box by printing your file to a laser printer (postscript printer) or, alternatively, to Laser Writer 8 (on a Mac). 3. Search for an option to Print to File versus printing to the printer. 4. You may have to name the file and select from postscript choices. If available, check EPS. If not, go with default settings. 5. Click ‘OK.’ Postscript file will be generated. Open Postscript file in Photoshop** and set parameters for dimensions and resolution (300 dpi). Photoshop does not always open file. CorelDraw is far more universally accepting. * See quickphotoshop.com/making_tiffs.pdf for more information on Adobe Acrobat. * * You may only obtain lettering without image files.

Differences Between a Drawing Program (Vector Graphics) and a Paint Program (Image Files) If you have been accustomed to using such programs as PowerPoint and Microsoft Word, you have been using what are called vector programs in programmer’s parlance. These programs are made with the intent of going to a laser printer, or at least with the intent of going through a postscript interpreter. The postscript interpreter, in the simplest terms, provides a way for the computer to talk with the printer and to assign it font descriptions. VECTOR FILES

It also interprets so that the contents of your document can be scaled up or down in size without a loss in detail. Image files, on the other hand, are intended for photographic printers, ink jets, and 35mm slides. These are comprised of pixels, and the number of pixels is fixed by the file’s resolution. Some differences are listed below:

IMAGE FILES

Vector is meant to be printed to a laser printer.

Image files are meant for photo printers or slide writers.

Vector uses pixellated fonts and lines (on the computer screen) to make smooth fonts and lines in printouts.

Image files print fonts and lines exactly as they appear on the screen (at ~100% to 120% magnification).

Vector needs fonts (which may or may not exist on another computer).

Image files contain lettering that is not associated with a font library.

Vector files use fonts and lines as guides for printing.

Image files are pixels only.

Vector files contain virtual pages upon which text and images are placed.

Image files include the "page" as part of the file.

Vector files typically can't handle large images.

Image files are intended to handle large images.

Vector typically doesn't need so much RAM or disk space.

Photoshop does.

Vector is scalable (prints to any size without loss of detail).

Image files are scaled and given resolutions manually (except when a “fit to page” option is given).

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Either vector or image file, not both. Interpreting a digital file as vector and as an image comes closest to coexisting in Photoshop 6x and 7x. Most the time, either you have representations of letters and lines NOT meant to look good on your screen, but meant to print out well on a laser printer (vector), or it is made up of pixels (image files). Software programs are intended for specific output devices. In the broadest view, vector files are meant to be printed to laser printers. Image files are meant to be printed on photo printers or slide writers. When image files are printed to laser printers, results are unpredictable and not representative. Conversely, vector files usually cannot be printed as images unless RIPped (Raster Image Processed: made from vector to image—or raster—files). The best solution is had with ink jet printers, though these are slower than laser printers. Ink jets do a good job of interpreting text, lines and images. Remember, however, that these printers depend upon the quality of the paper to which files are printed, and these devices cannot print out as broad a range of color (gamut) as photographic printers (see page 84). Rasterizing, When vector programs like Powerpoint are used for making slides, each file must be made into a bit image) file before it can be written to a slide Again, the file needs to be RIPped.

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Pagemaker, Quark, Framemaker, and Illustrator (all page layout programs, all vector) are meant to deal with both text and image files. In each of these programs, the image is not opened or imported into the program, it is PLACED: the image, in this instance, is simply linked or referenced so that it can be found when printed (thus, image files must be included with these page layout program files when submitting these to a publisher, along with fonts). What you see on the computer screen in these programs (with the exception of Quark) is a low resolution representation of the PLACED (or linked) image.

Dots vs. Pixels when printing. Note that a laser printer must form images from dots, not pixels. Ink jet printers use irregular dots, and these generally print “photo quality.” Photo printers, on the other hand, melt dye onto paper (thermal or dye sublimation printers) or expose a silver emulsion with lasers (such as the Fuji Pictrography printer). In both cases images are not separated into so many dots. Because images are not formed into dots—which are typically larger than individual pixels — even the highest resolution laser printers, which now print at 1440dpi, do not match the resolution of 400 pixel per inch photo printers.

Ethics and Photoshop It only stands to reason that the sorts of enhancements that could have been made in the photographic darkroom for the last 100 years or so of research should also be changes that can be made in Photoshop. The difference is that, in the former instance, a negative (and less often a transparency, or a positive) was used when making those enhancements when using an enlarger, and in the latter instance no “negative” exists from which to make these enhancements. And yet, the negative DOES exist. That the “original” is a digital image viewed as something “virtual” on a computer screen does not mean it is less a “negative.” Because the digital image must be transferred to some kind of hard copy (a 35mm slide, a photographic print, published material, etc.) by definition it becomes a “negative.” The digital image becomes an intermediate image—just like a film negative—for a hard copy. Thus, it is entirely appropriate to view the original digital image as a negative. And it is also entirely appropriate to print that negative differently to the various kinds of hard copy so that the final product looks very much like what was once seen by the researcher. Obviously, what is “seen” by the researcher can be subject to his or her desire to over-enhance, but those desires had also been satisfied at one time in the darkroom. But instances also exist in which what had been “seen” simply cannot be interpreted by the hard copy because of inherent limitations in the technology. A case in point is the way fluorescent dyes appear on hard copy. Often these dyes are compared to one another to view side-by-side experimental conditions, and one or the others are compared in terms of brightness. It turns out that our eyes, the computer screen, and photographic printers often pick up shades of green better than red or blue. If the experimental condition shows that green, red, and blue are equally bright, then a straightforward, non-enhanced print would yield falsely printed results. It would seem only appropriate to show red and blue as equally bright on hard copy. Unfortunately, the only way to do that is to intensify red when post processing the image and to change the hue of blue more to cyan; or, conversely, to dim the green. In the end, how an image is shown and what that tells those who look at that image really lies in the hands of the researcher, and it depends entirely on his or her reputation and integrity. The integrity can be found out because research—at least in academics—to some degree is self-monitoring. If experiments done in one lab cannot be corroborated in other labs, then it becomes clear that images had been

made with too little of “what was seen by the eye” and too much of “what was seen by the researcher.” It is important to note in this discussion that it is a moot point whether images are corrected or enhanced by the traditional photographic process, or by Photoshop. Both methods lend themselves to making corrections based upon interpretation. The only difference is that it is faster in Photoshop, and more available to a larger number of people. It’s the latter instance that creates so much fear, and it provides the nuclear bomb argument: the more it’s available, the more potential it has for being abused. While that argument contains truth, it does not make using Photoshop wrong, unless data is intentionally added or enhancements are deliberately done to make data fit the hypothesis. Photoshop is an electronic darkroom in the hands of all but researchers desperate to fabricate results. While they may be able to do that in Photoshop, the likelihood of doing it well enough to fool reviewers is rare. Few learn Photoshop well enough to create convincing visual data (and this manual won’t tell you how), probably only as many as had formerly been able to in the darkroom. To protect against what might be called “enhancement that is too energetic,” several protocols in this manual are saved as Photoshop actions. These actions can be referenced in a paper in the same manner as methods are cited for image analysis protocol using popular programs such as MetaMorph, Image Pro Plus, NIH Image, IPLab, PixCell, etc. The only other issue that remains unresolved is that of adding or subtracting the number of pixels that make up an image, or what is called re-sampling in the digital world. For microscopists, re-sampling introduces the danger of adding or subtracting not just pixels, but data. Out of respect for the desire to avoid re-sampling, and because it’s a good idea until hard copy is actually produced (then the printing device will potentially change the number of pixels to fit the page), protocol for maintaining original numbers of pixels is also included in this manual. In any event, the original image taken by the camera or scanning device should be saved in the same manner that Polaroids or negatives have been saved in the past. By saving that digital image, researchers can keep a record in the event that their research is contested. Careful notes must also be taken regarding the date the Continued on next page >

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picture was taken and the nature of the material photographed, all of which constitutes good laboratory practices, and all of which can be defensible and legal for such matters as FDA approval. Notes in the form of annotations to the image itself can be included, if the acquisition software allows for it (Photoshop 6.0 gives the option for annotation). That image, or a second copy, can be watermarked so that it cannot be altered. These can be saved to a CD for permanence (at least the degree of permanence necessary in most of research: 10 or so years, or until the next technology takes over, at which time these images are transferred to the newest archival material). More details on this can be found on page 83, “Saving Files.”

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Let those who view your images know when images have been enhanced in Photoshop. A simple, “Images Enhanced in Adobe Photoshop” will do that.

Computer Requirements for Running Photoshop Lots of RAM memory. Photoshop is a RAM (Random Access Memory) intensive program. The caveat is: the more, the better. The speed of Photoshop processes increases dramatically with an increase in RAM. Photoshop needs at least 64 megabytes of RAM, and would do far better with 128-256 megabytes of RAM. Hard disk space. It also needs three times more hard disk space than the file size it is working on. Or, it can potentially need as much hard disk space as the number of undos set in the History window (5x, 6x, 7x). The space Photoshop needs is referred to as “scratch disk space.” Thus, a 30 megabyte file often requires 90 megabytes or more of free space on the hard disk. On multi-user computers in which files fill up hard disk space rapidly, it’s not unusual to encounter the message from Photoshop thatreads “...not enough space on the scratch disk.” That message refers to extra hard disk space that is not available to complete Photoshop functions. It’s important to note, too, that Photoshop writes and reads faster from disks that are not fragmented into too many short tracks, something sure to occur after lots of computer crashes. Windows allows users to defragment the disk, but more capabilities for both Macintosh and Windows can come with utility programs (such as Norton Utilities). The processor. Processor speed, or clockrate—what is called so many “megahertz”—doesn’t seem to make as much difference as RAM. In this instance, more is not necessarily better for most functions in Photoshop. Again, it is better to put money toward RAM than it is to get the fastest processor. The kind of chip used, whether Celeron®, Pentium®, Motorola®, and so on, seems not to make as much difference.

Video display cards. The video card (or the display card) is what interprets the information from your file and re-interprets it so that the information becomes so many tri-colored dots on a CRT (Cathode Ray Tube) computer screen. Obtaining a video card that quickly interprets information for immediate display used to be a problem in the past, but, with the advent of more video RAM on currently available cards (more is not necessarily better, since video card RAM is often used for games that contain video), slow interpretation is no longer a problem. (Older cards on older computers usually required the user to watch the screen while row after row of the image file appeared on the screen. Users could not proceed to the next step until the display was through “writing out” the image). Video cards can cause troubles. Sometimes one image is moved over another and a white or black rectangle is left behind. Either the card or the computer lacks the resources to fill in the rectangle with an image. Simply zooming out and then back in will correct the problem. Speed of file transfer. Finally, a hard disk or CD-ROM that uses a SCSI controller performs better than the EIDE controller (SCSI comes with a Mac, EIDE with a PC— though EIDE is getting faster). Hard disks send information far more quickly to Photoshop when opening and saving files using a SCSI controller. For saving to external drives (ZIP drives, JAZ drives, etc.), firewire and newer SCSI cards operate far faster than a parallel port. The advantage of firewire is the ability to plug and unplug devices while the computer is running. The computer can “see” the new device immediately, eliminating the need to restart the computer.

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Computer Screens (Monitors) Large screens. Perhaps the most desired quality of a monitor is its physical size. Many users prefer a larger screen. Just remember that the video card needs to be able to drive a larger screen to provide adequate resolution (at least 1024x768), and a good refresh rate. The higher the refresh rate, the less blinking of the screen, and the easier the screen becomes on the eye. A refresh of 80 is minimum; 100 or better is ideal. Buying for color matching. Computer screens also need to provide us with information about the color, contrast, and overall lightness or darkness of an image. The drawback to computer screens, however, is in how differently each displays, even when these are made by the same manufacturer. Thus, an image may look different on one computer than on another. Worse yet, the image on the screen may poorly match the print you eventually make, or what is published. This is because of limitations in cathode ray tube technology. (Monitors using LCD’s—Liquid Crystal Displays—perform no better. In fact, the range of colors these monitors display is less than the cathode ray tube monitors). Having said that, monitors can be calibrated by buying software and a calibration tool to calibrate color and contrast to a particular printing device, or to several devices, for better matching. However, imaging professionals in the publishing

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industry cannot agree, as a whole, about the merit of spending the money to calibrate in this manner. That’s because they have had trouble matching their published material to these calibrated screens. Files converted to the Adobe Acrobat format seem far less likely to shift in color, and this file format is increasingly desired by publishers. The best monitors and imaging systems. Monitors can, however, be matched so that what you see is closer to what is printed or published. Part of that depends upon the monitor and the imaging system. Two helpful solutions are as follows: 1. Buy a Macintosh. Macintosh computer systems paved the way for electronic publishing. Much of that technology does a better job with color and contrast matching, especially when it comes to publications. 2. Buy a self-calibrating monitor, or a monitor designed for graphic arts professionals. These monitors are far more expensive than the norm, but they’re worth it. The self-calibrating monitors self-adjust as the cathode ray tubes lose energy because of wear, thus keeping color more accurate. The absolute best are Barco monitors which are calibrated in hardware at the factory.

Calibrating Your Monitor Whether or not you have the extra cash to spend on a “professional” monitor, you must adjust the monitor you currently own. The greatest trouble with monitors insofar as color- and contrast-matching from the monitor to the resulting print (when using a dye-sublimation or photographic printer vs. ink jets or laser printers) is in their ability to display detail in the black or shadow end of images when the screen itself is set too bright (often arising from a human desire to see things better, or from a desire to make out information from the web, where images are often dark). The recommendations that follow are made with the idea that a darker and more contrasty screen more accurately resembles what becomes hard copy (the ambient

1. Using controls on your monitor itself, adjust brightness so that it is at the halfway point, and contrast so that it is all the way up (as contrasty as possible). 2. If not done already, set up computer screen for color and contrast using Adobe Gamma: Windows: 1. Click on Start. 2. Select Settings/Control Panel. 3. Double click Adobe Gamma. 4. Choose Step by Step Wizard (See steps at right for details). 5. When setting gamma, choose 2.2 (Europe: 1.8); this is the author's suggestion.

Mac: 1. Click on Apple symbol (upper left). 2. Highlight Control Panels/Adobe Gamma. 3. Choose Assistant. 4. When setting gamma, choose 2.2 (Europe 1.8).

light of your room should also be on the dim side to match the darkness of the screen. A closer match of ambient or room light and monitor results in better interpretation of image details and less eye strain). These contrasty settings work well for publication, most digital printers, and 35mm slides. These do not work, however, for matching some ink jet printers, and for previewing what is put on the web (as long as your web audience has set their monitors to “web” settings: gamma of 1.8).

Step 1: Description or Profile. Your Profile is found by Adobe Gamma. Change to another setting if you are certain of the brand (many Mac monitors are Trinitron monitors, and should be set, therefore, to Trinitron) by choosing Load. In your monitor manual, the manufacturer may suggest appropriate “icm”—internal calibration for monitors—as a starting point. Otherwise, use the default description. Step 2: Brightness and Contrast. The contrast and brightness controls refer to the controls that come with your monitor. Older monitors have dials, newer ones have buttons usually situated below the screen. Once adjusted correctly, you will see a dark grey box against black box(es). Typically, the contrast dial or button is turned to its most contrast setting and the brightness dial or button is up about halfway. Step 3: Phosphors. Again, use the manufacturer’s recommendations if you wish to change Phosphors settings. Otherwise, go with Phosphors that are indicated.

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Step 4: Gamma. Uncheck View Single Gamma Only. You will be able to set the three color channels, red, green and blue. The gamma setting determines how bright the values between white and black are (the midtones). Adjust sliders until the box inside matches, as best you can, the brightness of the inner box to the brightness of the patterned frame. Desired Gamma. Set to Windows Default so that your gamma is 2.2. At this more contrasty setting, you will be best situated to judge the output from 35mm slide making devices, from the web, and from many digital printing devices. Ink jet devices (as of this printing) tend to print less contrasty. If you consistently print to a less contrasty output device, you may want to set this to 1.8. The European standard is also 1.8 (Macintosh). Note: this option is not available on Windows systems that cannot control the monitor. Step 5: White Point. Use manufacturer’s recommendation, or click on “measure” and follow directions. You will be asked to choose the most neutral square. A “cool” square tends toward blue or green, a “warm” square toward red or yellow.

Note: If, at 5000k, the calibration looks dull, dark or yellow, you may need to replace monitor. Use 5000k specifically if you wish to print or make slides. Use 7500k to 9300k when working on images for the web. Various settings can be saved and then loaded depending on how you plan to ouput the image.

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Setting Memory for Photoshop on a Macintosh Windows vs. Mac. The Windows environment does not allow users to have much control regarding how computer memory functions, both in terms of what is called virtual memory, and in how much space you will set aside for a particular application program. In Windows, those functions are set automatically. On Macintosh computers, however, those settings are determined by the user.

Note: Mac system software will complain if you set Photoshop memory too high when trying to open another program. If you wish to have both open at once, iteratively set memory for Photoshop until the desired “other” program opens.

Setting up Memory on a Mac Control Panel 1. Under the Apple, pick Control Panel and then Memory. 2. Make sure the virtual memory is OFF! On Desktop (Finder) 1. Make sure no other application programs are open. 2. Double click on hard disk, and find the Photoshop application file (not any alias icons that may be on the desktop). 3. Under File, find Get Info. Choose Memory from the Get Info selections. 4. Under the Macintosh Apple at the upper left corner of the screen, select About this Macintosh. 5. Look at the memory available and set Photoshop: A. To 5 megs under the total memory if you wish to run Photoshop only. B. To 10 - 20 megs under if you wish to have another program (such as PowerPoint) open at the same time.

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Visual Definitions

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Overview of Toolbar Toolbars. Image programs use tools for functions. Some—but not all—tools are duplicated in the menu. Not all tools are shown.

Marquee Tool. The default tool is the Marquee tool in the uppermost left corner. When not using another tool, click on this.

Click and hold to see more tools on those tools with small arrowhead at bottom right.

Hand Tool for navigation Foreground/Background. The bottom of the toolbar contains two squares; the one in front is foreground, behind is background. All tools use the foreground color. If you click on two smaller boxes under the two squares, you will make the foreground pure black and the background pure white.

Click and drag on solid bar to move. Move Tool. To save time going back and forth between the Marquee tool and the Move tool, simply remain on the Marquee tool. When you want to use the Move tool, hold down the control/command key and the Marquee tool will become the Move tool.

Photoshop 6x, 7x: The Photoshop 6.0 and 7 Toolbar contain all of the same tools as well as some more, and they are arranged slightly differently.

Line Tool. If you were used to using Photoshop 4x, the Line tool was found at another position. It now existshere for 5x. More than likely, you ll have to click and hold on the Pencil tool to choose it.

Eyedropper Tool. The Eyedropper is good for finding a color in your sample and clicking to transfer that color to the foreground. The Eyedropper automatically activates in dialogue boxes for other functions, like when adjusting contrast. Thus, the foreground color can potentially change often when using Photoshop. If you click on the "two arrowhead" curved line, the foreground/ background will change position.

Line Tool (behind Shape Tool in 6x & 7x)

Make Invisible/Visible. You can make the toolbar invisible by pressing the tab key. To make it visible, press tab key again.

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Setting up the Photoshop Workspace

In Menu Under View highlight the Following • Show Rulers so that rulers appear alongside your image • Show Guides so that guidelines will be visible (6x & 7x find Show, then Guides)

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Troubleshooting “Cannot complete request because the file layer is locked” or “Cannot complete because area is empty.” 6x & 7x auto-locks the background or 1st layer. Duplicate layer by dragging background layer into page icon at bottom of dialogue box and make changes on the duplicated image. Or, choose the layer you intended to work on. Computer crashes often in Photoshop. Usually that means not enough space exists on the hard disk. Free up space by eliminating files, or set scratch disk space to take advantage of a second or third hard disk (see page 28). You may also want to defragment your hard disk. Crop tool magnetizes to the edge. See page 38. Crossed line over circle icon appears. You are on a text layer. Choose another layer in the Layers window. Illustrator and Acrobat files cannot be saved as anything but the Photoshop (psd) format. An invisible layer is introduced into illustrator and acrobat files. Flatten the layers by choosing that option in the Layers dialogue box drop down list, or by choosing that option under Layers in the menu (5x, 6x & 7x), then save the file to the format of choice. Function is chosen, but nothing happens. A selection has been accidentally drawn. Turn off selection by using Control/Command + d on the keyboard, or under Select, choose Deselect. Or, a selection has been hidden. Reveal the selection by using Control/Command + h; or, under View on the menu choose Show Guides in 4x and 5x, Show and then Selection Edges in 6x and 7x. Line tool doesn’t make a line. Be sure that the foreground color on the toolbox is not the same color as the line you are drawing (such as a white foreground color and thus a white line - on a white background). If that is not the case, make a new

layer (if it isn’t the top layer, click and drag the layer to the top of the Layers box) and try again. You may have to make more than one until the line tool works. Otherwise, save your file and re-start Photoshop. Menu selections are grayed out. Either the Mode is incorrect (it’s a Bitmap, Indexed Color or a 16-bit image) or a text layer is selected (in Photoshop 5x, 6x & 7x). Merge Visible is grayed out in Layers Menu when merging visible layers (layers with eye symbol). Click on a layer in which the eye symbol is visible, then Merge Visible from the Layers drop down list. No pixels are more than 50% selected. The selection edges will not be visible. When Feathering, this warning indicates that you will not be able to see the marching ants selection. Usually, you will need to make the selected area larger, or simply ignore the message (click Okay). Scratch disks are full. Photoshop requires at least three times the hard disk space than the size of your file. If you have more than one hard disk, make the least filled hard disk your primary scratch disk. Find Preferences (Control/Command + k) and then select Plug-Ins & Scratch Disks. Otherwise, you will need to delete or move files from your hard disk to make more room. You may want to reduce the size or resolution of your image using the Image Size dialogue box if the file is over 20 or so megabytes (under Image, choose Image Size); or you can Merge Layers or Flatten the image as layers add a great deal to the file size (use the Layers Window drop down box to find these). This action cannot be completed... Change the Mode of the file. The operating systems can also interfere with Actions. If it’s an action that came with this manual, check www.quickphotoshop.com for updates and/or to contact author.

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Load Actions in Photoshop Download the automatic actions to be used with this manual from quickphotoshop.com/downloads.pdf. These actions will comprise much of what will make Photoshop “quick,” and these are used for all the steps that follow. You will need to load these actions in Photoshop by doing the following: 1. Download actions from the www.quickphotoshop.com/downloads.html site. Select file to download based upon your operating system and version of Photoshop. 2. Open Photoshop. Under Window in the menu, select Show Actions (or locate Actions box on screen if Hide Actions is shown). 3. Click on upper right hand arrowhead. Select Load Actions from the drop down list. If Load Actions is grayed out (unavailable), select Default Actions at the top of the listings in the Actions dialogue box. 4. Select “Photoshop-for-research.atn” from the location into which you downloaded the file.

Under Window, select Show Actions. Click on arrowhead triangle at top right. Select Load Actions.

Set up Actions Dialogue Box Scroll down past Default Actions until you find “Photoshop for Research” and its sequence of buttons that follow. Click and pull on bottom right corner of box and stretch until 1 or 2 columns of buttons appear and button titles are readable. Click on top right arrowhead triangle. Select Button Mode.

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Don’t stretch so that button titles are not displayed in full.

Part 2 — Quick Photoshop

Steps for Single Images Whether images have been scanned from film or taken by a digital source, images should be duplicated in Photoshop to avoid saving over the original. Then most images need to be sharpened, color balanced, contrast enhanced, made free of dust, sized, and cropped (extraneous white space around the image needs to be removed).

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Marquee Tool and Guidelines Before using any tool, it is a good idea to know how to use the marquee tool and guidelines. The marquee tool may take some practice if you are not used to drawing with a mouse. Remember to click, hold down the mouse, drag, and then release when you are at your finish point. This creates an outline, or what is called a selection, which is marked by an animated marquee referred to as “marching ants.”

Marquee tool

Marquee tool along with choices for its shape

Guidelines help with the alignment of images and text. Guidelines appear only on the screen; these do not print. To Use the Marquee Tool

1. Click on the Marquee tool at the top left of the toolbar, or type the letter “m.” 2. Hold down the Marquee tool to reveal other shapes you may want to use. 3. Click on a starting point for your selection, hold and drag to outline the area of interest, then release. 4. To get rid of a selection, you can click just outside the selection once, inside the selection twice, or in the menu choose Select and choose Deselect. Alternatively, use the keyboard: Control/Command + d.

The Marquee tool should always be selected after using other tools. In that manner, the Marquee tool functions as “home.”

Click and drag to position marquee over area of interest.

Be aware of accidentally drawing tiny selections.

To Use Guidelines

1. In the menu under View, make sure Show Guides and Show Rulers are

selected (4x, 5x). It should not read Hide Guides or Hide Rulers. In 6x & 7x, under View, Show Extras should be checked. 2. To include a guideline, simply click on the ruler and drag. A guideline will appear. Drag down or across to the desired location. 3. To remove a guideline, hold down the control/command key, or click on Move tool, and drag the guideline back to the ruler area. Guidelines are useful for setting borders for areas of interest. Once guidelines are in place, these serve as constraints for either the Marquee or Crop tools.

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You can choose View in the menu and then Snap to Guides if you want to outline an area of interest using guidelines to constrain the selection.

Open Image/Duplicate Open Image File — Standard Image Files (TIFF, PICT, GIF, PSD, PDF, JPEG, etc.)

On Mac or PC: Do NOT double click on files in hopes they will open Photoshop. First open Photoshop, then find files under File/Open or File/Open As.

1. Launch Photoshop, in menu choose File then Open. 2. Click on file, click Okay.

When File Does Not Appear Files often don’t appear because of file naming problems, typically when going from Mac to PC or PC to Mac. It’s best to name files without slashes, colons, or multiple periods in the “Windows” convention. File names should be ended with a period followed by an extension (as in graph.tif).

Open As Window

To View All Files, and Then to Open

Click on triangle to expand list. Choose the file type you think it is.

1. On PC: Under Menu select File/Open As. On Mac: Under Menu select File/Open and check Show All Files (4x, 5x). On 6x & 7x “All Files” show without check mark. 2. Select a file type from the drop down list. Start with “Photoshop” file, then TIFF, GIF, JPEG, PICT, and so on. Often a “thumbnail” image will appear to let you know you found the correct file type. Files are TIFF files, but the software that created them is relatively new (or odd). Try opening these files in programs with broader definitions of what a TIFF file looks like. NIH Image on the Mac is such a program (or Image J in the “Windows” platform); or CorelDraw on the PC. Re-save as TIFF and as another image format (such as bitmap—.bmp) just to be sure.

Corrupted File

RAW. You can also open as RAW. If you know the values, you can either enter values for pixels across in X and down in Y, indicate whether the image is 8-or 16-bit, leave count and header where Photoshop sets it; or you can click Guess.

Files have been corrupted. Corrupted files sometimes open, but when they do the image looks like it has been cut in strips and the strips have been re-arranged. If the file does not open at all, open as RAW (see above). Grayed out menu functions. Some files open, but only a limited number of functions can be used. See section on “Mode” on page 39.

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Once Your File is Open Size of window. The file you open will create as large a “window” on your computer screen given the limits of the size of your monitor settings. The percentage on the bar at top only relates to the size of the image in pixels across and down in relation to the control panel setting for the display (e.g. 1024 x 768 pixels). It can bear no relation to the inch or centimeter dimensions of your digital image, nor can it bear a relationship to the dimensions of what is ultimately printed (see the www.quickphotoshop.com site for a more detailed explanation).

Duplicate 1. In Actions window, click Duplicate Image button (or, under Image, select Duplicate. 2. When dialogue box appears, re-name or click okay.

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It is strongly recommended to duplicate your image file so that you do not save over your original!

Rotate/Flip Especially when scanning artwork and negatives on a flatbed scanner, resulting images are tilted. These images can also be upside down or, in the case of negatives, scanned with a negative flipped in the wrong direction. The following functions will correct these problems. 1. In Actions window, click on Rotate 180°, Rotate 90°CW (clockwise), Rotate 90°CCW (counter-clockwise), Flip Horizontal, or Flip Vertical (these functions are repeated in the menu: under Image, select Rotate Canvas).

Arbitrary Dialogue Box Clockwise Counterclockwise

The degree of rotation or angle is typed in by user.

For Small Degrees of Rotation Click in ruler area and drag down guideline to provide “straight line” reference.

1. Drag a guideline down by clicking in rulers area and dragging down line (To obtain a view of rulers, see p.28, step 3, if rulers aren’t already visible). 2. In Actions window, click on Arbitrary Rotation (or, under Image, select Rotate, then Arbitrary). Enter the degree of rotation, usually less than an angle of 2. 3. Undo, if necessary, and start again (under Edit, select Undo, or use Control/Command key + z).

For Interactive and High Degree of Rotation (Useful for Rotating Parts of an Image) 1. Select area you wish to rotate by clicking and dragging with the Marquee tool. 2. Under Edit (Layer in 4x), highlight Transform, select Rotate (or use Control/ Command key + t) 3. Double arrow forms instead of crosshair for cursor. Click, hold and rotate. Unclick when desired rotation is acheived, then double click inside selection to complete the rotation (or click on Marquee tool in the Toolbar; then click Okay).

After rotating and cropping

Rotation Using Transform

Rotate when crosshair turns to double arrow

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Undo & History Photoshop can undo the previous step, and in versions newer than 4x, it can undo several steps (20 steps is the default setting). You can toggle between undo and redo to evaluate efficacy of your corrections.

History Window

Two Ways to Undo/Redo: Using keyboard: Control/Command + z (toggles between undo/redo)

In Menu: Under Edit, select Undo/Redo

History You can undo several steps back by opening the History window and clicking on the last step you wish to retain. This option is not available in Photoshop 4x.

1. Under Window, choose Show History (if you see Hide History, then History window is available on the screen). 2. Click on steps back to where you’d like to be.

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Click back to step you wish to retain

Zooming In & Out Before determining whether dust or scratches need to be removed, or to check sharpening effects, you may need to zoom in to parts of the image to examine it. This can be done with the magnifying tool on the toolbar, but it is best done with keyboard controls. That’s because you won’t always be able to select from the toolbar (when certain dialogue boxes are open).

Keyboard Commands For Zoom (Preferred) Zoom In: Control/ Command & + (plus key). Zoom Out: Control/Command & - (minus key). Zoom In: Spacebar + Control/Command key + click of mouse to zoom in Zoom Out: Spacebar + Alt key + click of mouse to zoom out

Moving Image While Zoomed In (Navigating) Keyboard Command: Spacebar + Shift key + click & drag.

Double click on hand tool to zoom back to “normal” magnification. This can be done if image is zoomed in accidentally when magnifying tool is in motion while simultaneously clicking (image is super-magnified).

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Crop Unlike a drawing, word processing, or graphing program, white space or the white background is part of the image. That’s because both the white space the image “sits” on and the image are made up of pixels (picture elements). Extra white space, in this scenario, comprises extra unnecessary pixels that need to be eliminated. Furthermore, only part of the image itself may be important. In both instances, the extra area needs to be “cut out,” or cropped. This function does not add or reduce the number of pixels making up the part of the image that remains. For those who wish to also crop to match two different images in terms of size, or for those who wish to adjust image sizes to match their output (for a page, a slide, an on-screen presentation, etc.), it is recommended that you do that after several other steps. It is best to adjust image size and resolution as one of the last decisions.

1. Click and hold on Marquee tool (“dotted” square icon) at top of toolbar. Select Crop tool (if you mistakenly double click on Crop tool and a dialogue box appears, simply close or ignore it for now). 2. Outline area of interest by clicking at corner and dragging. 3. Fine tune selection by readjusting outline. Move cursor over corner or between corners along the outline until arrows form ( ), then click and drag to reposition. 4. Press Enter or Return key, or click inside box to complete the crop (or click on Crop tool in the toolbar and respond to message that will pop up).

Cropping Off White Space

Crop tool outline - reposition after outlining by moving cursor over corner boxes. Click and drag to reposition around area of interest.

If the cropping outline is near the edge of your image window, it tends to jump. In that instance, zoom in before adjusting. Or, click on marquee tool, outline the part of the image you desire (middle of crosshair is the “writing” point), and move the outline into position, if necessary, pixel by pixel with the arrow keys. Re-shape the outline in 5x - 7x, if necessary, by choosing Select, then Transform. Under Image, select Crop.

Outline is drawn from center of Crop tool icon. Excess white space

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Mode The following functions in Photoshop will not work unless the image file has certain characteristics. In Photoshop parlance, that is called the mode of the image file. Only three modes can be worked on with all functions available: Grayscale (what is called a “black and white” in photography: all gray tones including pure black and pure white), RGB (Red, Green, Blue, or color files containing the three primary colors of light), and CMYK (Cyan, Magenta, Yellow, and Black, or color files containing the three primary colors of pigment plus black). Color files should be worked on as RGB until the end of the editing process, since computer screens work on the principle of mixing varying degrees of red, green, and blue light versus pigments. Furthermore, files also contain varying levels of gray tones from black to white. Typical files contain 256 gray values; these are known in programming parlance as

8-bit files. In the world of research, however, files can contain 65,536 gray values, what is known in programming parlance as 16-bit. Only 8-bit files can be worked on with all functions available. Note that Indexed Color files (those files, typically from the web, which contain 256 colors versus millions) and Bitmap files (those files which contain only pure black and pure white with nothing else) must be changed to RGB and Grayscale respectively. Other modes are rarely encountered. Photoshop will not open most 12-bit files. These need to be converted into 8-bit or 16-bit in the program that created the 12bit file.

Changing Mode to RGB (Color) 1. In Actions Window, Click on Mode RGB. Or, under Image, select Mode and highlight RGB Color.

Changing Mode to Grayscale (or 16-bit to 8-bit) 1. In Actions Window, click on Mode Grayscale/16 to 8-bit. Or, under Image, select Mode and highlight Grayscale. If necessary, select Mode again and highlight 8-Bits/Channel (near bottom of drop down list).

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Dust and Scratches Scanned slides, along with gels, blots, and films, can include specks of dust that look black on the image. These and slight scratches can be removed with the application of a filter. The filter also blurs slightly. This protocol uses the Dust and Scratches filter for smaller specks and the rubber stamp tool for larger specks. The rubber stamp takes on a “footprint” according to what “brush” is chosen in the Brushes dialogue box: a fuzzy-edged brush or footprint uses diminishing opacity to the edge to blend changes. Use either of the following steps to remove dust or scratches.

Dust & Scratches Dialogue Box

Smaller Specks of Dust 1. In Actions window, scroll down to gray Sharpen/Dust sequence of buttons 2. Choose Dust 1 for small specks, or Dust 2 for larger specks. 3. Under Edit, choose Undo if dust and scratch removal introduces too much dithering. Or, under Filter, select Noise, then Dust & Scratches. Use a radius of 1 or 2. A larger radius eliminates more dust, but also introduces more blurring.

Before changes

After changes

Change Radius to 1 or 2; if higher, too much blurring is introduced.

Rubber Stamp Tool Larger Specks of Dust or Scratches 1. In toolbar, click on the Rubber Stamp tool. In menu under Windows select Show Brushes for 4x & 5x(if Hide Brushes is shown then the Brushes dialogue box is already open); in 6x & 7x look for Brushes in the toolbar under the menu. 2. Select a brush or footprint that is slightly larger than the speck or scratch you wish to eliminate (by choosing Precise in Preferences—see page 28—an proportionately sized circle will form as the toolbar is moved onto the image). 3. Hold down the Alt/Option key and with the rubber stamp click on an area very close to your speck or scratch. (This allows you to borrow the color/ make-up of that area to cover your speck.) Release the alt/option key. Place your brush over your speck/scratch and click. 4. Repeat process for additional specks or scratches. It is best to borrow from various areas. The borrowed area “follows” the rubber stamp tool in the default setting.

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Place footprint over dust speck after selecting brush and borrowing an area with the Rubber Stamp tool.

Before changes

After changes

Sharpening Several actions have been provided for varying degrees of sharpening. The automatic sharpening actions use the Filter>Sharpen>Unsharp Mask function, which gives greater control over the sharpening effect. Almost every image can improve with the use of sharpening filters, but look carefully at the image to determine whether the effect has led to a pixelated or grainy image.

Do NOT sharpen images from which density/intensity measurements will be taken.

1. In Actions window, scroll down to gray Sharpen/Dust sequence of buttons. 2. Choose among several sharpening amounts, marked lowest to highest. 3. Under Edit, choose Undo (or use Control/Command + z) if sharpening introduces pixelation. Or, under Filter select Sharpen and Unsharp Mask. Set the slider Amount by eye (make sure Preview is checked), and keep the Radius between 1 and 2 and the Threshold at zero.

Higher resolution images can tolerate greater amounts of sharpening. JPEG images, on the other hand, can suffer from sharpening regardless of resolution.

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Contrast & Color: A Primer If images are grayscale, good scientific photographs often contain a full range of tones from black to white; if images are in color, they contain rich, deep hues. Either instance requires judicious use of contrast and color controls to not only enhance images, but to produce images that reproduce as they appear on the computer screen. Typically, images need to have shadow areas that do not contain blacks that are too black when images are reproduced. When it comes to the white end, only images destined for 35mm slides or graphs and line drawings should remain so white as to be transparent. All other outputs need to be the lightest of grays so that edges of images do not print at the same color as the white background. What is generally true about midtones (those tones between blacks and whites, or those colors and tones that are neither in the deep shadow range or in the brightest range) is that nearly every final use for the image improves when midtones are too bright overall versus too dark. The caveat for images destined for printing presses and the web is that they “can never be too bright.” Midrange tones are also the best to alter when desiring to match contrast of computer screen to hard copy. It is important to learn terminology for a better understanding of contrast and color: the most important of Photoshop functions. What follows are some terms and their meanings: Contrast (or Black Level): Contrast refers to how dark the black end, or the darkest tones in your image, appear. If your black end is very dark, everything else appears lighter in comparison. Thus, your image has an appearance of greater visual impact and greater contrast. Saturation: Saturation, in terms of color, refers to how instances in which a hue is at its maximum color intensity. Another way of conceiving saturation is to think of a hue furthest away from grey. Almost all colors used in research are saturated colors,

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which makes these images particularly unfit for reproduction. Overly saturated hues usually create problems when viewing on the screen and even more so when printing to a printing press. Note that the meaning of saturation changes somewhat when the reference is to untextured highlights. Brightness (or White Level): Brightness refers to how bright the white end, or the brightest tones in your image, appear. The danger with brightness is to set the whitest values so high that the detail disappears, which is called saturated whites (or untextured highlights). Hue: Hue is the correct reference to the specific shade of color.Color, on the other hand, is a combination of hue, saturation (or intensity), and lightness. Gamma (or Midtones): Gamma refers to the contrast or brightness of the midtones, or the grays and colors in between the whitest tones and the darkest. Gamma is what most improves the reproduction of an image. It also changes the linearity of image tones and makes them logarithmic.

If colors are altered on an image in which hues relate to intensities or some other measurement, be sure to include standards in the image BEFORE altering color.

Do not change contrast on images in which density/intensity measurements will be taken.

Contrast/Color Auto Actions Correcting Color and Contrast for Typical Images (generally from scanned negatives, slides, or digital cameras) 1. In Actions window, scroll down to blue Contrast/Color sequence of buttons. 2. Choose Contrast/Color Autobalance 1 to keep darkest shadow levels (but not too dark for output devices). Choose Contrast/Color Autobalance 2 for lighter shadow (black) levels. Choose Contrast/Color Autobalance 3 for lightest shadow (black) levels. * Under Edit, select Undo if you do not like the effect (or use Control/ Command + z).

If Auto-Balance Doesn’t Auto-Determine Hues Correctly and White Areas Exist in Image File 1. In Actions window, scroll down to blue Contrast/Color sequence of buttons. Select Color-White Level. Or, under Image select Adjust, then Levels. 2. Click white balance eyedropper tool on whitest spot in your image. Photoshop will auto-balance from the point you clicked on. 3. Set Output Levels to 254 on the white end if you are working on an image. If you are working on a graph, or in the white area above a gel, keep at 255. An output of 254 will make the white in the whitest areas slightly gray (to distinguish the edge of the image from the white of the page your image will be printed on). Under Edit, select Undo if you do not like the effect (or use keys Control/ Command + z).

Note: The contrast and brightness settings of your computer screenwill affect how well you see details in the darker areas of your image. If these were not set, please set computer screen (see page 23).

Levels Dialogue Box White balance Eyedropper tool

Click on whitest area on image.

Set White Triangle Output Level to 254 by sliding triangle to the left (only when it’s an image vs. a drawing or graph).

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Contrast/Color Manual Adjustments Manual Adjustment of Contrast 1. In Actions, click on Contrast-Manual Adjust (or, under Image, select Adjust, then Levels). 2. With images that are lackluster in contrast (histogram is narrow), move white and black triangles to either end of the histogram. This will expand the contrast. If images suffer from a lack of detail in the shadows, or from a lack of mid-range contrast when plenty of white and black values exist (a wide histogram), move grey triangle in middle to the left (to lighten shadows) or to the right (to bring in shadows or increase the gamma: mid-range contrast). 3. Set Output Levels on the white triangle to 254 for photographic type images. Set Output Level on the black triangle to at least 10 (to lighten deep blacks to printable levels).

Contrast Adjustment Using Levels

Move black slider to the right to increase the darkness of blacks in your image, typically to the end of the black “hill” of the histogram.

Move white slider to left to increase brightness of image, typically setting by eye (rule of thumb: digital images can never be too bright).

Move grey (middle) slider to left to increase brightness of shadows, to the right to increase contrast in mid-tones.

After adjusting top sliders, move black slider to right until Output Level reads at least 10 (set higher if blacks diminish fine details). Put white level on 254 for images (vs. drawings or graphs or white area around images) by moving Output Level slider to left (this makes white slightly greyer than its surrounding white page when printed).

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Manual Adjustment of Color Images generated in the course of research often don’t respond well to generic color correction. The reality is that, for some images, only manual corrections will create the correct colors. While these sorts of corrections are best left to experts, you may do well to try these yourself.

Narrow histograms comprise a narrow brightness range. In that instance, white and black sliders need to be set by eye in each channel iteratively.

1. In Actions window, click on Contrast-Manual Adjust (or, in menu, under Image select Adjust then Levels). 2. Click on Channel arrowhead to reveal drop-down menu. 3. Move white slider to right end of histogram and black slider to the other end in each channel (Red, Green, Blue). Do this for each channel before evaluating colors. 4. When done, if colors are still incorrect, go into problematic channel and back off on sliders (typically the white slider). For example, if image is too red after adjusting sliders in each channel, go to red channel and back off on the white slider until image looks more correct (this may have to be done in more than one channel). OR... If that doesn’t work, cancel and start again. The problem could be a saturation in one or two hues (e.g., the red in an H&E stain can be neon colored) use Hue & Saturation to reduce the saturation of the offending hue (see page 50). Then adjust again in Levels.

Do NOT set middle slider when adjusting gels, blots, autoradiographs, or any other kind of image in which linear dark to light values should be maintained for measurement or evaluation.

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Correcting Color Shift in Whites & Selected Areas Correcting for Hue-Shifted Whites Cross curves. Cross curves comprises a situation in which a correction in one color causes a shift in another and the two corrections work against each other. This can affect not only hues in the sample itself, but it can also introduce a hue into white backgrounds. In that instance, specific areas need to be selected before making color corrections. 1. In Actions window, click on Color- Color Range button (or, under Select, choose Color Range). In dialogue box, Select should be Sampled Colors, and Selection Preview should be checked. 2. Click on offending hue or hue-shifted white on image. Click first on one shade of that offending hue with eyedropper tool, then select the “plus” eyedropper tool and click on multiple ranges of color or hue shifted whites. The greater the range of color, and the more points clicked, the greater the exactness of selection (if you desire, for example, only the hue-shifted whites, click as many points as possible and you will be less likely to overlap into other parts of your image that contain other colors). 3. Adjust Fuzziness to select no more or no less than desired parts of the image. 4. If the selection is white, and it’s a photographic image, under Edit select Fill and Use: White. Go back into Levels to backoffOutput to 245 or so (to make white somewhat gray). If the selection is a particular element of the sample (say, blue-stained cells), use Levels to adjust contrast by going into pertinent channels and moving the middle slider, and use Hue & Saturation (see page 50) to change hue. If too much or too little area was selected, undo (under Edit select Undo) and adjust Fuzziness in Color Range to a different level. Sharply defined borders are softened best by using a higher Fuzziness value. Color Range will put a scrolling selection around areas of interest. This selection can obstruct detail. Use Control/Command + h to hide scrolling lines. When finished with Contrast/Color tools, don’t forget to deselect by using Control/Command + d or by clicking outside selection (make sure marquee tool is selected), or, in menu under Select, choose Deselect.

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Use Eyedropper tool to click on what should be white, then use Plus Eyedropper to click on more points.

Set Fuzziness after clicking on several points. Determine setting visually.

After using Fill to fill with white, set output slider in levels until white becomes light gray.

Be sure Sampled Colors is selected and Selection is checked underneath graphic image.

Before (top) and after (bottom) selecting with Color Range and then correcting color shift.

Contrast/Color Darkfield & Drawings or Graphs Contrast for Darkfield (Bright Objects on Black Background) When it comes to darkfield images, or images that contain brighter features on a black background, most often it is the black background that encroaches upon details. That may not be seen so clearly on the computer screen—you may see, for example, fine neurites against deep black on the computer screen—but when printed some details are lost. The other problem has to do with the brightest

features, some of which may not contain any detail. The problem with background that is too dark can be remedied using action buttons below; the problem with features without details cannot be solved except by reacquiring the image. A more exhaustive explanation of contrast correction for darkfield can be found at www.quickphotoshop.com.

1. In Actions window, scroll to violet-colored buttons. 2. Select button using following table. Fluorescence-brighter1

Brightens fluorescence and makes processes visible in dark areas by lightening blacks slightly. Changes gamma. Continue clicking on this button to keep increasing detail in black areas.

Fluorescence-brighter2

Slightly greater increase in brightness than previous option.

Fluorescence-up contrast1 Increases contrast by slightly darkening shadow end of image and brightening white end. Changes gamma. Fluorescence-up contrast2 Slightly more contrast than previous option.

Contrast for images only containing pure black and white (called “bitmap” in Photoshop)

Adjusting For Grayed Whites on Bitmap Images

1. In Actions, click on Contrast-Manual Adjust (or, in menu under Image choose Adjust, then Levels). 2. Use white Eyedropper tool and click on part of image you wish to be white. Click on various parts of image until white background is evenly white. If you overshoot, click Cancel and start again. 3. Keep black Output Level at 0 and white at 255. White Eyedropper tool

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Contrast: Measuring Pixels & Matching Background Gray Levels Contrast For Images with Gray or Neutral Backgrounds (such as gels or electron microscopy images) 1. Under Window, open Show Info. 2. Click on top right arrowhead triangle and select palette options. 3. Set 1st window to grayscale, 2nd to RGB, and 3rd to inches, centimeters, or pixels (your choice). 4. Under Actions, select Contrast-Manual Adjust (or, in menu under Image choose Adjust, then Levels. 5. Set white level by using white triangle slider. Measure level of background grey by moving cursor over representative background areas in your image and looking at info window. (Grayscale values are set by percentages, 0 is white, 100% is black). Find level that is agreeable (I use 10-12% for gels) and record for future contrast adjustments for similar images and/or to match similar images to the one you are presently working on.

Info Window

Click on arrowhead and select Palette Options.

Set First Color Read-

out to Grayscale. K: Previous Gray value percentage / Gray value percentage after adjusting slider

Set Second Color Readout to RGB Color.

Background Adjustment

Reducing Graininess of Films 1. In Actions window, select Contrast-Reduce Grain 1, 2, or 3. 2. Carefully compare original image with grain-reduced image to check for any loss of image information. 3. Undo, if necessary. 4. Proceed to steps outlined above if you wish to further adjust brightness of gray background or darkness of blacks. More information about this action can be found at www.quickphotoshop.com. Extreme graininess, such as that found in images of scanned phosphor screens can only be reduced using proprietary filters, such as those provided by Bio-Rad with their analysis software.

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Move slider to left to increase brightness. Slide black triangle to right for Output Level if blacks appear too dark. Do this by eye.

When moving cursor onto image, it turns into an Eyedropper tool. Info readout will give the values of gray (the K: value) at the dropper end of this icon. Find representative background and adjust slider to see before/after values. Check in a few places to get a mean value.

Manual Color Controls/Subtle Colors Variations (for Subtle Modifications of Color and Contrast) 1. Under Actions, select Color-Variations. Or, in menu under Image choose Adjust, then Color Variations 2. Look at window below to make sense out of this function.

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Color: Changing Certain Colors Hue/Saturation 1. In Actions, click on Color-Hue & Saturation. Or, in menu under Image choose Adjust then Hue & Saturation. 2. In dialogue box, choose the hue by clicking on the Edit arrowhead and choosing from the drop down list. 3. Expand or contract the range of colors in your selection by moving the slider at bottom, or by using the Eyedropper tools. 4. Change the Saturation first by moving slider to the left to de-saturate, because most color problems are related to colors that are too bright or gaudy. 5. Change Hue if the color is incorrect, or change Lightness ifthe color is too dark or too light. If you see no change, chances are you have chosen the wrong color to manipulate. For example, you may have chosen red when it should have been magenta.

Changing the Color Blue Scientific images often contain a handful of hues, or shades of only one. The blueto purple-blue hue can be seen on the computer screen but is likely to print as almost black. Thus, color changes are necessary for reformulating a blue with red in it to one that contains green (more of a cyan hue). 1. In Actions window, select Blue-Correct If that doesn’t select all blues in your image, use Hue/Saturation method and change hue to make greener (more cyan). Shift Hue slider to the left to -30 or so.

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Adding Color Adding Color to Grayscale Images (Darkfield) In Actions Window, select desired button: Make-Red, Make-Green, or Make-Blue (shifted to cyan). Manual method: The manual means for adding color is outlined at www.quickphotoshop.com.

Adding Pseudocolor to Grayscale images (for visualization purposes) Pseudocolor images resulting from this action follow the visible spectrum from the short wavelength of violet (deepest black parts of your image) to the longest wavelength red (brightest parts of your image). Your image becomes an RGB color image. This action takes a while to complete! 1. In Actions window, select Pseudocolor-Spectrum. 2. Image can be edited with Contrast-Manual Adjust. Manual method: see www.quickphotoshop.com

Minimizing Grayscale/Color Values (for visualization purposes) This action breaks down the image into eight gray or color levels using the posterize function. Used as an aid in visualizing and segmenting images. 1. In Actions window, select either Pseudocolor-8 Grays or Pseudocolor-8 Colors. Manual method: see www.quickphotoshop.com

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Subsequent Steps for Single Image Save Image If you haven’t saved your image file yet, you should do so now. Save the file, for the meantime, as a Photoshop file. Go on to subsequent steps before saving as other file types.

In Actions window, click the Save As button (or, in menu under File select Save As). Subsequent steps for Single Image To add Lettering go to page 61. To Save existing file in several formats for several purposes go to page 83. To insert file into PowerPoint go to page 96. To add symbols go to page 78. To combine or include with other images read through Part 3. To set final resolution see page 53

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Final Resolution and Dimensions for Single Images Setting resolution and dimensions comprises the final step in the imaging process. Again, resolution is the number of pixels across and down, usually expressed in terms of how it is outputted to a printing device as pixels per inch or pixels per centimeter. The dimensions in inches or centimeters must also be determined along with output resolution (the two go hand in hand) when printing to digital photographic printers and to publications. These kinds of output devices print only at defined output resolutions. The resolution is not so important, however, when printing to ink jets and laser printers. That’s because no particular gain in resolution is had by arbitrarily adding more pixels to your image (what you do when you increase the number of pixels, or resample) with these devices. Ink jets and laser printers reinterpret pixels to make dots, and, without going into an exhaustive explanation, it’s fair to say that dots, by their nature, provide less resolution than pixels. To state it another way, you’ll always get more resolution from a digital photographic printer which has, say, a resolution at 300 pixels per inch than you will from an ink jet that provides 1400 dots per inch. Dots, at this printing, cannot be made as small or as well defined as pixels, except, perhaps, in the instance of high end jet printers such as the IRIS. Ink jet

printers only look at the output dimension of the print: resolution settings are meaningless, unless the number of pixels across and down is low: at less than 500 pixels or so at one-half page. 35mm slide-making devices, on the other hand, use software that fits the image to the slide, so neither the dimensions nor the resolution is important. These print at extremely high resolutions. In that instance, even poor resolution (72 ppi) can be printed upon so many thousands more pixels to “add” resolution (though poor resolution from the get-go can never be adequately corrected: chippy or aliased lettering is evident). Nothing is gained by arbitrarily adding pixels to the image file itself (thus changing resolution), or by changing the image dimensions. In any case, a target resolution can be set to fit nearly all output devices. That setting is what prints best to publication. If the image then needs to be printed to a printing device at a different dimension, re-set just before printing.

Setting Image Dimensions and Resolution 1. In menu, under Image, select Image Size. 2. Enter Height or Width, depending on the most important dimension (Constrain Proportions should be checked so that image will retain its width/height relationship, also known as aspect ratio). 3. Enter Resolution of 300. Uncheck Resample Image when subsequently printing to ink jet or laser printer.

Go on to Making Multi-Image Plates (next section) or to “Cropping Several Images to the Same Dimensions” (page 57) if more than one image is to be included in a figure or plate.

Image Size Dialogue Box

Set most important dimension in inches or cm. Set Resolution in pixels/ inch. This is OUTPUT resolution. Check Constrain Proportions. Check Resample Image unless going to ink jet or laser printer.

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Part 3

Combining More Than One Image and Adding Lettering

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Layers For the following steps, an understanding of the layers capability in Photoshop is absolutely necessary. The beauty and bane of Photoshop is in its ability to keep images on separate layers. While layers keep images from interfering with each other, as though on transparent sheets one above the other, the sheer multiplicity of images, along with text, can make Photoshop difficult to navigate. Action buttons have been included to make it easier to keep the number of layers down to one or two, depending on the action. For most purposes, only two layers need to exist: 1. In Actions window, click on Show Layers (or, under Windows in menu, select Show Layers. If Hide Layers is listed, then the Layers window is already open somewhere on your screen.

one for the image (typically the background layer) and one for text and symbols (what is labeled “text” in the Photoshop for Research action buttons). Images that contain layers can only be saved in the Photoshop format (.psd). These cannot be opened by very many other programs, but that number is rising. It‘s useful to save your images with layers so that the image can be edited later, if necessary. Photoshop 6x & 7x Layers window looks quite similar. There are some added functions, and some things are in different places, but the basic window is the same.

Layers Window Click on arrowhead to reveal layering options. Normal is most often used (other options are discussed elsewhere in this manual). Eyes can be clicked on or off. When off, the layer disappears. Chains can lock one layer to another, so that if one layer is moved, both move (this doesn’t always work). Layers can be dragged to new positions by clicking and dragging. The higher layer covers the layer below. New layers can be made by clicking on paper icon. Layers can be deleted by dragging them into the trash icon, or by simply selecting the layer and clicking on the trash icon.

Click on arrowhead to activate drop down list. Ability to flatten layers or to merge visible (only those with “eyes” on) is selectable from drop down list and also from Layers in the menu selections (5x, 6x). Transparency or opacity of layer is set by clicking here. Layers are made more translucent if opacity is reduced. Capital “T” indicates that this layer is a nonrendered text layer (5x-7x). Thus lettering is able to be edited by double clicking on the layer, but images cannot be added to it.

Adjustment Layers can be made only for contrast/color adjustments. A new adjustment layer is found in the drop down list. Black cutouts indicate a masked layer. New images/text cannot be added to black part of mask.

Clicking on layer while holding down the Control/Command key selects the objects on that layer. You will see selections, or what are called “marching ants,” surrounding your objects.

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Layers: Actions & Flattening Layers Action Buttons Layer- Flatten: flattens all layers. This is necessary when saving to formats other than the Photoshop (.psd) format, and when desiring to make the file size smaller. Or, under Layers (or in the Layers drop down menu) choose Flatten Image. Layer- Flat Except Background: Flattens all layers except background layer. This is useful for reducing the number of layers to make working with the file more manageable. Layer- Render Text: Makes text layer into “normal” layer. Text layer is no longer editable by double clicking, but text can be easier to move around (the text layer need not be double clicked to move text around on the image). Because limited text is typed in Photoshop, rendering text should be considered a useful function. Also available under Layers when selecting Type, then Render (5x) or in 6x & 7x under Layers, then Rasterize and select Type. Merge Visible: Too many layers can make navigation difficult, and so it is advisable to merge layers of one type into a single layer (such as all the text or all the images). This can be accomplished manually by clicking on eye icons to make layers that are not needed invisible, then by merging what is visible. Under Layers, choose Merge Visible (or look for this function in the drop down box). Merge Visible is grayed out (unavailable) when a visible layer (a layer with the “eye” visible) is not selected (clicked on).

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Cropping Several Images to the Same Dimensions Several images may need to be cropped to the same dimensions, and, additionally, may need to be cropped in the same location. Thus, if you acquired two images, a control and an experimental, and you wished to crop out the unnecessary detail, you will need to crop not just to the same size, but you will want both images cropped in the same place. This can be done as long as both images are the same size in pixels across and down, which would be true if you collected from a fixed

digital imaging system, such as from a digital camera. You can find how to set cropping with the “fixed image size” description below. If your image files are different sizes in dimension and/or resolution, then you will need to choose “varying image sizes.” In that instance, the cropping tool is used. That tool precludes the ability to transfer the crop to the exact same location from one image to another, and so landmarks on the image itself will have to be used.

Cropping Image Files that are at the Same Resolution and Size 1. Open all images you wish to crop to the same size. From these images, determine what would be the largest dimension for cropping. 2. Select the Marquee tool from the toolbox. Outline region of interest from image with largest dimension. If your first attempt at drawing an outline over the region of interest fails to outline correct area, click outside the outline to eliminate “marching ants” (scrolling outline) and start again. Or, under Select choose Transform Selection (not available in 4x). Selection can be reshaped. 3. Save the outline or selection. In Actions window, click on Selection- Save (or, in menu under Select, choose Save Selection). 4. Go to 2nd image and load the selection you saved. In Actions window, click on Selection- Load (or, in menu under Select, choose Load Selection). 5. In Load Selection dialogue box, click drop down arrowhead to choose image from which you saved the selection. On a Mac, choose also Alpha One from Channel. 6. Repeat steps 4 and 5 for remaining images. 7. In Actions window, click on Crop button (or, in menu under Image, select Crop) for all images. Be aware that saving selections results in what are called alpha channels. These will need to be “trashed” before saving file as a TIFF file (or it may not open in other programs). In menu under Window, select Show Channels. Drag alpha channels to the trash icon.

Transferring selections from one image to another Make selection with Marquee tool. Save to new channel.

Load selection from 1st file/ alpha 1 channel.

Selection transfers to 2nd image in same location

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Cropping Images that are the Same Resolution but Different Dimensions Sometimes images are scanned at the same resolution, but are slightly different in size, or the locations of related images have shifted from one scan to the next. In that instance, an outlined area of interest (a selection) can be made on one image file and then dragged to the related image file. The location of the selection can be eyeballed by landmarks on the image itself. 1. Open all images you wish to crop to the same size. From these images, determine what would be the smallest dimension for cropping. 2. Select the marquee tool from the toolbox. Outline region of interest from image with smallest dimension. If your first attempt at drawing an outline over the region of interest fails to outline correct area, click outside the outline to eliminate “marching ants” (scrolling outline) and start again. Or, under Select choose Transform Selection (not available in 4x). Selection can then be reshaped. 3. Crop that image. In Actions window, click on Crop button (or, in menu under Image, select Crop). 4. Select the entire cropped image. In Actions window, click on Select All (or, in menu under Select, choose Select All. Alternatively, you can use Control/Command + a). 5. Click and drag selection outline to the related image. Align by eye (hopefully shared landmarks exist in the images), then crop. Repeat for additional images.

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Cropping Images of Different Sizes and Resolutions to a Specified Size In some instances, you are aware of the final output size, and fitting all images to the same dimensions and resolution guarantees that two or more images side by side will be identical. Again, it is best to shoot for a target resolution that will yield the largest file at the most ideal resolution. Other files can be made from this master file at smaller and smaller resolutions. This method for determining dimensions requires a bit of simple math. If, for example, you are using a U.S. letter page size as your output, and you are printing two images side by side at a half page in the portrait (taller-than-wide) dimension, then the total length will be about eight inches (U.S. letter page size in the shorter dimension is 8.5 inches, but 1/2 inch is generally white margin). Thus, each of the two images will be about four inches wide with a small border between the two. In that instance, you would set your width to slightly less than four inches and the height to whatever fits (one dimension is typically considered more important than the other). Your Resolution is at 300 ppi. 1. Click and hold on Marquee tool to reveal crop tool (4x, 5x) or, in 6x & 7x simply select from toolbar. Double click on it to reveal dialogue box (4x, 5x). 2. Check Fixed Target Size. Type in dimensions for width and height, and set resolution to 300 pixels/inch. Note that one dimension is more important than the other, and the latter may have to be determined by clicking and dragging to make crop outline to see whether it falls short or long (by trial and error). Note that when crop selection is enlarged or reduced, it maintains its aspect ratio (ratio of width to height). 3. Double click inside crop outline to complete crop, or click again on Crop tool. Return to Marquee tool when done.

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Merging Images Merging Images/Colocalization In this instance, merging is considered to be the combination of two or more RGB color images, one on top of the other: an equal percentage of color from different channels. This technique is commonly used in fluorescence to show coexistence or colocalization of two fluorophores labeling similar cell features. Red and green colored labels merge best since the combination of the two produces the easily recognized color of yellow. 1. Close and save all images. In the Actions window, select Merge Red & Grn-Start on Red. 2. You will be prompted to open the first image, which needs to be the redcolored image. 3. You will then be prompted to open the second image, which needs to be the green-colored image. 4. Optional. In some instances, the images can be slightly misaligned because of color shift from one wavelength to another. This action puts your green image on a layer above the red image. Simply hold down the Control/Command key and move green image over red image or use Move tool. While holding down, use arrow keys for fine movements. Manual methods can be found at www.quickphotoshop.com.

Continued on next page >

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Combining >1 image Combining 2 (or more) Images, or Parts of Images, into 1 Making extra canvas (either white space or the color of the background) Each image should already be “cleaned up” via previous steps in this manual. 1. Open top left image file. In Actions window, select Combining- Add Space (Canvas). Or, in menu under Image select Canvas Size. 2. In Canvas dialogue box, choose a unit of measurement and estimate amount of extra space you’ll need to accommodate extra image(s). Give plenty of room, since extra space can be cropped later. 3. In Canvas dialogue box, choose Anchor grid square to give direction for additional space. Click okay. The image will expand beyond the boundaries of its window. You’ll have to zoom out to see what you have done, or pull on corner of image file window to expand its size.

Cutting and Pasting Into First Image File 4. Open image(s) you plan to combine with first one. 5. In Actions window, click on Combining- Cut & Close (or, in menu choose from several functions: under Select choose Select All; under Edit choose Cut; under File choose Close—don’t Save). 6. Now first image should be the active window (or you may have to select the first window, the one to which canvas has been added). 7. In Actions window, click on Combining- Paste (or, in menu under Edit, select Paste). 8. Hold down Control/Command key—cursor turns to 4-way arrow—click and –drag pasted image into position. If, after you click, you also hold down the shift key, the image will move only on the x or y plane (horizontally across or vertically down) 9. Repeat for additional images. Continued on next page >

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Resizing Image, if Necessary Images that are pasted in may need to be enlarged or reduced. 1. In Actions window, click on Combined- Transform button (or, in menu under Edit (Layer in 4x), select Transform then Scale). 2. While holding down Shift key (so that proportions don’t change), place cursor over a corner (to get double arrow). Click and drag until desired size is reached.

Make Final Contrast/Color Adjustments, Crop, and Flatten Layers 1. In Actions, you may need to go back to Contrast- Manual Adjust to match contrast and brightness and color of your images (or, in menu under Image, select Adjust, then Levels). 2. If necessary, select area of interest with crop tool and crop out extra white space. 3. You will want to flatten your image when finished, since each image sits on a separate layer. In Actions, click Flatten Layers (or, in menu under Layer, select Flatten Image).

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Adding White Space For Lettering in Photoshop If you want to complete lettering in Photoshop (as opposed to lettering in a drawing program like Canvas, CorelDraw or Illustrator), you may need to add more white image area above, below, and/or to the sides of your image. It isn’t as easy as simply adding space, however, because the number of pixels across and down (resolution) affects the quality of your lettering (if insufficient pixels exist—usually <700 pixels across or down at half page dimensions—lettering can look pixelated).

Adding White Space Using Canvas Choose unit of measurement from drop down by clicking on arrowhead. Put in values for ample space.

Thus, two steps may need to take place: •

White space needs to be added using Canvas command.

•

Resolution must be made at appropriate size for publication and presentation, especially important when images are downloaded from the web, or when items at <10cm have been scanned incorrectly.

The chosen square on grid defines the position of the image. Extra canvas is added in the direction of arrows.

Adding White Space and Resolution 1. In Actions window, select Combining- Add Space (Canvas). Or, in menu under Image select Canvas Size. 2. In Canvas dialogue box, choose a unit of measurement and estimate amount of extra space you’ll need to accomodate lettering. Give plenty of room, since extra space can be cropped later. 3. In Canvas dialogue box, choose anchor grid square to give direction for additional space. Click Okay. The image will expand beyond the boundaries of its window. You’ll have to zoom out to see what you have done, or pull on corner of image file window to expand its size. 4. In Actions window, select Image Size (or, in menu under Image, select Image Size). Increase resolution to 300 pixels/inch if necessary (Note that original data will be changed. Make sure Resample Image is checked).

As a general rule, pixels should NOT be added when images were obtained using a digital camera or PMT/laser system at a fixed resolution. Otherwise, data is added to the original image.

Move cursor over corner, click and drag to reveal more image. You may also have to zoom out after making canvas larger.

Image Size Dialogue Box Find pixels across and down here (only visible when Resample Image is checked).

Put in resolution of 300 at desired height or width when working with lower resolution images

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Manual Lettering of Images (4x, 5x) Good lettering depends upon the resolution of the image. Because Photoshop must place lettering over so many pixels, a user should expect lettering to look on paper close to how it appears on the screen (at a reasonable zoom: zoomed about as far away as it would be when reading at a reasonable viewing distance). Photoshop puts a ghost image at the edges of lettering to fool the eye. That is called antialiasing (see page 15). Users should be aware of a few features of the Type tool. Each time you type and then click okay, you create another layer. When the Layers window shows a T, that layer can be double-clicked and you can edit the lettering (not in 4x, however). You cannot edit the lettering after the layer has been rendered, which occurs when layers are flattened (5x - 7x). To prevent layer after layer from building from the addition of letters, I find it best to flatten text layers. The small amount of text on

images can be re-lettered rather painlessly at a later time, if editing is necessary. The Text tool dialogue box is rather clunky. Photoshop has improved the text tool in version 6x - 7x by eliminating the dialogue box so that a user can type on the image itself. Photoshop is useful for small amounts of lettering. Extensive lettering should be done in Photoshop’s companion program, Illustrator, or in other page layout programs such as Canvas, CorelDraw, etc. Text from other programs can be cut and pasted into dialogue box, but formatting may be lost.

Type Tool Dialogue Box (5x)

Adding Text in 4x - 5x 1. 2. 3. 4.

Drag guidelines from ruler area for alignment of lettering. Select the T from the toolbar, or type the letter “t.” Click on place where you’d like to letter. Dialogue box appears. Type into this box. Text appears on your image as you type in 5x, but not in 4x. Use Helvetica or Arial text for better readability after images are shrunk in publication. Choose Regular, Bold, Italic, etc., and color (by clicking on the color box or by changing the foreground color at the bottom of the toolbar) as desired. 5. To change size or typeface of text, highlight it (by clicking and dragging over it) and type in new size. These changes can be previewed in 5x, but in 4x the lettering must be placed on the page (by clicking Okay). In 4x, if changes need to be made to the type size, typeface, etc., it is best to simply remove the text layer on which changes need to be made (drag layer into trash icon in the Layers dialogue box), and start again. 6. Move text in place by dragging with cursor while in dialogue box (5x), or afterward by holding down Control/Command key (4x, 5x). 7. Click okay when done. Returns have to be put in manually.

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Choose Arial or Helvetica.

Set Size of Type here. Color is set to the same color as the foreground; or, by double clicking on box, you can set a different color. Lettering must be highlighted before changes can be made. Be sure Fit in Window is checked so that you can view all lettering.

(5x - 7x) Leading is the spacing between lines, Tracking is the width between letters, and Baseline is for setting superscript/subscript. Leave at default for the most part.

Lettering in Photoshop (6x, 7x) Applying lettering in 6x and 7x is not so different than doing so in PowerPoint: Simply click on the Text tool in the toolbar (or type the letter “t”) and then either click at the point where you’d like to place lettering, or click and drag to make a text box. In the latter instance—when making a text box—formatting can be changed by dragging the box to the desired width and length, and the text can be rotated (hold down the shift key to constrain rotation to 15 degree increments). Changes can be made in the typeface, color, size, etc., in the Character Palette (available when

clicking on Palettes below the menu). Text along with their respective layers can be auto-found by simply clicking on existing text when text tool is selected. You cannot, however, continue to click to find more text on other layers unless the check mark or “T” box is clicked first. When first using 6x or 7x (after being accustomed to 4x and 5x), new work habits need to be incorporated. These are indicated below.

Text in 6x, 7x 1. Select T from the toolbar, or type the letter “t.” Click at the desired spot for placing lettering or click and drag to make a text box. 2. Click on Palettes below the menu to reveal the Character Palette (if not already visible). Choose Helvetica or Arial for the typeface, along with attributes such as Regular, Bold, Italic, etc. Choose color by doubleclicking on the color window, or by changing the foreground color at the bottom of the toolbar. Type in text. 3. If changes need to be made, the text need not be highlighted. Instead, simply click on the T in the Layers dialogue box, then make changes to the typeface, color, size, etc. in the Character Palette. Re-positioning text. Before clicking on T in the Layers dialogue box (when starting at a click point only and not within a text box): move cursor away from the text area. The text symbol will change to a move symbol. Click and drag to move. After clicking on T in Layers box: Hold down the Control/Command key or use the Move tool. When finished with applying text: Click on the check mark below the menu on the right (or click on the Marquee tool). Clicking on the X eliminates text. Returns at the ends of lines must be included with “click point” text, but not with text boxes.

Set size of type Choose Arial or Helvetica Choose attributes here

Set paragraph style Indicate color by clicking on color box

Set strength of anti-aliasing (Strong is recommended)

Click to open Character Palette

Click check mark when done; click X to eliminate text.

Character Palette “T” box in Layers

Kerning

Leading Tracking

Click on “T” box before making changes in the Character Palette

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Enhancements to Lettering Outlining Black Lettering in White, or White Lettering in Black 1. Select your text layer(s). In Actions window, click Show Layers then click on text layer (if Layers box doesn’t appear, it is already open). 2. In Actions Window, click on Stroke- Black (Thin), Stroke- Black (Thick), Stroke- White (Thin), or Stroke-White (Thick). This actions will make text into rendered and uneditable text. Or complete several steps for each layer (5x): under Layer, choose Type then Render Text; hold down control/ command key and click on text layer to make selection around text (4x); under Edit, choose Stroke, enter Width of 2 - 8 pixels. Stroke uses the foreground color. For 6x and 7x, in menu under Layer choose Layer Style, then Stroke. Enter Size of 2 - 8 pixels, click on Color: box to choose color. “Stroke” is when a line follows a selection or the edge of letters. 3. In menu under Edit choose Undo (or use Control/Command key + z) if you do not care for effect, and repeat with a thicker or thinner stroke.

Placing a Box Behind Text 1. In Actions window, click on Show Layers button. Select the layer upon which images are placed (should be below text layers). 2. Make a new layer: in Actions window, select Layer- Make New (or, in Layers dialogue box, click on paper icon). 3. Select Marquee tool from toolbox. Click and drag behind text to the size of box desired. If you are working on more than one image, drag guidelines down from ruler area (or turn on grid under View: 5x - 7x only) for alignment purposes. 4. In Actions, select Fill- With White or Fill-With Black (or, in menu under Edit, select Fill and click on arrowhead at top to get drop down list. Choose white, black, foreground or background). 5. Click inside “marching ants” selection and move selection to next position by clicking inside the marquee and dragging (hold down the shift key while dragging to move in horizontal or vertical direction). Repeat step four. If you accidently lose the “marching ants” outline, it can be retrieved by finding that step in the History window (5x - 7x only), or by clicking Undo under Edit.

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Alignment and Orientation of Type Alignment (5x - 7x) Already much has been said about using either guidelines or a grid for aids when aligning text and images. To allow for yet another choice, Photoshop included another method for alignment of several image or text layers in versions 5x - 7x. To take advantage of this method, text and images need to be on their own separate layers versus placing all text or all images into one layer. 1. In Layers dialogue box, select a desired text or image layer. Choose layers to which chosen layer is associated, and place lock symbols by clicking in the boxes to the right of the eye symbol. 2. In menu under Layer choose Align Linked if you wish to align text or images from several layer along an axis. If you wish to make spacing consistent between text or images from several layers, choose Distribute Linked.

Rotating Text 90 Degrees Labeling in research can often mean setting type at an angle. This can be done in Photoshop, but it requires a few steps: 1. Click on T icon on toolbar. On image, click on approximate place where you’d like lettering located. 2. Type lettering in dialogue box (4x, 5x) or on image itself (6x, 7x). 3. In Actions window, click the Transform- Rotate button. Or, in menu under Edit (Layers in 4x), select Transform, then Rotate. If a text box is made in 6x or 7x, move cursoroutside box area and wait until the cursor becomes a rounded arrow symbol. 4. Hold down Shift key, move cursor to corner (arrow becomes rounded), click and drag to rotate. Text will rotate in 15 degree increments. 5. Double-click inside outlined area to complete the rotation, or click on Marquee tool and Apply.

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Rows or Columns of Lettering at 45 Degrees (5x - 7x) Start by making 45 degree line, type text 1. In toolbar, click on Path tool. 2. Draw a line approximately the length of your row of lettering by clicking at the top point, holding down shift key, then clicking at the bottom point at 45 degrees to the right of the top. A line will form that is transparent to printers, like guidelines. 3. Click on T icon in toolbar. On path, click the start point for the first row. 4. Type text/numeric entries in dialogue box (5x) or on the image itself (6x, 7x). Type all entries on the same layer using the space bar to align entries with the path. Use only a single space between text/numeric entries. Rotate and Align Text 5. Double click on Path tool. Paths dialogue box appears. Click arrow at upper right to get drop down list. Select Delete Path (6x. 7x) or Turn off Path (5x). Select Marquee tool! 7. In Actions window, click Transform- Rotate button (also located under Edit in menu). 8. Move cursor to corner (arrow becomes rounded), click and drag to rotate into horizontal position above image area. Use guidelines to aid positioning. 9. Double-click inside outlined area to complete the rotation, or click on the Marquee tool and click Apply. 10. If the spacing between the entries are not great enough, double click on text layer (5x) or use the Character Palette (6x, 7x) to reset the Leading. Either choose from existing values from the drop down list, or type in your own values. 11. Rotate text/numeric entriesagain to align horizontally. Fine tune alignment by clicking at beginning of entry and changing values for kerning. In 4x, it is best to type all lettering/numeric entries with returns and then rotate the entire column 45 degrees. One by one, outline entries with the lasso tool (under the Marquee) and move into place above image area. Line up by eye or by using guidelines.

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Part 4 Finalizing Figures

If you need to put two or more images on a page, you may want to choose a template. Actions to complete templates can be downloaded from www.quickphotoshop.com. These templates fall into two categories: templates for similarly acquired images from a digital camera or PMT/laser system at fixed resolutions and everything else. Everything else. “Everything else” includes what has been scanned, drawn in other programs, or gathered together from different sources. These are images in which several different image resolutions are put onto the same page. In this instance, it behooves the user to choose a page size that matches a target size. While no particular target size fits every circumstance, the most universal size would be one that complies with the needs of publishers AND satisfies the need to make a print or 35mm slide. Thus, you are given the choice of U.S. letter format (8x10.5 inches) and A4 size (210 x 297mm), in both instances allowing for room at the top and bottom for printer margins. In this instance, the resolution is still defined by pixels across and down, but it is more commonly understood in terms of how many pixels are crammed into an inch or centimeter, since the image eventually gets printed to paper. Three hundred pixels per inch is ideal, for it translates well to publisher guidelines, it provides enough pixels so that images look clean and non-pixelated without a loss of detail, and these images look stellar on 35mm slides. For the web, laptop presentations, and e-mail, these images can be compressed so that large files (files that take up a lot of memory and hard disk space) can be made more manageable (see “Saving Files,” page 83).

Digital Imaging Systems. Digital camera systems and PMT/laser systems operate by using a sensor with certain number of photon collection points going across and down, as in 640 x 480. This correlates to the number of pixels across and down in your image. The number is fixed--the system can take no more or no less than the number across and the number down. This defines the resolution of the camera. Every newly manufactured camera improves upon the old by multiplying this number, and new cameras are available just about every month, so the templates currently available cannot possibly cover every camera system made. For updated templates, check www.quickphotoshop.com,or contact the author with the number of sensors across and down for your digital device, and you will be provided with a template for your use. Choose your template by the way in which you acquired your images. Find out how in the pages that follow.

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Auto, Single & Multi-Image Figures (or Plates) Automatic page layout actions have been included with this manual. Once you have completed work on your single images, you are now ready to put all your images together on a layout or figure at a target resolution of 300 pixels per inch. Several options are available using U.S. Letter, A4, and “publication” templates. The templates for a publication page fit most journals. A slight difference in column size can be changed using the Image Size dialogue box under Image to fit any specifica-

tions. Note that only single images are fit to publicationsizes. That’s because of difficulties in estimating heights from various sources. You can use the templates for U.S. Letter and then re-size to “publication” dimensions using Image Size under Image (see page 98). These templates are lettered A, B, C, etc.

From Image Files at Various Sizes 1. Save and Close all images: leave screen empty. 2. In Actions window, choose button from the series of Multi-Image layout (variety) buttons. Choices within these buttons include grayscale or RGB (color) and USA Letter Size or A4 (European, etc.). 3. You will be prompted to open images. The 1st image is the template image, and the following image files will start with the top left image, then either the image to the right or the image below, and so on left to right or up to down.

Some images may look pixelated if image was from web (less than 500 pixels in both dimensions), or if it was scanned at low resolution (say, at 72ppi). You will need to re-acquire the image, or locate the original.

single image 1/4 page

single image 1 column

single image 1/2 page

single image 2 column

single image full page

single image full “publication” page

Continued on next page >

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From Image Files at Fixed Resolutions 1. Save and Close all images: leave screen empty. 2. In Actions window, scroll down to find Fixed Size Layouts. Find pixelsacross-by-pixels-down size that matches the camerasystem you used. 3. You will be prompted to open images. The first image is the template image, and the following image files will start with the top left image, then either the image to the right or the image below, and so on left to right or up to down. All of the following templates assume six images total. If you have less than six images, simply cancel the action mid-stream, and then crop to size (see Crop, page 38).

Some Templates for Image Files at Fixed Resolutions

Guideline dividers will not look evenly spaced even though the spacing is perfect. That is both a screen resolution and a “work-around” bug in the action.

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Auto-Layout Corrections For Lettering Lettering is in the wrong position. Lettering is normally placed in the corner in which the least amount of image information exists. 1. Select the Text Layer in Layers dialogue box (in Actions window, use Show Layers button, then select text layer). 2. While holding down the control/command key, click and drag lettering to re-position. Lettering is Too Large or Small or it Needs to be Re-Done in a Different Way. 1. Select the Text Layer in Layers dialogue box. 2. Drag into Trash icon or click on Trash icon. 3. Type in your own letters using instructions on page 68.

Lettering Needs to be White. 1. In Actions, click on Lettering- Make White (Renders) button. This action renders the text into pixels, which means that the text cannot be edited by double-clicking on its layer in the Layers window (in other words, if you wish to change type later, you will need to trash text layer and start fresh).

Lettering Needs to be Outlined in Contrasting Black or White (to separate letter from its background) See outlining lettering on page 66.

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Changing Position of Lettering Former Position

New Position

Re-Positioning Both Images and Lettering Your image files may not exactly fit into auto-templates when images come from various sources and resolutions (the images fit into their respective positions using a “best fit” function). Your images may be either wider or taller than the height and width of each “panel” these do not conform to what is called the aspect ratio (ratio of width to height). Moving Images Along with Lettering

1. In Actions window, click Show Layers button (or, in menu under Window, select Show Layers) 2. In Actions window, click on Layers- Flatten (or, in Layers dialogue box, select from drop down list) This will place lettering on image so that the lettering becomes part of the image and is no longer editable. 3. Click on Marquee tool on toolbar. 4. Click and drag to outline area you wish to move on the image. 5. Hold down Control/Command key, click and hold mouse button, and hold down shift key to move images with lettering in a straight line. Alternatively, layers can remain separate. First the image can be moved, and then the text layer can be selected and it can be moved. These two layers can also be linked (by selecting the image layer in the Layers dialogue box and clicking grey box next to the text layer to create lock symbol), but both layers don’t always link together when re-positioning.

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Contact Sheet II In Photoshop 5.5 and above you can also make auto-layouts using the Contact Sheet II function (if you have Photoshop 5.0, you can upgrade to 5.5 by visiting the Adobe web site for the upgrade software). This is an automated function that

requires you to place the image files you wish to include in the layout in their own folder. Your screen needs to be empty of images.

1. Under File, select Automate, then Contact Sheet II. 2. Click Choose button and find directory in which your image files are located. 3. Set width, height, and resolution to your specifications 4. Under Thumbnails, set columns and rows. You cannot set the width of the borders, so white space surrounding images will not be consistent. Use technique on previous page to reposition images. You will have to apply lettering on your own.

Contact Sheet II dialogue box

Click Choose to find directory in which your files are located. Set Width, Height and Resolution. You cannot specify width of borders between images. Indicate how many images you wish to place across (Rows) and down (Columns ).

Uncheck this box if checked.

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Manual Layout of Multiple Images to Make a Figure or Plate For ease, you may use templates that are provided, already replete with guidelines for placing images, or you may wish to make your own template. In the latter case, a template is made based either on the premise that images need not be fixed to any

one size (described as “everything else” earlier in this manual on page 69), or on the basis of a fixed size in which magnification often needs to be maintained.

New Image Dialogue Box

Templates Based Upon Images at Non-Fixed Resolutions 1. In Menu under File, select New 2. Set Width and Height in inches or centimeters based upon preferred page size (see table on page 8 for commonly used sizes). Set Resolution to 300 pixels/inch. 3. Set Mode to Grayscale or RGB.

Indicate Width and Height in inches or centimeters. Set Mode to Grayscale or RGB.

Templates Based Upon Fixed-Resolution Images (e.g., from digital camera systems) 1. In toolbox, be sure to make background color white (or the color you prefer for the background color). 2. In Menu under Image, select Canvas Size. You will be adding extra space to your existing image to give yourself more “canvas,” as in a painting. 3. In Canvas Size dialogue box, enter in height and width the appropriate number of pixels to allow for remaining images plus extra pixels for borders. It's best to estimate more than what you need. For example, if your image is 640 x 480 and you wish to have a total of four images in your figure, you'll need 2 x 640—1280—for the width and 2 x 480—960—for the height PLUS room for borders between pictures—20 pixels. Final canvas is 1300 x 980, estimated at 1400 x 1100). 4. Select square of grid from which you’d like extra space to be added.

Canvas Size Dialogue Box

Enter Width and Height in Pixels.

Click on desired box. White space is added in direction of arrows. In this instance, image is on top, left: canvas is added to the right and below image.

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Manually Adding Images to a Page Now that you have made space or canvas for images, you can add images to the template or page. Photoshop allows for images to be dragged from one window to another by selecting the Marquee tool and holding down the Control/Command key (or by selecting Move tool), and clicking on desired image to drag to the page. While that is an appropriate way to move images, the author’s suggestion when two windows are open on your screen is to select all of one image, then to cut and close the image. The image can then be pasted into the other image window. That way, image files don’t clutter the screen to make navigation confusing. An action button is provided to shorten the process. 1. Open image file that will become the top, right image. In Actions, click on SelectAll/Cut/Close, then click on file to which canvas space has been added (your template) and click on Paste. OR... In toolbar, be sure Marquee tool is selected. Hold down Control/ Command key, click and drag desired image onto template, or use Move tool. If images are too large, too small, or at an odd aspect ratio, re-size the image using Transform- Scale button (or, in menu under Edit, select Transform and then Scale). Hold down Shift key (to keep proportions) when pulling on corners to re-scale You may need to eliminate extra image area. 2. When you have two images on a page, roughly line up by eye with what seems to be a good border in between. Place guidelines at the bottom and sides of the images for lining up images and for making borders by dragging guidelines from ruler area at the side of the image (if rulers aren’t visible, in menu under View, select Show Rulers). Hold down Control/ Command key when dragging guidelines. Alternatively, You may prefer using a grid overlay for layouts, thus avoiding the use of guidelines. In menu under View, select Show Grid (4x, 5x); in 6x & 7x under View choose Show then select Grid. 3. Fine adjust your images so that they line up along guidelines by moving them one-by-one until they “magnetize” into place. Make sure Snap to Guides is checked in menu under View. These will be on different layers, and so you will have to select the appropriate layer before moving in the Layers dialogue box.

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Dragging 2nd Image onto Template to Which Canvas Has Been Added 1st image

2nd image is moved and eye-balled into place on template after using Select All/Cut/Close then using Paste to paste onto template.

Drag guidelines to define edges and borders.

Eliminate extra image area by outlining with Marquee tool and pressing delete button (make sure background color in toolbar is white). Zoom in to point at which ruler readings are interpretable (you may have to change units (keyboard command: Control/Command + k, choose Units and Rulers). Determine distance and make new borders for subsequent images at the same distance apart. Zoom back and remove images to align to guidelines, if necessary.

Continued on next page >

Manually Adding Images to a Page, Continued.

4. If you are laying out more than two images, zoom in and use the ruler to determine the width of your border between the first two images. Drag guidelines into place for additional images along with borders, matching the width of your first border. 5. Repeat steps to add additional images.

Add new images.

Crop entire image to remove extraneous image area.

Deleting Extraneous Image Area 1. In toolbar select the Marquee tool. 2. Outline (make selection) over area you wish to eliminate 3. Making sure in toolbar that background is desired color (normally white: click on black/white boxes at bottom of toolbar), press Delete key. Final Image

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Symbols Symbols include the addition of anything other than text to the image, including dashes (or “tick” marks), arrows, asterisks, boxes, circles, etc. More complicated shapes and drawings can be done in Photoshop, but that kind of activity normally takes place in a drawing program (the program that most resembles the drawing protocol in Photoshop is its sister program, Illustrator. Drawings done in Illustrator

can be opened in Photoshop. See page 16). For your convenience, a set of symbols and patterns has been included for downloading at quickphotoshop.com. You can simply drag symbols onto your image from these image files, or make these using manual Photoshop functions.

Symbols from Symbol.psd file 1. Open the image file upon which you wish to put symbols. 2. Open the downloaded symbols file. Zoom in and scroll down to find symbol desired. 3. Each symbol is on a different layer. Open Layers window (in menu, under Window, select Show Layers. If you see Hide Layers, then Layers window is open). Select layer and symbol of interest. 4. Hold down Control/Command key and drag symbol from the downloaded symbols file to your image file. 5. If size is too large or too small, in Actions window, select Transform- scale button (or, in menu under Edit, select Transform then Scale). Place cursor over corner of outline, hold down shift key, click and drag. Some symbols may fit your intention better by NOT holding down shift key and scaling file in one dimension (by width only or height only). You may wish also to rotate the symbol. In Actions window, click on Transform-rotate button (or, in menu under Edit select Transform then Rotate). Make White or in Color 1. White. Select desired layer in Layers window. Click a second time while holding Control/Command key to make selection around feature of interest. 2. Click on Make White (Layer) button in the Actions window. Or, in menu under Edit choose Fill: choose White from the Use: drop down box. 1. Color. Start by double clicking on foreground window on the toolbar. 2. Select a color from dialogue box (or select color from your image itself by clicking on Eyedropper tool and then clicking on desired contrasting color in your image). 3. In Actions window, click on Fill- foreground color button. Or, in menu under Edit choose Fill: choose Foreground Color from the Use: drop down box.

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Symbols and patterns sizes are matched to an image size that is approximately 300ppi. Symbols can be scaled down; patterns, however, may not work as well. Dragging Symbol from Downloaded Symbols File

Symbol is dragged from downloaded symbols file.

In Actions click on TransformScale. Re-scale image to desired size while holding down Shift key. In Actions click on TransformRotate if you wish to rotate symbol. Copy and Paste (under Edit ) to make additional symbols at same orientation and size. To make symbol white select layer symbol lies on and click a second time on that layer while holding down Control/Command key. In Actions window, click on Make- White.

Making Symbols Manually If the symbol you are looking for does not exist in the “symbols” file, you will have to make the symbol manually.

If line or arrow does not appear, your foreground color is the same color as the image area you are drawing on. If that is not the case, make another new layer in Layers window and try again (a “bug” in Photoshop).

Arrows and Dashes 1. Make a new layer in Layers Window. (click on top, right arrowhead to reveal drop down list. Select New Layer or use “page” icon at bottom of dialogue box). 2. In toolbar, set foreground to the color desired for the arrow or dash. Then find the Line tool. 3. Double click on Line tool to reveal dialogue box. 4. Set thickness of line in dialogue box under Weight for both lines and arrows (Undo and repeat, if necessary until correct width of line is ob tained). For arrows, click on Start or End next to Arrowheads. In Arrowheads dialogue box, set the Width, Length, and Concavity (or leave concavity at 0) to make arrowhead longer or shorter. An equal width and length would make an isosceles triangle as an arrowhead. 5. Hold Shift key down, click and drag to draw arrow or dash. Do not hold down Shift key if you desire to draw arrow or line at an angle. 6. Undo and repeat with new values if dissatisfied. 7. Rotate, if necessary, with Transform- Rotate button in Actions window. Or, in menu under Edit (Layers in 4x) choose Transform, then Rotate. Copy and Paste existing arrows and lines to duplicate (remember that each will be on its own layer).

Making Arrows Using the Line Tool In Photoshop 5x, Line tool is beside Pencil tool.

In Photoshop 6x, the Line tool is under the Text tool.

Set thickness of the line by placing value in Weight, usually between 4 and 8. Check Start or End for Arrowheads, then click Shape to set values for arrowhead.

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Boxes and Circles Making Square Outlines Outline area you wish to make box or circle around.

1. Make a new layer in Layers Window. (Click on top, right arrowhead to reveal drop down list. Select New Layer or use “page” icon at bottom of dialogue box). 2. In toolbar, set foreground color to desired color. Choose Marquee tool. Click and hold Marquee tool to reveal circle shape if you desire a circle or ellipse. 3. Make outline (selection) on desired part of image. Hold down Shift key while drawing if you wish to constrain outline to a square or a circle. 4. In menu under Edit, choose Stroke. This function will draw a line along your outline. 5. In Stroke dialogue box, set width to desired size. (2 - 8 is typical). Do not set too small or line may disappear when published or reproduced. 6. Undo and repeat, if necessary until correct width of line is obtained.

In menu under Edit, select Stroke. Set width of line you wish to trace along square or circle. (2 8 is typical).

Keyboard Symbols Symbols such as prime, double prime, degrees, copyright, and so on are best found in fonts. Many of these can be found in the Symbols and Verdana fonts. You can find these symbols in Windows 2000 by clicking on Start, then going to Programs > Accessories > System Tools > Character Map. Other Windows operating systems require that the application (charmap.exe) be installed from your Windows CD using Add/Remove Programs in the Control Panel. On a Macintosh, special characters can be found under the apple icon in Keyfinder or Keycaps.

Photoshop 6x & 7x Symbols A nice addition to 6x and 7x is that of more extensive tools for making shapes. These operate like text insofar as they are vector shapes. These are rather painful to use until figuring out how to get rid of the automatic fill (that can be done by clicking on check mark at far right of menu). Working with the Marquee tool, as described in this section, may be the better option, but you can decide for yourself.

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Stroke follows outline.

Filling Image Areas with Patterns Patterns are normally placed into a shape or into an existing feature in your image file. In order to do that, you must first select a region of interest. This can either be done manually by using the marquee or lasso tool, or, more than likely you can automatically select that region of interest with the wand tool. The wand tool selects an area radiating out from where you click on the feature. The selectable 1. Open any one of several patterns files downloaded from www.quickphotoshop.com. 2. Select all of the pattern file (under Select, choose Select All or Control/ Command + a) and, under Edit, choose Define Pattern. 3. Open your image file. 4. Double-click on Wand tool from toolbar. Leave Tolerance at default of 32. Click on image file feature, such as the interior of a rectangle. If you wish to paste over a complex feature containing multiple color or grayscale values, use the Marquee tool or the Lasso tool to make or alter a selection. If you wish to make several selections, hold down Shift key while making selections on several areas. 5. In Actions window, click on Fill with Pattern (or, in menu under Edit, select Fill, then, in dialogue box, click arrowhead for drop down and use Pattern).

area is “stopped” by some kind of border, in the same way that the edge of a pan stops the addition of water. That border can be jumped over by setting tolerance higher in the wand tool’s dialogue box. You can download “patterns” files from www.quickphotoshop.com/downloads.html.

Wand Tool and Dialogue Box Start with Tolerance at 32. Increase or decrease depending on efficacy of Wand tool to outline desired area of interest.

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Magnification and Scale Bars In the world in which the size of objects is important when presenting data, some means must be employed to provide scale. When a ruler or recognizable object cannot be included in images, a scale bar provides the best way to give a reference to units of measurement. Other instances occur, as in the world of microscopy, when magnification is denoted without the use of scale bars. The rule of thumb is to multiply the lens power by the eyepiece, so that a 10X lens is multiplied by a 10X objective to give a magnification of 100X. This way of determining magnification is incorrect because what is imaged by a digital camera is not always at the same magnification as the objective multiplied by the eyepiece, and magnification is never

1. Make an image of a ruler or calibration grid using your fixed camera system. 2. In menu under Window, select Show Info (if already shown, you will see Hide Info). 3. In Info dialogue box, click on top, left arrowhead to reveal drop down list. Select Palette Options. In Ruler Units, choose Pixels. 4. Use the Marquee or Measure tool (not available in 4x) to stretch from one measurement mark to another. The Info box will show how many pixels equal your ruler units. If you are also using the calibration for subsequent calibration in quantitation software, be sure to measure in both the x and y axis in the event that your camera system does not produce square pixels. 5. Draw a scale bar using the Line tool while holding down shift key and looking at the Info box (see page 27 for location of Line tool). Draw the pixel length which equals the length of the ruler unit. For example, you may draw a line across two measurement marks that equal 10 centimeters. The info box may show that you have drawn across 250 pixels. If you wish to make a scale bar on an image taken under the same conditions, you can draw a 250 pixel line and label it as 10 centimeters.

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exact unless the final publication size is known and the original view of the image can be measured against the size of the published image. As long as some kind of ruler or grid can be imaged using a digital camera system, scale bars can be made. Or a scale is provided with some devices, such as electron microscopy devices. Calibration slides can be obtained in the microscopy world through microscope sales personnel. Once a ruler of some sort exists, the means for making a scale bar is fairly straightforward.

Saving Files It would seem that files could be saved in one format to make everything simpler. To date, the closest format to just one is not the TIFF format, but the Adobe Acrobat format. Adobe Acrobat files (.pdf) have several intrinsic advantages: these files are compressed, so they don't take up so much hard disk space; the fonts are saved in such a way that various computers do not need to have the same font for opening the file; and publishing agencies are quickly moving to use this format for output to publications (these reportedly have the advantage of little color shift). The compression on these files can be so incredible that cutting file sizes by onefourth can result in absolutely no visible change in image data! 1. Photoshop file (.psd). This is the layered image which can be edited later. 2. TIFF (Tagged Information File Format) file (.tif). This file format is currently the most universal and it is normally requested by publishers. It does not contain layers. Photoshop gives the option of LZW compression, but that should not be used because then the file can only be opened in Photoshop and a few other programs. 3. Adobe Acrobat file (.pdf). This file format is now being requested by editorial boards of journals for paperless reviews of submissions. This file format can also be e-mailed, put on a web page, and, increasingly, it can be for the submission of documents, such as grants. This file format is saved with ZIP compression, because it is non-Lossy (no image details are sacrificed). Choosing ZIP compression does not always result in file sizes

The reality is, however, more sobering. At this printing, it is best to save files not only in several different formats, but also in more than one mode (ideally in CMYK as well as RGB). Because hard disk space is currently cheap, and because CD recordable disks and the concomitant devices are also inexpensive, the suffering level for having to save in so many ways is at least minimized on the economic level. It is recommended to organize files in such a manner that each file has its own folder: One folder is for Original files, a second for Photoshop files, a third for TIFF files, a fourth for CMYK files and a fifth for PDF and JPEG files. Thus, action buttons are included to save as follows: that are significantly smaller. Files can also be saved with JPEG compression at several settings. Remember that some image information may be compromised. Check for yourself against the original image. 4. JPEG (Joint Photography Experts Group) file. This file format is ideally suited for web publication and for PowerPoint. Various levels of compression and image quality loss are selectable by the user. 5. TIFF or EPS (Encapsulated PostScript) CMYK (Cyan, Magenta, Yellow, Black) files. This mode is necessary for submission of color photos to publications. Its range of colors (colors it can print) is far less than the visible range that we can see by eye and the range we can see on our computer screen.

Saving by Using Action Buttons In Actions, choose buttons for several ways to save (Save as Photoshop, TIFF, JPEG button is recommended): Save as Photoshop, TIFF, JPEG (this saves in all current formats for several outputs, starting with Photoshop so that layering is preserved for subsequent editing). Other buttons include, Save as- Photoshop; Save as- TIFF, Save as- CMYK TIFF, or Save as-CMYK EPS; Save as- JPEG; and Save as- PDF (Adobe Acrobat file). You choose the compression for JPEG files: suggested compression is medium to maximum.

Note: If you wish to save manually, remember that only the Photoshop format contains layers. To save in other formats, layers need to be flattened (in drop down list in Actions dialogue box, select Flatten Layers. Also found under the Layers Menu in 5x - 7x). If you have opened a PDF, Illustrator file, or from some other formats, you will need to flatten layers though you may have made none (Photoshop, frustratingly, introduces invisible layers in these file formats).

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Prints and 35mm Slides From Digital Files The most frequent mistake made when working with images concerns settings that have to do with output resolution, or how many pixels per inch/centimeter. Output resolution values may be changed or left alone, depending on the printer. If you used templates provided with this manual, your sizes are set to half- or full-page, regardless of the printing device (except in the instance of photographic printers,

like the Fuji Pictrography in which output resolutions must be matched to the printer’s resolutions when desiring matched dimensions). When making 35mm slides from image files made in Photoshop (rather than from PowerPoint), the target output resolution of 300 pixels per inch typically provides plenty of pixels.

Overview of Printers Setting dimensions and resolution by the kind of printer. Printers fall into five broad categories: laser printers (most of which are postscript printers); ink jet printers and their larger version: poster printers; and pre-press printers. A fourth category defines slide writers—those devices which expose digital files to 35mm film—and a fifth includes photographic printers. Laser printers. These printers do very well with drawing or vector files. Images, however, suffer from being re-formed into so many dots. Sometimes, as with high resolution printers (defined by dots per inch), images do not suffer much at all in terms of clarity. In any case, the detail in the shadow areas and in the whites usually is not preserved and blacks aren’t deep. Photoshop typically does not print well to laser printers. Lettering can look fuzzy when it is re-formed into dots. These printers do not print well with large files, so reduce the size or make JPEGs so that the printer does not “hang up.” Unless printers require CMYK, you can print RGB files to these printers. Ink jet printers. These printers have shown the most promise as versatile, high resolution printers with good tonal range. These can also print sharp lettering, depending on the printer and choice of paper. Their drawback is in how slowly prints are made. It is a good idea to experiment with printer settings to obtain the kind of output that matches your computer screen. For example, on a PC, the Epson® printer makes the best images from Photoshop when using the Advanced Setting, and then choosing ICM (Internal Calibration of the Monitor). Choose paper stock carefully to obtain the best reproduction. Paper stock should be thick and somewhat shiny (coated) for best results. Large files can be printed to these printers with a fairly good tonal range. Some colors, however, do not have good range at all, such as shades of red. As time goes

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on and more ink colors are added to ink jet cartridges (the higher end Epsons now have 6+ colors), reproduction of all colors should improve.

RGB files can be printed to ink jets. Many of these use drivers that either convert files to CMYK transparently, or require CMYK files for output. In either case, it is best to make CMYK files yourself so that you can predict the outcome. Pre-press printers. Several companies make CMYK printers. These include dye-sublimation printers, wax transfer, and a plethora of ink-based systems. The dye-sublimation prints, such as those made by Codontics, require CMYK files. These printers are meant to give you a preview of how you should expect these to appear in publication, but don’t expect exact color matching unless you have gone through a great deal of work calibrating colors. These printers also anticipate that you have set both resolution and dimensions before printing: 300 pixels per inch at your desired dimensions (or you may be able to choose the option Fit to Page). Slide writers. Unlike Pre-press printers, these devices anticipate that files are RGB, not CMYK! Lettering, graphs and drawings are transparently made into image files, using a software process known as rasterizing or RIPping (Rastor Image Processing). See page 18 for more information on RIPping. In this scenario, lettering will print as it appears on the computer screen. Maintaining a high resolution of the file is only important insofar as lettering does not become pixelated. Resolution that is too high (higher than 400 pixels per inch at page size) creates resolution that exceeds the device: empty resolution. Files that are higher than 15 megabytes exceed the resolution of film, and thus do not provide any more information than slides can resolve.

Photographic printers. These include the Fuji Pictography and Durst® printers, both of which are RGB printers. These print to continuous tone photographic paper using lasers for exposure. The print quality typically exceeds what can be obtained from other printers. Resolution is set to match printer resolutions, and then dimensions are set.

These printers typically take output from Photoshop, and not vector programs, unless RIPping software is also purchased (results are not always expected: some files lose information after rasterizing. See other methods on page 16). Expect resolution, detail, color and contrast range to be extraordinary on these devices.

Features of Various Printing Devices

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Setting Resolution and Dimensions For Output When desiring that your image prints to an exact dimension—as opposed to making a print that simply fills the page (by checking the Fit to Page option in the printer’s dialogue box)—you will need to reset dimensions and, possibly, the resolution setting, using the Image Size dialogue box. How this box is set depends on the output device. See below for instructions on the output device you use.

Great care can be taken to make sure no pixels are added or subtracted from images when using this dialogue box by unchecking Resample Image. If images are enlarged or reduced when printing to those printers which allow users to “fit to page,” the printer software will add or subtract pixels from the image and pixels will be added or, less often, subtracted, in printer software.

For Pre-press printers, and Photographic Printers Laser Printers and Ink Jets 1. Set Height and Width to desired dimensions. 2. Set Resolution (this is really the output resolution) to 300ppi or to resolution settings of printer. In the latter instance, match to printer’s resolution so that file size remains about the same (check with manual that came with the printer). 3. Make sure Resample Image is checked.

Type in desired Height and Width Uncheck Resample Image

Laser Printers and Ink Jets 1. Type in desired Height and Width dimensions. Ignore Resolution values. 2. Uncheck Resample Image

Pre-Press Printers, Photographic Printers

For Publication 1. Set Height and Width to desired dimensions. Normally these sizes are indicated in the publisher’s guidelines. If unsure about the ultimate dimensions, make image two times the likely size of reproduction. Set Resolution as follows: 300ppi Images Images & Text 600ppi Text/Drawings 1200ppi If scanned images or text started as low resolution files (<800 or so pixels), these will need to be re-scanned.

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Set Height and Width to desired size. Set Resolution (this is really the output resolution) to 300ppi or to resolution settings of printer. Make sure Resample Image is checked

Resolution Values from Digital Systems and Scanned Films Files that come from digital systems, including digital cameras, typically show up at a resolution of 72 - 96 dpi at huge width and height dimensions. IN THAT INSTANCE, DO NOT BE MISLED BY THE RESOLUTION! Remember, the word “resolution” in this box refers to output resolution. The inherent or real resolution of the image is simply its measurement in pixels across and down (the Width and Height settings at the top of the box). Simply reset Resolution values to what is desired while leaving the Resample Image box unchecked. If the dimensions are not right after changing values for Resolution, and you wish to change dimensions, check the Resample Image box and type in desired dimensions (you will add or subtract pixels from the original image).

For example, if Resolution shows a value of 72 ppi, but the width and height dimensions show 24 by 24 inches, simply uncheck Resample Image and change the resolution value to 300. The dimensions will change to a width and height of 5.76. If you wish to print your image at a “full page” size, place a check next to Resample Image and type 8 inches for width or 10.5 inches for height (the largest print possible on an 8.5 X 11 inch sheet, allowing for one-quarter inch on each side for a border). In like manner, files that were acquired with a slide scanner show a high output resolution value (e.g., 1200 ppi) and small height and width dimensions (e.g., 1.3 inches in width). Use procedure above to set values.

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Working With Color CMYK Files (for Publication and Pre-Press Printers) From dynamic to dull. Perhaps the most frustrating process in the imaging world is to go from RGB files—those files that look good on a computer screen—to CMYK—those image files that immediately lose pizazz to become lifeless and drab. Worse, contrast can decrease. And then, if these shifts weren’t bad enough, colors can shift once more from what you see on your computer screen to what is seen on the screen at the publishing agency and then to what is published.

cathode ray tubes age and lose capacity) are recommended by many print shops. The printing and graphic arts industry was built upon technologies developed by Apple.

Saturated colors. In the former instance, the greatest contribution to this problem is the world of research itself: so many colors in research are saturated. They are completely green, completely red, completely blue, and so on. Research colors tend to be more primary versus colors in the real world which are more pastel and less concentrated. That phenomenon is especially true when coming from the world of Silicon Graphics® computers and their screens, in which deep and saturated colors display richly, but the colors translate to no other output.

Other solutions. Having said that, the next best solution is to make your best print from your RGB file (if possible), or make your best print on a CMYK printer. Send the print with the digital file and ask the publisher to match the colors. When viewing your own print, you might consider buying a viewing booth, or setting aside an area you can light with 5500 degree Kelvin fluorescent lights (available at professional photography retailers or graphic arts suppliers). These lights provide a “standard” color temperature for evaluating prints. If prints are evaluated under other lighting conditions, colors can shift to green (most fluorescent lighting), to red or yellow (under incandescent lighting), to blue (in shadows near window light), or a mixture of two or all three.

From your monitor to publication. In the latter instance—regarding computer screens and how poorly one a computer screen matches the output at the publisher—the best advice is to use a high end hardware calibrated monitor (such as the Barco® line of monitors). On the more affordable level, Macintosh® computers with Apple’s® self-calibrating computer screens (these monitors adjust color as the

Adjusting colors. You can adjust colors that have faded or lost contrast after changing from RGB to CMYK. Three methods are suggested, depending on the degree to which colors are “out of gamut.” In all situations, color changes are made using Hue & Saturation. Remember that each image has its own peculiarities, and the RGB to CMYK process requires trial, error and experimentation.

Check To See How Much Color Shifts If colors are unacceptable, start with the following steps: 1. Under View, select New View. A second image will appear. Arrange image windows so that both, or important parts of both, can be viewed (image can also be duplicated). 2. Under View, select Preview then CMYK. Evaluate whether significant color shifts have occurred. 3. If colors look okay, select one of the two images. Under Image, select Mode then CMYK.

1. Close New View image window. 2. Duplicate RGB image (under Image, select Duplicate). 3. Convert duplicate image to CMYK (under Image, select Mode then CMYK). Flatten the image. Align so that important parts of both images are visible. In general, if the color shift is small, work with the CMYK image In general, if the color shift is great, work with the RGB image.

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Method 1. Color and Contrast Restoration on CMYK Images with Small Color Shifts 1. Select CMYK image. 2. In Actions window, click on Color- hue & saturation (or, under Image, select Adjust then Hue & Saturation). Work on colors one by one. 3. In dialogue box, click drop down box next to Edit. Choose a color that has shifted. Or... Use Eyedropper tool to select offending color. 4. Slide Saturation slider to the right to increase saturation, to the left to decrease. You may also want to lighten or darken with the Lightness slider.

These tools, by themselves, may bring color into acceptable range. If these do not solve the problem with color shift, restore color and contrast with Method 2.

Remember that blues and purple-blues will print as dark blobs. Move Hue slider to left to add green.

Hue & Saturation Dialogue Box Most problems with CMYK have to do with saturation of hue. Move slider to right to brighten, to left to affect contrast. Subtle adjustments in Hue and Lightness may also help.

Click on arrowhead to select color, or use Eyedropper tool.

You can edit your selection further by using the Eyedropper tool to click on the features in your image that contain the colors you’re interested in changing. Click on the first feature with the unmarked Eyedropper tool, and then on additional features with the plus (+) Eyedropper tool. If you accidently choose an unwanted color, use the minus (-) Eyedropper tool to click on the same point, or start over again. Note: When using Eyedropper tool, be sure to select numerous points, including bright to dark values within each hue. Or... expand or contract range of colors by adjusting sliders (available when a color is selected).

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Method 2. Color and Contrast Restoration on RGB Images with Large or Problematic Color Shifts 1. Select RGB image. Duplicate RGB image and align on screen so that important areas are visible. 2. Under View, select Gamut Warning (or, better yet, use Control+Shift key + y). 3. In Actions window, click on Color- Hue & Saturation (or, under Image, select Adjust then Hue & Saturation). Work on colors one by one. Select colors by using eyedropper tools as described in the previous method. Keep gamut warning overlay on, especially when selecting bright objects against a dark background (as in darkfield microscopy). Be sure to use plus (+) Eyedropper tool to select to edges of objects, much easier seen when gamut warning is active. 4. Adjust the Saturation slider first. Keep in mind that all colors will print if saturation is set low enough (to the left). Adjust slider until a fair amount of the gray overlay disappears. 5. Adjust Lightness slider by increasing (moving to right) shy of eliminating too much contrast. Now increase Saturation as much as possible (move slider toward center) without increasing gray overlay. The idea is to work between the two, lightening as much as possible without losing contrast, and keeping saturation as close to the center as possible. Don’t be surprised if the color you are working on needs dramatic desaturation. 6. Adjust Hue carefully to determine if a shift in one direction or the other might increase chances of eliminating gray overlay. Play with all three to get close to your desired color and contrast. You may need to turn gamut warning off and on. That is best done with the keyboard command while the Hue & Saturation dialogue box is open (Control/Command + Shift + y). You need not remove all the gray overlay, just 90 percent or so. 7. Make image CMYK: in menu under Image choose Mode, then CMYK Color.

Use the gamut warning indicator to decide which colors need to be altered. The gray overlay dissapears when colors are adjusted correctly. That may not happen with the saturatedcolors used in research. Expect to remove 90 - 95% of gray overlay for any hue: the remaining unbalanced color will be at edges of bright or dark samples. These small areas of unbalanced color are imperceivable when printed.

Use Gamut Warning to Preview Out-of Gamut Colors Everything covered in gray represents what will not transfer well to CMYK. Gray overlay shows edges where too dark to be seen by eye. After clicking on the first point with the Eyedropper tool in the Hue & Saturation dialogue box, be sure to use the plus (+) Eyedropper tool to click on multiple points, including dark peripheries. Image on left is how RGB image appears as uncorrected CMYK; on right is the corrected CMYK image in green hue only. Desaturation using the Saturation slider eliminates most of the gray overlay.

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CMYK Correction for Blue Shades of blue appear bright on a computer display, but tend to print dark and without contrast, especially after being changed to CMYK mode. This means for correcting blue tends to print close to what is seen on the screen, but, for exact matches of hue, you’ll need to use the method on the following page.

Example of RGB Image (on left) Corrected for CMYK in the Blue Hue (on right)

1. Move the Hue slider to the left to add green (make more cyan). Move to about -30. 2. Adjust Saturation and Lightness by eye. Evaluate the color after printing to a hard copy, as opposed to evaluating by looking at the display on the computer screen. 3. When satisfied with the result, in menu under Image choose Mode, then CMYK Color. Only in certain colors, such as cyan-blue, does an increase in saturation help.

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Steps to Follow When Color Matching is Near-Impossible If Method 1 or Method 2 do not give you good color matching from RGB to CMYK color space, then use the steps that follow. These use Pantone Inks® as color guides. Because colors contain a range of hues from dark to light, and because only one particular ink color is chosen, color changes may have to take place in 1. In menu under Window, select Show Color. Click on arrowhead at top right and select CMYK Sliders. Leave this window open. 2. Click on representative area of image with eyedropper tool containing the color you’d like to match. 3. Double click on foreground image at bottom of toolbar icon. In Color Picker dialogue box, click on Custom. Click Book drop down list and choose Pantone Coated (journals are typically printed on what’s called coated stock: the shiny paper found in magazines). 4. Choose ink color that best represents color you’d like to match. 5. Write down the CMYK values.

stages. In that manner, a bright green may be selected using Color Range, and then a mid-green, and so on. These steps require trial and error, and fiddling is a requirement.

Show Color Window Click on arrowhead to select CMYK Color.

Continued on next page >

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Color Range Dialogue Box Set to Sampled Colors. Make Selections Around Desired Colors Using Color Range

6. In Actions window, click on Color Range button (or in menu under Select, choose Color Range). 7. In dialogue box, use eyedropper tools to select color you wish to work on. Use the leftmost eyedropper tool to select the first color, then use the plus (+) Eyedropper tool to add more shades of the same color. Set Fuzziness high (50 -100). For bright objects against a dark background, keep gamut warning overlay on to be sure you are selecting to the edge of your objects. You may wish to hide the moving outline (selection) while making changes. To do so, press Command/Control key + h. It’s a lot easier to see your corrections, but the downside is that you can forget that you’ve hid the selection later.

Fuzziness should be set 50-100 or so.

Check Selection.

White areas indicate what has been selected.

Use leftmost Eyedropper tool to click on first point in desired color. Use plus (+) Eyedropper to select additional shades of the same color.

Continued on next page >

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Adjust Colors Using Curves

8. In Actions window, click on Contrast Adjust- Curves (or in menu under Image, select Adjust then Curves). 9. Click on representative color in image with Eyedropper tool (identical or close to the position chosen when determining ink color). 10. By clicking and pulling line in Curves dialogue box to the left across the top, or down along the right, adjust Red and/or Green and/or Blue Channels individually while looking at the Show Color Dialogue box to match CMYK values to those you have written down (see table on the following page). Turn View Gamut off and on using Command/Control + Shift + y keys to test the efficacy of your changes. If the hue doesn’t look right, use Curves line across and down to fine tune by eye while ignoring CMYK values in Show Color. For example, if representative value reads 65% Cyan, 0% Magenta, 100% Yellow and 0% K (black) on the closest match of a Pantone ink value, adjust Red channel and Blue channel to achieve a similar reading. Values will change on the Color Palette as adjustments are made in the Curves dialogue box. Readjust by eye. Do not expect to see a perfect match of hues! Some colors are outside the gamut (breadth of color available) of what can be printed by a printing press. Do expect, however, to improve upon the RGB image with a slightly different hue.

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Final Steps

11. Once satisfied with adjustments while in RGB color space, change Mode to CMYK Color: in menu under Image choose Mode, then CMYK Color. Flatten image if prompted to do so (in menu under Layers choose Flatten Image). Be sure to have saved the RGB image before doing so. 12. For final adjustments and fine tuning, use Hue & Saturation or Curves to adjust colors and contrast.

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Reviving Contrast in the CMYK File Once you’ve made the CMYK file using methods outlined in previous pages, and once you have corrected the color in CMYK color space so that it matches the RGB hues fairly well, the image may yet be devoid of contrast Often contrast can be revived by simply moving the bottom of the line in the Curves dialogue box to the

right, thus introducing more black in the darker parts of the image. Less frequently, more dramatic steps must be taken. These concern ways in which contrast can be introduced into ranges of tones, whether these are shadow areas, mid-tones or highlights.

Reviving Contrast In Highlights Using Curves Overall Contrast

Original RGB Image

CMYK image after color corrections

1. Use Color Range to select areas of interest according to procedures oultined earlier. 2. In Curves dialogue box, click Channel drop down box. Select Black. Drag line across bottom to increase black values (and contrast).

Adjustments Within Tonal Ranges (Shadows, Midtones, Highlights) 1. Use Color Range to select areas of interest according to procedures oultined earlier. 2. In Curves dialogue box, determine where values of interest lie along line. To do that, click and hold with eyedropper tool on areas of interest. A circle should appear along the line. 3. Make 3 points at that location along the line. Bend line so that hills and valleys create shadows and highlights within narrow color range.

Application of curves to green areas. Extra points added to bend line.

Green and purple after Curves adjustments

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Working with PowerPoint To Make JPEG Images for Importing into PowerPoint PowerPoint generally requires small file sizes, best accomplished by making files into the JPEG (Joint Photographic Experts Group) format. This format compresses the file using what is called Lossy Compression. That means some image information is inevitably lost with this format. The amount depends

upon how much compression is desired. When set at maximum, the amount of loss is typically imperceivable by eye; at low, image deterioration is best seen at edges of elements in your image or in an overall fuzziness.

1. In Actions, choose Save As JPEG (if making JPEGs manually, duplicate image and then make sure layers are flattened. In the Layers menu drop down box, select Flatten Layers. Under File, select Save As. Choose JPEG). Early versions of PowerPoint on Macintosh computers do not take JPEG files. Make PICT files instead.

To Correct for Darkened Images in PowerPoint Images tend to become more contrasty and darker in Powerpoint. You can remedy this problem one of two ways: If you are printing to 35mm slides, you can ask your service agency change the gamma (typically the gamma is increased). Software made to write digital files to 35mm slides often provides that. You simply need a willing technician who will experiment until an ideal gamma is found for your images. Otherwise, increase brightness and reduce black levels in Photoshop before importing images to Powerpoint (use Levels, page 43).

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Setting Dimensions of Images to Match PowerPoint PowerPoint defaults to an On-Screen aspect ratio when creating new presentations. That aspect ratio fits the height to width of a computer screen, assuming you wish to make a “live” presentation from your laptop. The 35mm aspect ratio must be chosen under File/Page Setup when desiring to make PowerPoint presentations to 35mm slides. That aspect ratio fits the width and height of a 35mm slide, more a rectangle than a square. Both aspects are given inch dimensions (in the American versions) without resolution settings. PowerPoint acts like a laser or ink jet printer insofar as the resolution is not important: only the dimensions of the image. 1. Set the dimensions of the image using Image Size dialogue box (in menu under Image, select Image Size). For side-by-side images, re-size by calculating how much space you’ll need given the dimensions below while allowing for room at the top for titles and at the sides (allow extra room at sides when making 35mm slides to allow for space taken by the slide mount). The cropping steps can also be used (see page 38). Be sure to uncheck Resample Image. Fit both dimensions or either the width or height to desired aspect ratio: 11.25 X 7.5 inches (landscape) 35mm Slides On-Screen 10 X 7.5 inches (landscape)

PowerPoint Files to Photoshop Early versions of PowerPoint saved to low resolution, pixelatedfiles. The latest versions allow for exporting to TIFF at higher resolution. The latter should be chosen when desiring to open a PowerPoint file in Photoshop. If that option does not exist on your version, see page 15 to determine how to get high resolution files into Photoshop.

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Making White-on-Blue or Yellow-on-Blue Slides to Match PowerPoint History. The typical colors used in PowerPointscientific presentations comprise a graduated blue background with yellow lettering. The use of these colors came out of the period of time before widespread use of computer graphics. At that time, when color was desired, a diazo was made. That process involved exposing black on clear high contrast negatives (Kodaliths) onto blueprint film. The film was then developed by placing it in a jar with a concentrated solution of Ammonium Hydroxide. The result was a deep blue-purple background with white lettering. These were typically mounted on 35mm glass slides. Diazos made sharp edges and projected better than any other color combination even in rooms that were not completely dark. The disadvantage was that diazos faded over time.

Gradients. Those colors have been borrowed for modern computer graphics, with the exception that a complementary color (yellow) comprises the lettering. The ability to place a darker blue-black at the top of the slide graduating to a lighter blue causes the viewer to look at the area of highest contrast (the top of the slide where the title exists) and then to naturally move down.

To Make White or Yellow Drawings, Graphs & Text:

1. Open or select drawing, text or graph file you wish to paste on desired blue image file. In Actions, click on any of the following buttons: White on Blue 35mm, Yellow on Blue 35mm, White on Blue for On-Screen, Yellow on Blue for On-Screen. These actions auto-select what is black and paste it over the background. Make sure these are high contrast (see page 44). 2. If the image is cut off at the top or bottom, re-size. In Actions, choose Resize or Transform. (Or, under Edit select Transform, then Scale). Hold down Shift key while dragging the corners of the outline to re-size the image.

Slides in Photoshop. In that spirit, the following procedures will create White-onBlue or Yellow-on-Blue graphics for 35mm slide format or for on-screen presentations (at 1024 x 768 pixels) in the landscape dimension. These are meant for blackon-white graphs and drawings with text. You can try to select only the black parts of the image using Color Range (page 93), copy and then paste into PowerPoint, but the results are typically unsatisfactory (either the text is inadequately selected, or the white background is included with the selection). If you wish to place images versus text or graphs on a blue, graduated background, choose the action buttons in the first step below. In any case, remember that the shade of blue from the template/actions will not be an precise match to the dark blue color of PowerPoint. The manual method for making slides to match PowerPoint can be found at www.quickphotoshop.com.

Yellow Lettering on Blue Background from a Black on White Illustration

Use appropriate Actions buttons to auto-create yellow-on-blue image from a white-on-black image.

To Make a “PowerPoint” Type Background:

1. Make a solid blue or blue gradient image file by clicking the Blue Gradient 35mm Slide, Blue Without Gradient 35mm Slide, Blue Gradient On-Screen, and Blue Without Gradient On-Screen buttons in the Actions dialogue box.

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Color Shifts with Grayscale Photographs The digital revolution has left us primarily with color digital printers. These tend to shift in color, and mostly toward blue, in contrast to photographic prints from the darkroom, which often shift toward a brown color. This shift in color is not to be regarded as trivial. The overall color can affect our perception of resolution and detail. Once, as an experiment, I made several slides with different color casts, 1. Make a typical print in the darkroom using the photographic paper of choice. 2. Scan negative from which typical print was made or choose similar negative. 3. Make print without any color adjustments to your digital printer. 4. Compare digital print with print made from darkroom and determine which way the gray color has shifted. 5. Change mode of grayscale image to RGB color (or CMYK Color if you have a CMYK printer). Make several copies of the same image on a single page (see page 74). 6. On the menu under Image choose Adjust, then select Curves (Control/ Command + m). Click on the Channel arrow to get drop down of Red, Green or Blue channels. 7. Change color of each channel in degrees, by 5-10 points, in the direction of the color you hope to achieve. For example, if the color of an RGB image has shifted to blue on the digital printer, and you hope to achieve a browner tone, you will want to decrease blue, therefore adding yellow and increase red (to get brown, which is a combination of red and yellow). The 1st print could be -10 yellow, the 2nd -10 yellow +5 red, the 3rd -10 yellow and +10 red, and so on. This is called a ring-around print. You may need to do more than one ring-around print to arrive at a satisfactory result. 8. Evaluate prints using typical lighting in the room. Even better, evaluate prints under 5500 degree kelvin lighting (available as fluorescent tubes at professional photographic and graphic arts supply stores). 9. Save the result by clicking on Save in the Curves dialogue box and apply to future images (using Load). This may not be foolproof, since factory runs of digital print paper may shift differently. This procedure may have to be repeated after receiving new shipments of paper, but the shift should be small.

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going from a greenish-gray to a bluish-gray. Nothing else was changed but the color. A radiologist to whom these were shown told me that the bluish-gray images were best because these were highest in resolution. The caveat is that the color shifts inherent to grayscale images printed on a color printer should be seamlessly maintained from what was printed in the darkroom to what is now done on a digital printer. Here’s a method:

Matching Grayscale Colors to Photographic Prints

Photographic Print

Select Channel of interest Click arrowhead so that dark gradient is on the left bottom.

Digital RGB Print

Slide top of Curves line to the left or down to increase or decrease channel’s color. Record Input and Output Values; type these on ring around print. Determine which Curves correction works best. Click on Save to save the Curves correction and apply to subsequent images using Load.

Organizing & Archiving Files Archiving. As of this printing, many experts agree that film is yet the cheapest and safest way to archive material. Of course, that also depends on how the film is stored (in moist conditions mold can form) and in how well the image is fixed to the film (if fresh fixer is not used when processing the film, it can yellow and fade after 20 - 30 years). If images begin as digital files, then it is wise to use current technology and update as technology changes. At this time, that would mean burning files to CD-ROM (do not use smelly inks to write on the CD itself) and, in the next couple years, transferring those files to DVD-ROM. When the next technology arrives, transfer again.

Organizing. Photoshop provides two ways to organize files: as hard copy on paper and electronically. On paper, what are called contact sheets can be made. These are pages of small pictures (called “thumbnails”) along with their file names. Electronically, you can have images printed as JPEGs (compressed images) to the web as both a one-page contact sheet for low resolution, and as high resolution by clicking on the file name. In this scenario, your files can be reviewed via a web browser, such as Netscape or Microsoft Explorer. These functions are not available with versions earlier than Photoshop 5x.

Contact Sheets

1. Under File, select Automate, then Contact Sheet II 2. Choose the directory from which you wish to make contact sheets. The dialogue box gives several options for page size, resolution, and number of images across and down. Size of lettering, unfortunately, cannot be made small enough for long file names, except in 7x. 3. Click Okay. This function will continue processing images and making new images until it finishes with all files in the directory you chose.

Example of Image Files made into a Contact Sheet

Electronic Images in Web Browser

1. Under File, select Automate, then Web Photo Gallery. 2. Choose a Source (the directory from which you wish to have image files made to a slide show) and choose a Destination (where web file is placed). 3. You can include a Site Name, Photographer and Date. These are placed in a banner above your images. 4. Unless you have specific sizes you wish to make, leave Thumbnails and Re-size Gallery Images on default settings (where they are). 5. Click Okay. After all files are processed, your default web program will open and display the file. 6. Rename file to better describe nature of image files, and leave the extension .htm or .html (Photoshop will only make a file named Index.htm. If a file with that name is uploaded to your website, it will become your home page. Renaming the index.htm file will solve that problem). Because Photoshop only makes a web page named “index.htm,” any new image files must be included with existing files when making additions.

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Summary of Steps

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Index Symbols 12-bit 38 16-bit 39 3200 degrees kelvin 9 35mm slides 84 3D objects 7 5500 degrees kelvin 9 72 dots per inch 15 8-bit 39. See also Bit-Depth

A A4 70 A4 size 69 Accumulate 14 Actions 30 Adding color 51 to grayscale images 51 Adding white space. See Lettering Alignment 67 Anti-aliased lettering. See Lettering Anti-aliasing 64 Archiving 101 Arrows. See Symbols Artifact 40 Auto-balance 43 Automate 74 Autorads 8

B Binary 2 Binning 12 Bit-depth 11 Bitmap 2, 15 bitmap 47 Black level 5, 12 Blank field 10 Box under text 66 Boxes. See Symbols

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Brightfield 9 Brightness 5, 12, 42

C Calibrating monitor. See Monitors Calibration tool 22 Canvas 61 anchor 61 CD 20 Character palette 65 Circles. See Symbols CMYK 2, 83. See also Mode color/contrast restoration 89 reviving contrast 96 CMYK color 88 color shifts 88 correcting blue 91 difficult corrections 92 large corrections 90 Colocalization 60 Color picking 92 Color range 46, 92 fuzziness 46, 92 Color shifts. See CMYK color Color temperature 9 Color wheel 13 Combining images 61 two or more into one 61 Complementary filtering 13 Computer screens. See Monitors Contact sheet 74 Contrast 5, 12, 42 Coomasie blue gels 8 Correcting color 43 changing certain colors 50 changing the color blue 50 manual corrections 45 selected areas 46 subtle colors 49

whites 46 Correcting contrast 43 manual correction 44 Corrupted files 33 Crop 38 different dimensions 58 fixed target size 59 several to same size 57 Crop tool 59 Cropping white space 38 CRT (Cathode Ray Tube) 21 Cursor icons 26 Curves 95 adjusting colors 94

D Darkfield 9, 47 Dashes. See Symbols De-interlace 13 Defragmenting 21 Descreen 5, 7 Digital camera 9 Dimension 53 Disk space 21 Display card. See Video card Dots per inch 5 Duplicate 33 Dust. See Dust and scratches Dust and scratches 40

E Ektachrome 6 Electron microscopy films 8 EPS 83 Ethics 19 Export options 15 Eyedropper tool 89

Index F

H

FDA approval 20 Figures 70. See also Templates File formats Acrobat (pdf) 16 BMP 15 EPS 16 GIF 33 JPEG 33 PICT 15, 33 PostScript 16 RAW 33 TIFF 15, 33 Film scanner 6 Flatbed scanner filter 2 scanning from books 7 Flatfield image 10 Flatten layers 83 Flip 35 Fluorescence 47 Frame average 12 Frame-average 9 Fuji Pictrography 18 Fujichrome 6

Histograms 44 History 36 Hue 42 Hue & Saturation 45, 50 Hue & saturation 89

I IDE controller 21 Illumination, Kohler 10 Illumination, uneven 9 Illustrator 16, 18 Image files 17 Image size 53 resample image. See Image size Image type CMYK 2 Image types. See Film scanner Image window 26 Indexed color. See also Mode Ink jet printer 18 Ink jet printers 18 Integration 14

J

G

JPEG 83

Gamma 5, 12, 23, 42 monitor's gamma 24 Gamut 18 Gamut warning 90 Glare 13 Grayscale. See Mode. See also Mode color shifts 100 Guidelines 32

K Keyboard symbols 80 Kodak Gold 6

L Laserjet printer 18 Layers 55 adjustment layers 55 flatten 56

merge visible 56 render text 56 LCD (Liquid Crystal Displays) 22 Lettering 45 degree rotation 68 add white space 63 alignment and orientation 67 anti-aliased lettering 15 corrections for auto-layout 72 manual (4x, 5x) 64 Manual (6x, 7x) 65 outlining 66 rotating 67 Levels 44 channel 45 Levels dialogue box 43 output levels 44 Line tool 29, 79 Lines disappear 7 Lines per inch 5 LUT (Look Up Table) overlay 10, 12

M Magnification 82 Marquee tool 32 Matching gray levels 48 Median filter 5 Memory setting for Macintosh 25 Menu 26 Merging images 60 Microscope slides 7 Microsoft Word 17 Mode 39 Moire patterns. See also Laserjet printer Monitors 22 self-calibrating 22

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Index N Navigating 37 Nitrocellulose 8 Noise 14 Noise, dark noise 12 Normal viewing distance 7

O Open 33 Open As 33 Open as 33 Optical Character Recognition (OCR) 2 Optical resolution. See Resolution Organizing 101 Orientation 67 Outlining. See Stroke Output levels. See Levels Output resolution 53. See also Resolution Output size 3 Over-sampled. See Sampling Over-saturation. See Saturation

P Pagemaker 18 Palette 26 Patterns 81 pdf 83 Phosphors 23 Photo printer 18 Photons 69 Pixelated Image 15 Pixelated lettering 7 Pixels measuring 11, 48 Plates 70. See also Templates Polarizing 13 Postscript interpreter 17

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PowerPoint 17, 53, 97 correct for darkness 97 make yellow on blue 99 making JPEGs 97 setting dimensions 98 Pre-press printers 86 Preferences 28 Print to file 16 Printers 84 features 85 inkjet 84, 86 laser 84, 86 poster 84 pre-press 84, 86 slide writers 84 Prints 84 Processor speed 21 Profile. See also Monitors Profiles 28 Pseudocolor 51 Publication 86

Q Quark 18

R RAM (random access memory) 21 Reducing graininess 48 Refresh rate. See Video card Resolution 6, 15, 17 adding 63 by file size 6 for output 86 from digital systems 87 setting by file size 3 setting for images 53 setting resolution 3 RGB 83

RGB color. See Flatbed Scanners; Mode RIP (Raster Image Processed) 18 Rotate 35 Rotation Arbitrary Rotation 34 Rubber stamp tool 40 Ruler units 82 Rulers. See Preferences

S Saturation 42 over-saturation 11 Scale bars 82 Scaling 3 Scratch disk space. See Disk space Scratch disks 29. See Preferences Scratches. See Dust and scratches SCSI controller 21 Shading image 10 Sharpening 5, 41. See also Flatbed scanners Show color 93 Stroke 66 Summary 102 Symbols 78 manually making 79

T Templates 69, 71 manual assembly 75 Text arial 64 helvetica 64 The Image Processing Handbook 5 TIFF 83 Toolbar 27 Transform 35 Troubleshooting 29 True color. See Flatbed Scanners

Index Type tool 64

U Undo 36 Unsharp mask 41 USA letter size 70

V Variations 48 Vector 17 Video card 21, 22

W White balance. See Digital camera White Level 42 Word Processing Programs 15 Working With Color CMYK Files (for Publication and 88 Working with PowerPoint 97 35mm slide aspect ratio 97 on-screen aspect ratio 97 PowerPoint files to Photoshop 99 rasterizing 18 setting dimensions of images to fit PowerPoint 97 to make JPEG images 97

Z Zoom Tool 37 moving image while zoomed in (navigating) 37 zooming in & out 37

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