quick photoshop for research a guide to digital imaging for photoshop 4x 5x 6x 7x

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

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

Jerry SedgewickDepartment of Neuroscience

University of MinnesotaMinneapolis, Minnesota

KLUWER ACADEMIC PUBLISHERSNEW YORK, BOSTON, DORDRECHT, LONDON, MOSCOW

eBook ISBN: 0-306-47938-9Print ISBN: 0-306-47375-5

©2002 Kluwer Academic PublishersNew York, Boston, Dordrecht, London, Moscow

Print ©2002 Kluwer Academic/Plenum Publishers

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: http://kluweronline.comand Kluwer's eBookstore at: http://ebooks.kluweronline.com

New York

PrefaceQuick Photoshop for Research: A Guide to Digital Imaging contains essential information for the use ofPhotoshop specific to researchers. This step-by-step guide is the only book published for users whose needs arenot for the purpose of graphic or web design: instead, this book only addresses the tools and functions necessaryfor 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 theimaging process to its end. Additional information about scanning and acquiring images via a digital camera orlaser/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 lifesciences, though it can be applied to forensics, astronomy and engineering. It can also be distributed from coreimaging facilities from within universities and corporations for those who use microscopes, scanners, confocalsystems, and other imaging devices. Actions can be downloaded from quickphotoshop.com to automatefunctions covered in this book.

Part 1 of the book covers what users should know about imaging, ethics, computer requirements, differencesbetween vector and imaging programs, setting up preferences in Photoshop, and calibrating computer screensbefore 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 addingcolor. Part 3 addresses what functions are used when working with several images to make a plate. You'll learnabout layering, cropping several images to the same dimensions, combining images, adding white space forlettering, using the text tool, and changing the orientation of text. Part 4 covers the lay-out of multi-image figuresor plates; the addition of symbols such as boxes, arrows, and circles; how to fill with patterns; the construction ofscale bars; what file formats need to be saved; how to set resolution for output; the steps for converting RGBfiles 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 mostpopular and widely-used imaging program. It speaks to what researchers know intrinsically in a world that hasbecome far more visual: images often convey more than words; and, like it or not, images leave an impression inrecipients' minds, often making the difference between a grant that receives more points rather than less, orbetween a paper that is accepted in a prestigious journal versus one that is less desirous. Like it or not, imagescan make or break careers.

AcknowledgmentsThanks 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.

ContentsPart 1 Before Getting Started In Photoshop

Flatbed ScannersTypical Choices When ScanningImage TypeResolution/Output Size (or Scaling)Resolution: Setting by File SizeFilter OptionsContrast, Gamma, Brightness

Film ScannersScanning TipsDigital Camera Systems

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

Making Non-pixelated, High Resolution Images fromGraphing, Drawing & Word Processing Programs

Differences Between a Drawing Program (VectorGraphics) and a Paint Program (Image Files)

Ethics and PhotoshopComputer Requirements for Running PhotoshopComputer Screens (Monitors)

Calibrating Your Monitor

Setting Memory for Photoshop on a MacintoshVisual DefinitionsOverview of ToolbarSetting up The Photoshop WorkspaceTroubleshootingLoad Actions in Photoshop

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

Dust and ScratchesSharpeningContrast & Color: A PrimerContrast/Color Auto Actions

Correcting Color and Contrast for Typical Images (generallyfrom scanned negatives, slides, or digital cameras)

If Auto-Balance Doesn't Auto-determine Hues Correctly andWhite Areas Exist in Image File

Contrast/Color Manual AdjustmentsManual Adjustment of ContrastManual Adjustment of ColorCorrecting Color Shift in Whites & Selected Areas

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Part 2 Quick Photoshop: Steps for Single ImagesMarquee Tool and GuidelinesOpen Image/Duplicate

Open Image File -- Standard Image FilesWhen File Does Not AppearOnce your File is OpenDuplicate

Rotate/FlipFor Small Degrees of RotationFor Interactive and High Degree of Rotation (useful forrotating parts of an image)

Undo & HistoryZooming In & Out

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

Crop

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

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 BackgroundGray Levels

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

Reducing Graininess of Films

Manual Color Controls/Subtle colorsVariations (for subtle modifications of color and contrast)

Color: Changing Certain ColorsHue/SaturationChanging the Color Blue

Adding ColorAdding Color to Grayscale Images (Darkfield)Adding Pseudocolor to Grayscale images (for visualization

purposes)Minimizing Grayscale/Color Values (for visualization purposes)

Subsequent Steps for Single ImageFinal Resolution and Dimensions for Single Images

Part 3 Combining More Than One Image andAdding LetteringLayersLayers: Actions & FlatteningCropping Several Images to the Same Dimensions

Cropping Image Files that are at the Same Resolution and SizeCropping Images That are the Same Resolution, but DifferentSizes

Cropping Images of Different Sizes and Resolutions toa Specified Size

Merging ImagesCombining >1 Image

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

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

Outlining Black Lettering in White, or White Lettering in Black(for lettering against "noisy" backgrounds)

Placing a Box Behind Text

Alignment and Orientation of TypeRows or Columns of Lettering at 45 degreesRotate and Align Text

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Part 4 Finalizing FiguresAuto, Single & Multi-Image Figures (or Plates)

From Image Files at Various SizesFrom Image Files at Fixed ResolutionsAuto-Layout Corrections For LetteringRe-positioning Both Images and LetteringContact Sheet II

Manual Layout of Multiple Images to Make a Figureor PlateTemplates Based Upon Images at Non-fixed ResolutionsTemplates Based Upon Fixed-resolution Images (e.g., fromdigital camera systems)

Manually Adding Images to a PageDeleting Extraneous Image Area

SymbolsSymbols from Symbol.psd file

Adding Symbols ManuallyArrows and DashesBoxes and CirclesKeyboard SymbolsPhotoshop 6.0 Symbols

Filling Image Areas with PatternsMagnification and Scale BarsSaving FilesPrints and 35mm Slides From Digital Files

Overview of Printers

Setting Resolution and Dimensions For OutputFor Pre-press printers, and Photographic PrintersLaser Printers and Ink JetsFor PublicationResolution Values from Digital Systems and Scanned Films

Working With Color CMYK Files (for Publicationand Pre-Press Printers)Check To See How Much Color ShiftsMethod 1. Color and Contrast Restoration on CMYK Imageswith Small Color Shifts

Method 2. Color and Contrast Restoration on RGB Imageswith Large or Problematic Color Shifts

CMYK Correction for BlueSteps to Follow When Color Matching is Near-ImpossibleReviving Contrast in the CMYK file

Working with PowerPointTo make JPEG images for Importing into PowerPointTo Correct for Darkened Images in PowerPointSetting Dimensions of Images to Match PowerPointPowerPoint Files to PhotoshopMaking White-on-Blue or Yellow-on-Blue Slides to

Match PowerPoint

Color Shifts with Grayscale PhotographsOrganizing & Archiving FilesSummary of StepsIndex

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Part 1

Before Getting Started In PhotoshopWhile an extraordinary number of image problems can be solved by usingPhotoshop, the frustration of correcting these problems can be far more time-consuming than avoiding the problems in the first place. This is even more truewhen considering that only a handful of Photoshop experts exist to do the job rightin 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 aboutacquiring images than you are about enhancing those images in Photoshop. Thissection should be as carefully read as any of the Step-by-Step sections that follow.

1

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 grayscalegraduations, especially in pastel versus primary colors. Also, be awarethat the advertised resolution of a scanner is normally twice the trueoptical resolution. The advertised resolution is interpolated (pixelsare duplicated to double resolution).

Image Type

Color materials. Set at True Color or RGB color (whether 16.8 million, 32-bit, 24-bit, etc.) for scanning color materials.

Photographic type materials (with a range of grays). Set at Grayscale. If a choiceof “bit-depth” is shown (8-bit, 10-bit, 12-bit, and so on), use default setting or 8-bitunless 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 onceprinted 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. Youshould let Photoshop convert RGB to Indexed Color after scanning yourdocument. CMYK records color intended only for printing presses and isused primarily by graphic designers. Your computer screen shows colors asa 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 avoidhaving your choice changed by automation, either change the automaticimage type option in a preferences or options menu that may have comewith your software; or, after previewing and outlining your area of inter-est — and before your final scan — select the image type.

2

Resolution/Output Size (or Scaling)

“Resolution confusion” defines most situations in the digital world. In the photo-graphic/microscopy fields, resolution is thought of as the ability to discriminateclosely set details in space, and, in the past, films with different resolutions werechosen depending on how finely resolved the image needed to be. It means aboutthe same thing in the digital world, but that ability depends on how many pixelscomprise your image. The caveat would seem to be, "the more the better," but thereare times when the number of pixels chosen EXCEEDS either the resolution of whatis being digitized (when you scan a newspaper photo, for example), or the outputdevice to which these images will eventually be printed. Publishers normally printimages at resolutions no higher than 133 lines per inch (but 300 pixels per inch isrequired on the original image), where text and line drawings are printed at 1200 dotsper inch (dpi). Thus, scans at resolutions higher than that are pointless. Even 35mmfilm is super-pixelated (or what is called over-sampled) when it is scanned atresolutions higher than 1500 pixels per inch for digital duplication.

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

inch (used synonymously with dots per inch). One usually need not consider dotsor pixels per inch until printing to the output device, but resolution at the beginningof the process is thought of in terms of what comes at the end: outputting for aprinting device. In the end, the real resolution of an image is not pixels or dots perinch, but simply a measurement of pixels across and down. When resolution isexpressed as so many dots or pixels per inch, the reference is to OUTPUT resolu-tion.

For the sake of making resolution choices easier, the highest possible resolutionbecomes your starting point: the resolution required by a publisher. Resolutions formaking 35mm slides, on-screen slide presentations, digital prints, web images, andimages to be sent to reviewers all can either remain at the same resolution, or bemade into images with less pixels across and down (the latter comprises typicalchoices for on-screen presentations and web images). Choose from the followingtables when you set resolutions on a scanner.

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: 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)EM negatives 800-1200 dots per inch (dpi) 3.3 inches wide(8.5cm)/6.85incheswide(17.4cm)Text and Black/White Drawings (bitmap) 1200 dots per inch (dpi) 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 “filesize” method on the following page. Also use the “file size” method for a lessdetailed 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 12point type). Print out on an ink jet before re-scanning to make a judgement aboutquality, or scan at 600 dpi as grayscale. Whites can be whitened and blacksblackened using contrast adjustments in Photoshop (see Page 48), and the imagewill contain smooth edges.

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Resolution: Setting by File Size

The number of pixels that comprise an image (the more pixels, the higher theresolution) 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 fornearly 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 sizeof > 1200 pixels is adequate for most scanned images without text. These files can

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

Setting Resolution According to the “File Size” Approach:

Image Type

Color Scans without textColor Scans with textGrayscale without textGrayscale with textBinary

File Size

6-8 megabytes~10 megabytes2-3 megabytes4-6 megabytesIgnore 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 atthese resolutions can be made much larger in file size and dimensions withoutlosing detail. If only a part of a scanned image is used, as in an EM negative, butthe part has not yet been determined, scan at a higher file size.

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Filter Options

Often scanner software includes several options for filtering your image. The mostcommon include descreen (often median filtering) and sharpening functions.

Sharpen. Sharpen functions work far better in Photoshop, so it is best to turn offany sharpening in the scanning software.

Descreen (or median). This filter reduces or nearly eliminates the dot pattern inpictures from magazines and journals. These dots are typically at a density of 133dots per inch(dpi), also called lines per inch (1pi). Newspapers are less dense at 80dpi. Scanner software can do a great job of nearly eliminating the dot pattern tosolutions 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 blureverything else). The best descreen functions use a fourier transform in theirprocess. More information about fourier space can be found in “The ImageProcessing Handbook” by John Russ, or in the software manual that accompaniesthe “Image Processing Toolkit,” (a software program that provides Photoshopplug-ins) by Chris Russ (members.aol.com/ImagProcTK/).

However, most descreen functions use a median filter, so be aware that descreenmay not always give you the results you want.

Contrast, Gamma, Brightness

Most scans turn out looking like the original when contrast, brightness and gammasettings are set to automatic. However, in situations where contrast is too broad oruninterpretable by the software, you’ll have to set it manually. What’s mostimportant is to make sure detail is visible in the blackest and whitest parts of yourimage. The best way to do that is through adjusting the gamma, which is a way toaffect mostly the midtones, though these changes can also affect the blackest andwhitest 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, exceptthat film scanners allow for a far greater selection of image types, and the methodfor setting resolution is different.

Image types. Film scanners allow for making a selection of image type on the basisof the kind of film used. For the most part, slide film is Ektachrome or Fujichromeand color negative film, as of this printing, is typically Kodak Gold. Genericsettings that may be entitled “raw negative” and “raw slid e” exist if you areunaware of the film type.

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 re-lettered, or you can sharpen in Photoshop (but don’t expect great results).

Setting Film Scanner Resolution by File Size

Color scansGrayscaleFull resolution

6 - 8 megabytes3-4 megabytes15 megabytes for entire image

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Scanning Tips

Moire patterns. When scanning pictures from books and magazines, often you willsee a moire pattern on the computer monitor, which looks a lot like two screens thatare positioned at cross axis. This is NOT a resolution problem, it is simply thephenomenon of the dot pattern that makes up the computer screen crossed againstthe 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 isbecause the laserjet also screens another dot pattern over your image to print. Theeasiest way to get around this is to print out on an inkjet.

Disappearing lines. Also, when images are zoomed out, horizontal lines candisappear. Always zoom in to check to see that everything was scanned to yoursatisfaction.

Pictures and text from books and journals. Whenever scanning from books andjournals, you need to place a black piece of paper behind the page you are scanningto 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 maywant to Descreen to eliminate dot pattern if it is a picture (drawings usually don'tget 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 ofthe 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 aflatbed scanner, useful up to about 2x - 4x. You will need to scan at ~1800ppi orbetter at true optical resolution. Certain slide scanners can be adapted to scanmicroscope slides. If you can adapt, these scanners give even more resolution, withthe advantage of perfectly even illumination in background

Pixelated lettering. Lettering that is zoomed up too far will look pixelated. Zoomout to a normal viewing distance to evaluate the quality of your scan; remember thathorizontal lines may disappear. Print out results to evaluate on an ink jet printer, ifyou wish to be sure.

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Coomasie blue gels. These gels can be scanned dry or wet (as long as care is takento 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 sheetof 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 avoidthe accumulation of more bubbles due to the heat from the scanner.

Autorads. Autorads, or x-ray films, are fairly straightforward. Scanner software canread 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, andthen 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 timidabout de-focusing bands to eliminate grain.

Electron microscopy films. Beware of Moisture rings if placing on glass. Do notpress on glass. I haven’t found a good way to prevent these rings from occuringwhen in contact with glass, except to raise the film slightly off the glass using stripsof tape.

Nitrocellulose. Any material is easier to scan than nitrocellulose. The stain is so dimthat every last wrinkle in the paper must be brought out to see bands. Place thenitrocellulose blot on a piece of darkened glass and raise the glass off the surfacewith coins or spacers and then raise contrast and lower brightness. Note thatnitrocellulose is notorious for moisture rings (see above).

Newer scanners typically do not use glass for large format films.

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Scanning of Gels

Moisture Rings

Arrangement of gel andglass on scanner

Moisture ring on EM Film

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 proto-cols. However, white balancing can often be done manually, as well as avoidingover 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 towhat is white, and images take on an overall cast of color. Thus, many digital andvideo systems provide—either in the camera (using an internal auto-white balancefeature) or in the software—the white balancing option.

Off-Microscope white balancing. Take an image of a white card before takingimages of your samples. Be sure that the card is appropriately lit in a position inwhich the card reflects the “average” amount of light.

How to white balance on microscope. When the camera is on a microscope, micro-scope slides need to be removed so that the camera can read off the light comingfrom the light source. Once these cameras are white balanced, they read colorscorrectly until the camera is turned off or the light level is turned up or down (thuschanging the color temperature of the light).

If you can't white balance, set color temperature. If your digital camera does nothave the option for white balancing (or if you use film), then you need to set yourlight either to daylight or to tungsten lighting. (On a microscope, markings shouldexist to guide you regarding where to set the slider control for the light, typically aPHOTO indication). If no markings exist, then the light should be turned all the wayup (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 ofcolored light will exist in the background. The DIC prisms do what prismsdo: separate light into its color components.

White balancing is done with brightfield images (dark objects on a whitebackground) NOT with darkfield, such as fluorescence (bright objects ona black background). With darkfield, not only do pure hues exist, makingcolor interpretation unmistakeable; but the lamps and lasers do not pro-vide 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 degreeskelvin. 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 towardblue. If you think of an iron pot being heated by a fire, a cooler pot would beyellow, where a much hotter pot would be bluish. It’s precisely that concep-tion which gave rise to this way of thinking about and referring to light.

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

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Correct for Uneven Illumination

More than anything else, correcting for uneven illumination is critical for goodimaging. It is especially important with low magnification imaging: the world inwhich 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 mannerdescribed on previous page. When using a microscope, take a picture of the lightsource with microscope slide removed from the image area (be sure sample undercoverslip was in focus before doing so). Take a picture of that, making sure no partof the image is oversaturated (too white). If your camera adjusts brightnessautomatically based upon the brightest point, oversaturation witll not occur. If,however, the digital system is not automatic, manual controls will have to be set sothat a slight gray can be observed at the brightest area. Many systems allow for aLUT overlay to show oversaturated pixels (see page 12 for more info).

Auto-correction for uneven illumination. With many digital systems, the option oftaking what is called a "blank field," a "flatfield," or a "shading" image can beintroduced into an automated process so that uneven illumination is auto-correctedwhile acquiring subsequent images: look for this possibilty. In acquisition software,the flatfield image can be incorporated into an automated imaging operation inwhich the flatfield image is subtracted from subsequent images.

More ways to correct for uneven illumination. Software solutions can only solveminimal problems with uneven illumination. Frosted glass, clouded plexiglass, or afrosted cut out from transparent film can be put in the light path to even the lightfrom edge to edge to avoid spotlight effects or any other illumination problems. Ifuneven illumination still exists, you can purchase a gradient-type filter available atprofessional camera stores. Gradients come in different styles. Choose the gradientthat fits your lighting defect and place in the light path (effective only at a fixedmagnification).

Manual shading correction: take blank field image for subtraction later. If nooption exists internally in the camera or in software, then take a picture of a flatfieldimage. 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 website www.quickphotoshop.com.

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

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Avoid Over-Saturation

Avoid saturating white- and black-end of images (especially important with confocalimages). Avoid increasing brightness, or lowering black level so that image detailsare lost. Nothing is more unsightly than blobs of gleaming, flat, fluorescent-stainedcells on a puddle of pure black.

Rule of Thumb: In Photoshop you can always INCREASE contrast (makeblacks blacker and whites whiter), but you cannot create detail where itnever existed (where pixels are over-saturated).

Choose highest bit-depth available. Your camera system may include a higher bit-depth than the standard 8-bit-per-channel. Choose a higher bit-depth for greaterresolution of gray values and somewhat more ability to make out details in theshadow and bright areas. Your software should also have the option of saving as16-bit. Photoshop can only open 8- and 16-bit image files.

Human eyesight doesn’t see all grays. More detail exists than we are ableto 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'sbetter to keep detail in blacks — in case you need it — than it is to throw itaway by oversaturating.

Pixels can be measured. By placing the cursor over representative pixels onyour computer screen, pixel values often display at bottom of the screen inyour acquisition sofware. In Photoshop, the values are indicated in the InfoBox. In menu under Windows, select Show Info (see page 48) A pixel value of0 indicates over-saturation in the blacks; a pixel value of 255 (8-bit), 4096 (12-bit) and 65,536 (16-bit) indicates over-saturation on the white end. It may beopposite on a Macintosh.

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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 pseudocoloroverlay 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 theblack region, and red where the image is over-saturated in the white region. Byadjusting both the white level (alternately called brightness) and the black level(alternately called contrast), you can bring pixels “in range.” By providing thisoverlay, our limited vision, and the variances of how the computer screen is set interms of brightness, no longer play a part in determining where to set the black andwhite levels (this is especially important when subsequent intensity or densitymeasurements will be made).

Look for pseudocolor overlays, often called LUTs, in your image acquisitionprogram.

Change gamma when whites/blacks are out of range. Some instances occur inwhich whites are so intense in parts of your image, and dark values so dark, thatyour only option for capturing both within a single image is to change the gammavalue of the camera (not always possible on a CCD camera). Gamma settings“bend” the midtone grays, typically increasing the brightness of the darkest pixelsin your image. If a gamma setting does not exist, take two separate pictures: oneunderexposed to capture the light part of the image, and the other overexposed tocapture the dark parts. Recombine in Photoshop or Image J (seewww.quickphotoshop.com).

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

LUT Overlay

Image or Frame Average: Darkfield (Fluorescence)

The camera chip can become hot while acquiring, and it produces what is calleddark 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 averagethem together. A frame average amount of 8 to 16 is a good bet. The effectivenessof frame averaging is exponential. A frame average of 2 increases signal to noise bya factor of 1. An average of 4 increases signal to noise by a factor of 2, 8 increasess/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 (seewww.quickphotoshop.com/imaging_corrections.pdf).

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Other causes for noise. Some cameras allow for the option of setting electronicgain. 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 theCCD chip are grouped together so that, say, four sensors are used to collect onepixel (versus one sensor per pixel). In that manner, CCD chips become significantlymore 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 beplaced in the light path, or in front of a lens. The most useful way to acheivedarkening of a color is to choose the filter that is its opposite, or complement. Thedegree of darkening depends the intensity of the hue. Thus, a blue filter willincrease darkness and contrast in its complement (yellow or brown). Conversely, itwill decrease contrast and lighten any color of blue. If you wish to make certainstains stand out to be measured, or if you wish to diminish non-specific hues, filterscan often accomplish that end. These are best used with camera systems yieldinggrayscale images.

De-interlace (for video). Video frames occur at 30 frames per second. To acheive 30frames most efficiently, essentially only 1/2 the image is collected per frame. Sincevideo is read out in horizontal lines, each frame can include every other horizontalline 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-interlacingoptions, to "fill in" the missing horizontal lines. If it isn't available, Photoshop canbe 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, includingthose that are 3-dimensional.

• If possible, submerge in a bath of water.

• Polarize the light. Purchase polarizing filters for BOTH the light sourcesAND a rotatable filter for your imaging system. Place filter in front of yourlight source on its own stand. If more than one light source exists, setpolarizing filters at the same axis (axis should be marked). Then turn filterin front of camera lens while viewing through camera. Glare shoulddisappear 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 exception-ally dim signal. Accumulate adds one frame to another when acquiring an image.Thus, brightness builds as images are acquired. Electronic noise also increases, butthis can be used with frame averaging to improve signal to noise.

Integration (a cheaper alternative to buying a cooled camera: cooling is to reducedark current noise as a result of heated sensors on the camera's chip). Another wayto increase signal from dim samples is to “integrate on the chip.” This is really nodifferent than increasing shutter speed on a 35mm camera. Integrating allows thechip to collect photons over longer periods of time, but it, too, increases noise andrequires 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 theoption for making high resolution image or text & drawing files. With the exceptionof a few notable programs like CorelDraw, Canvas, Illustrator, and the newestversion 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 whitedrawing), or PICT can only be done at 72 dpi. The problem with a resolution thatspreads 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 andpixelated.

• No provision is made for creating text with a ghost image behind it forconfusing the eye (what is called anti-aliased lettering).

Only programs that allow you to save or export files at higher than 72dpi, and alsoallow you to check "anti-aliased lettering," will produce high enough resolution.Images saved as JPEG can look good, but these are compressed files, and someimage information is thrown away (judge for yourself).

Please note that the indication of an output resolution of 72 dpi does notalways indicate low resolution of the image itself. If the source of theimage 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 columnColor 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%) Method Three (Mostly works: postscript fileswork intermittently)

Print the File/Scan Result1. 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. Roughedges will disappear,

Save as Illustrator File, EPS (EncapsulatedPostScript), PostScript, or Adobe Acrobat1. Look for some way to either Save, Export, or

print the file to an Adobe Illustrator file,available on some software programs. Betteryet, buy the full version of Adobe Acrobat*and print to an Acrobat file (PC), or drag tointo the Acrobat printer icon (Macintosh).These files open in Photoshop. Be sure to setresolution/size accorting to chart (p. 18) ifprompted in Photoshop.

2. If no option exists, then open your printdialogue box by printing your file to a laserprinter (postscript printer) or, alternatively,to Laser Writer 8 (on a Mac).

3. Search for an option to Print to File versusprinting to the printer.

4. You may have to name the file and selectfrom postscript choices. If available, checkEPS. If not, go with default settings.

5. Click ‘OK.’ Postscript file will be generated.Open Postscript file in Photoshop** and setparameters for dimensions and resolution(300 dpi). Photoshop does not always openfile. CorelDraw is far more universallyaccepting.

* See quickphotoshop.com/making_tiffs.pdf formore information on Adobe Acrobat.

* * You may only obtain lettering without imagefiles.

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

Choose postscriptprinter.

Check Write or Printto file and EPS (ifpossible).

Rename file with .eps or .ps(for postscript file).

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 MicrosoftWord, you have been using what are called vector programs in programmer’sparlance. These programs are made with the intent of going to a laser printer, or atleast with the intent of going through a postscript interpreter. The postscriptinterpreter, in the simplest terms, provides a way for the computer to talk with theprinter and to assign it font descriptions.

It also interprets so that the contents of your document can be scaled up or down insize without a loss in detail.

Image files, on the other hand, are intended for photographic printers, ink jets, and35mm slides. These are comprised of pixels, and the number of pixels is fixed by thefile’s resolution. Some differences are listed below:

VECTOR FILES IMAGE FILES

Vector is meant to be printed to a laser printer.

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

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

Vector files use fonts and lines as guides for printing.

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

Vector files typically can't handle large images.

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

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

Image files are meant for photo printers or slide writers.

Image files print fonts and lines exactly as they appear on the screen (at ~100% to120% magnification).

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

Image files are pixels only.

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

Image files are intended to handle large images.

Photoshop does.

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 animage comes closest to coexisting in Photoshop 6x and 7x. Most the time, eitheryou have representations of letters and lines NOT meant to look good on yourscreen, but meant to print out well on a laser printer (vector), or it is made up ofpixels (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 beprinted on photo printers or slide writers. When image files are printed to laserprinters, results are unpredictable and not representative. Conversely, vector filesusually cannot be printed as images unless RIPped (Raster Image Processed: madefrom vector to image—or raster—files). The best solution is had with ink jetprinters, though these are slower than laser printers. Ink jets do a good job ofinterpreting text, lines and images. Remember, however, that these printers dependupon the quality of the paper to which files are printed, and these devices cannotprint 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.

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

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

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Ethics and Photoshop

It only stands to reason that the sorts of enhancements that could have been madein the photographic darkroom for the last 100 years or so of research should also bechanges that can be made in Photoshop. The difference is that, in the formerinstance, a negative (and less often a transparency, or a positive) was used whenmaking 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 assomething “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 35mmslide, a photographic print, published material, etc.) by definition it becomes a“negative.” The digital image becomes an intermediate image—just like a filmnegative—for a hard copy.

Thus, it is entirely appropriate to view the original digital image as a negative. Andit is also entirely appropriate to print that negative differently to the various kinds ofhard copy so that the final product looks very much like what was once seen by theresearcher. Obviously, what is “seen” by the researcher can be subject to his or herdesire to over-enhance, but those desires had also been satisfied at one time in thedarkroom.

But instances also exist in which what had been “seen” simply cannot be inter-preted by the hard copy because of inherent limitations in the technology. A case inpoint is the way fluorescent dyes appear on hard copy. Often these dyes arecompared to one another to view side-by-side experimental conditions, and one orthe others are compared in terms of brightness. It turns out that our eyes, thecomputer screen, and photographic printers often pick up shades of green betterthan red or blue. If the experimental condition shows that green, red, and blue areequally bright, then a straightforward, non-enhanced print would yield falselyprinted results. It would seem only appropriate to show red and blue as equallybright on hard copy. Unfortunately, the only way to do that is to intensify red whenpost processing the image and to change the hue of blue more to cyan; or, con-versely, to dim the green.

In the end, how an image is shown and what that tells those who look at that imagereally lies in the hands of the researcher, and it depends entirely on his or herreputation and integrity. The integrity can be found out because research—at leastin academics—to some degree is self-monitoring. If experiments done in one labcannot 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 seenby the researcher.”

It is important to note in this discussion that it is a moot point whether images arecorrected 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 largernumber of people.

It’s the latter instance that creates so much fear, and it provides the nuclear bombargument: 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 makedata fit the hypothesis. Photoshop is an electronic darkroom in the hands of all butresearchers desperate to fabricate results. While they may be able to do that inPhotoshop, the likelihood of doing it well enough to fool reviewers is rare. Fewlearn Photoshop well enough to create convincing visual data (and this manualwon’t tell you how), probably only as many as had formerly been able to in thedarkroom.

To protect against what might be called “enhancement that is too energetic,”several protocols in this manual are saved as Photoshop actions. These actionscan be referenced in a paper in the same manner as methods are cited for imageanalysis protocol using popular programs such as MetaMorph, Image Pro Plus, NIHImage, IPLab, PixCell, etc.

The only other issue that remains unresolved is that of adding or subtracting thenumber of pixels that make up an image, or what is called re-sampling in the digitalworld. For microscopists, re-sampling introduces the danger of adding or subtract-ing not just pixels, but data. Out of respect for the desire to avoid re-sampling, andbecause it’s a good idea until hard copy is actually produced (then the printingdevice will potentially change the number of pixels to fit the page), protocol formaintaining original numbers of pixels is also included in this manual.

In any event, the original image taken by the camera or scanning device should besaved 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 theirresearch is contested. Careful notes must also be taken regarding the date the


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picture was taken and the nature of the material photographed, all of which consti-tutes good laboratory practices, and all of which can be defensible and legal forsuch matters as FDA approval. Notes in the form of annotations to the image itselfcan be included, if the acquisition software allows for it (Photoshop 6.0 gives theoption for annotation). That image, or a second copy, can be watermarked so that itcannot be altered. These can be saved to a CD for permanence (at least the degreeof permanence necessary in most of research: 10 or so years, or until the nexttechnology takes over, at which time these images are transferred to the newestarchival material). More details on this can be found on page 83, “Saving Files.”

Let those who view your images know when images have been en-hanced in Photoshop. A simple, “Images Enhanced in AdobePhotoshop” will do that.

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Computer Requirements for Running Photoshop

Lots of RAM memory. Photoshop is a RAM (Random Access Memory) intensiveprogram. The caveat is: the more, the better. The speed of Photoshop processesincreases dramatically with an increase in RAM. Photoshop needs at least 64megabytes 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 itis working on. Or, it can potentially need as much hard disk space as the number ofundos set in the History window (5x, 6x, 7x). The space Photoshop needs is referredto as “scratch disk space.” Thus, a 30 megabyte file often requires 90 megabytes ormore of free space on the hard disk. On multi-user computers in which files fill uphard disk space rapidly, it’s not unusual to encounter the message from Photoshopthatreads “...not enough space on the scratch disk.” That message refers to extrahard disk space that is not available to complete Photoshop functions.

It’s important to note, too, that Photoshop writes and reads faster from disks thatare not fragmented into too many short tracks, something sure to occur after lots ofcomputer crashes. Windows allows users to defragment the disk, but more capabili-ties for both Macintosh and Windows can come with utility programs (such asNorton Utilities).

The processor. Processor speed, or clockrate—what is called so many “mega-hertz”—doesn’t seem to make as much difference as RAM. In this instance, more isnot necessarily better for most functions in Photoshop. Again, it is better to putmoney 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 muchdifference.

Video display cards. The video card (or the display card) is what interprets theinformation from your file and re-interprets it so that the information becomes somany tri-colored dots on a CRT (Cathode Ray Tube) computer screen. Obtaining avideo card that quickly interprets information for immediate display used to be aproblem in the past, but, with the advent of more video RAM on currently availablecards (more is not necessarily better, since video card RAM is often used for gamesthat contain video), slow interpretation is no longer a problem. (Older cards onolder computers usually required the user to watch the screen while row after rowof the image file appeared on the screen. Users could not proceed to the next stepuntil the display was through “writing out” the image).

Video cards can cause troubles. Sometimes one image is moved overanother and a white or black rectangle is left behind. Either the card or thecomputer lacks the resources to fill in the rectangle with an image. Simplyzooming out and then back in will correct the problem.

Speed of file transfer. Finally, a hard disk or CD-ROM that uses a SCSI controllerperforms 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 toPhotoshop when opening and saving files using a SCSI controller. For saving toexternal drives (ZIP drives, JAZ drives, etc.), firewire and newer SCSI cards operatefar faster than a parallel port. The advantage of firewire is the ability to plug andunplug devices while the computer is running. The computer can “see” the newdevice 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 beable to drive a larger screen to provide adequate resolution (at least 1024x768), and agood refresh rate. The higher the refresh rate, the less blinking of the screen, andthe easier the screen becomes on the eye. A refresh of 80 is minimum; 100 or betteris ideal.

Buying for color matching. Computer screens also need to provide us withinformation 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 lookdifferent on one computer than on another. Worse yet, the image on the screen maypoorly match the print you eventually make, or what is published. This is becauseof limitations in cathode ray tube technology. (Monitors using LCD’s—LiquidCrystal Displays—perform no better. In fact, the range of colors these monitorsdisplay is less than the cathode ray tube monitors).

Having said that, monitors can be calibrated by buying software and a calibrationtool to calibrate color and contrast to a particular printing device, or to severaldevices, for better matching. However, imaging professionals in the publishing

industry cannot agree, as a whole, about the merit of spending the money tocalibrate in this manner. That’s because they have had trouble matching theirpublished material to these calibrated screens. Files converted to the AdobeAcrobat format seem far less likely to shift in color, and this file format is increas-ingly desired by publishers.

The best monitors and imaging systems. Monitors can, however, be matched sothat what you see is closer to what is printed or published. Part of that dependsupon the monitor and the imaging system. Two helpful solutions are as follows:

1. Buy a Macintosh. Macintosh computer systems paved the way forelectronic publishing. Much of that technology does a better job withcolor and contrast matching, especially when it comes to publications.

2. Buy a self-calibrating monitor, or a monitor designed for graphic artsprofessionals. These monitors are far more expensive than the norm, butthey’re worth it. The self-calibrating monitors self-adjust as the cathode raytubes lose energy because of wear, thus keeping color more accurate. Theabsolute best are Barco monitors which are calibrated in hardware at thefactory.

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Calibrating Your Monitor

Whether or not you have the extra cash to spend on a “professional” monitor, youmust adjust the monitor you currently own. The greatest trouble with monitorsinsofar 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 thescreen 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 morecontrasty screen more accurately resembles what becomes hard copy (the ambient

light of your room should also be on the dim side to match the darkness of thescreen. A closer match of ambient or room light and monitor results in betterinterpretation of image details and less eye strain).

These contrasty settings work well for publication, most digital printers, and 35mmslides. These do not work, however, for matching some ink jet printers, and forpreviewing what is put on the web (as long as your web audience has set theirmonitors to “web” settings: gamma of 1.8).

1. Using controls on your monitor itself, adjust brightness so that it is atthe halfway point, and contrast so that it is all the way up (as contrastyas possible).

2. If not done already, set up computer screen for color and contrast usingAdobe 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, choose2.2 (Europe: 1.8); this is theauthor's suggestion.

Mac:

1. Click on Apple symbol (upper left).

2. Highlight Control Panels/AdobeGamma.

3. Choose Assistant.

4. When setting gamma, choose 2.2(Europe 1.8).

Step 1: Description or Profile. Your Profile is found by Adobe Gamma. Change toanother setting if you are certain of the brand (many Mac monitors are Trinitronmonitors, and should be set, therefore, to Trinitron) by choosing Load. In yourmonitor manual, the manufacturer may suggest appropriate “icm”—internal calibra-tion for monitors—as a starting point. Otherwise, use the default description.

Step 2: Brightness and Contrast. The contrast andbrightness controls refer to the controls that come withyour monitor. Older monitors have dials, newer ones havebuttons usually situated below the screen. Once adjustedcorrectly, you will see a dark grey box against blackbox(es). Typically, the contrast dial or button is turned toits most contrast setting and the brightness dial or buttonis up about halfway.

Step 3: Phosphors. Again, use the manufacturer’s recommendations if you wish tochange 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 thethree color channels, red, green and blue. The gamma setting determines howbright the values between white and black are (the midtones). Adjust sliders untilthe box inside matches, as best you can, the brightness of the inner box to thebrightness of the patterned frame.

Desired Gamma. Set to Windows Default so that your gamma is 2.2. At this morecontrasty setting, you will be best situated to judge the output from 35mm slidemaking devices, from the web, and from many digital printing devices. Ink jetdevices (as of this printing) tend to print less contrasty. If you consistently print toa less contrasty output device, you may want to set this to 1.8. The Europeanstandard is also 1.8 (Macintosh). Note: this option is not available on Windowssystems 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 toreplace monitor. Use 5000k specifically if you wish to print or make slides.Use 7500k to 9300k when working on images for the web. Various settingscan be saved and then loaded depending on how you plan to ouput theimage.

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Setting Memory for Photoshop on a Macintosh

Windows vs. Mac. The Windows environment does not allow users to have muchcontrol regarding how computer memory functions, both in terms of what is calledvirtual memory, and in how much space you will set aside for a particular applicationprogram. In Windows, those functions are set automatically. On Macintoshcomputers, however, those settings are determined by the user.

Setting up Memory on a Mac

Control Panel1. 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 anyalias 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, selectAbout 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 asPowerPoint) open at the same time.

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

25

Visual Definitions

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

Marquee Tool.The defaulttool is theMarquee toolin the upper-most leftcorner. Whennot usinganother tool,click on this.

Hand Tool for navigation

Foreground/Background. Thebottom of the toolbar containstwo squares; the one in front isforeground, behind is back-ground. All tools use theforeground color.

If you click on two smaller boxesunder the two squares, you willmake the foreground pure blackand the background pure white.

Click and hold tosee more tools onthose tools withsmall arrowheadat bottom right.

Click and drag on solidbar to move. Move Tool. To save time going back and forth be-

tween the Marquee tool and the Move tool, simplyremain on the Marquee tool. When you want to usethe Move tool, hold down the control/command keyand the Marquee tool will become the Move tool.

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

Eyedropper Tool. The Eyedropper is good for finding acolor in your sample and clicking to transfer that colorto the foreground. The Eyedropper automaticallyactivates in dialogue boxes for other functions, likewhen adjusting contrast. Thus, the foreground color canpotentially change often when using Photoshop.

If you click on the "two arrowhead"curved line, the foreground/background will change position.

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

Photoshop 6x, 7x: ThePhotoshop 6.0 and 7Toolbar contain all of thesame tools as well assome more, and they arearranged slightly differently.

Line Tool(behindShapeTool in6x & 7x)

Overview of Toolbar

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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 completebecause area is empty.” 6x & 7x auto-locks the background or 1st layer. Duplicatelayer by dragging background layer into page icon at bottom of dialogue box andmake changes on the duplicated image. Or, choose the layer you intended to workon.

Computer crashes often in Photoshop. Usually that means not enough spaceexists on the hard disk. Free up space by eliminating files, or set scratch disk spaceto take advantage of a second or third hard disk (see page 28). You may also wantto 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 anotherlayer 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. Flattenthe layers by choosing that option in the Layers dialogue box drop down list, or bychoosing that option under Layers in the menu (5x, 6x & 7x), then save the file tothe 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 usingControl/Command + h; or, under View on the menu choose Show Guides in 4x and5x, 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 isnot 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, IndexedColor 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 (layerswith eye symbol). Click on a layer in which the eye symbol is visible, then MergeVisible 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 themarching ants selection. Usually, you will need to make the selected area larger, orsimply ignore the message (click Okay).

Scratch disks are full. Photoshop requires at least three times the hard disk spacethan the size of your file. If you have more than one hard disk, make the least filledhard disk your primary scratch disk. Find Preferences (Control/Command + k) andthen select Plug-Ins & Scratch Disks. Otherwise, you will need to delete or movefiles from your hard disk to make more room. You may want to reduce the size orresolution of your image using the Image Size dialogue box if the file is over 20 orso megabytes (under Image, choose Image Size); or you can Merge Layers orFlatten the image as layers add a great deal to the file size (use the Layers Windowdrop down box to find these).

This action cannot be completed... Change the Mode of the file. The operatingsystems 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 fromquickphotoshop.com/downloads.pdf. These actions will comprise much of whatwill make Photoshop “quick,” and these are used for all the steps that follow. Youwill 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 ofPhotoshop.

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 thedrop down list. If Load Actions is grayed out (unavailable), select DefaultActions at the top of the listings in the Actions dialogue box.

4. Select “Photoshop-for-research.atn” from the location into which youdownloaded the file.

Click on arrowhead triangleat top right.

Select Load Actions.

Scroll down past Default Actions until you find“Photoshop for Research” and its sequence ofbuttons that follow.

Click and pull on bottom right corner of box andstretch until 1 or 2 columns of buttons appearand button titles are readable.

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

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Click on top right arrowheadtriangle.

Select Button Mode.

Set up Actions Dialogue Box

Under Window, select Show Actions.

Part 2 — Quick Photoshop

Steps for Single ImagesWhether images have been scanned from film or taken by a digital source, imagesshould be duplicated in Photoshop to avoid saving over the original. Then mostimages need to be sharpened, color balanced, contrast enhanced, made free ofdust, sized, and cropped (extraneous white space around the image needs to beremoved).

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Marquee Tool and GuidelinesBefore using any tool, it is a good idea to know how to use the marquee tool andguidelines. The marquee tool may take some practice if you are not used todrawing with a mouse. Remember to click, hold down the mouse, drag, and thenrelease when you are at your finish point. This creates an outline, or what iscalled a selection, which is marked by an animated marquee referred to as “march-ing ants.”

Guidelines help with the alignment of images and text. Guidelines appear only onthe 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 chooseDeselect. Alternatively, use the keyboard: Control/Command + d.

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

To Use Guidelines

1. In the menu under View, make sure Show Guides and Show Rulers areselected (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 willappear. Drag down or across to the desired location.

3. To remove a guideline, hold down the control/command key, or click onMove tool, and drag the guideline back to the ruler area.Guidelines are useful for setting borders for areas of interest. Onceguidelines are in place, these serve as constraints for either the Marqueeor Crop tools.

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You can choose View inthe menu and thenSnap to Guides if youwant to outline an areaof interest usingguidelines to constrainthe selection.

Marquee tool

Marquee tool along withchoices for its shape

Click and drag toposition marquee overarea of interest.

Be aware of accidentallydrawing tiny selections.

Open Image/Duplicate

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

1. Launch Photoshop, in menu choose File then Open.

2. Click on file, click Okay.

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

When File Does Not Appear

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

To View All Files, and Then to Open

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 willappear 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 filelooks 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.

RAW. You can also open as RAW. If you know the values, you can either entervalues 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 theimage looks like it has been cut in strips and the strips have been re-arranged. If thefile does not open at all, open as RAW (see above).

Grayed out menu functions. Some files open, but only a limited number of func-tions can be used. See section on “Mode” on page 39.

Click on triangle to expandlist. Choose the file typeyou think it is.

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Open As Window

Corrupted File

Once Your File is Open

Size of window. The file you open will create as large a “window” on your computerscreen given the limits of the size of your monitor settings. The percentage on thebar at top only relates to the size of the image in pixels across and down in relationto the control panel setting for the display (e.g. 1024 x 768 pixels). It can bear norelation to the inch or centimeter dimensions of your digital image, nor can it bear arelationship to the dimensions of what is ultimately printed (see thewww.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.

It is strongly recommended to duplicate your image file so that you donot save over your original!

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Rotate/FlipEspecially when scanning artwork and negatives on a flatbed scanner, resultingimages are tilted. These images can also be upside down or, in the case of nega-tives, scanned with a negative flipped in the wrong direction. The followingfunctions 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 RotateCanvas).

For Small Degrees of Rotation

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, selectRotate, then Arbitrary). Enter the degree of rotation, usually less than anangle of 2.

3. Undo, if necessary, and start again (under Edit, select Undo, or useControl/Command key + z).

For Interactive and High Degree of Rotation (Useful for RotatingParts of an Image)

1. Select area you wish to rotate by clicking and dragging with the Marqueetool.

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 selectionto complete the rotation (or click on Marquee tool in the Toolbar; then clickOkay).

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Clockwise

Counterclockwise

The degree of rotation orangle is typed in by user.

Click in ruler area and dragdown guideline to provide“straight line” reference.

After rotating andcropping

Rotate whencrosshair turns todouble arrow

Arbitrary Dialogue Box

Rotation Using Transform

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

Two Ways to Undo/Redo:

Using keyboard: Control/Command + z (toggles between undo/redo)

In Menu: Under Edit, select Undo/Redo

HistoryYou can undo several steps back by opening the History window and clicking onthe 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 Historywindow is available on the screen).

2. Click on steps back to where you’d like to be.

Click back to stepyou wish to retain

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History Window

Zooming In & OutBefore determining whether dust or scratches need to be removed, or to checksharpening effects, you may need to zoom in to parts of the image to examine it. Thiscan be done with the magnifying tool on the toolbar, but it is best done with key-board 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 inZoom 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. Thiscan be done if image is zoomed in accidentally when magnifying tool is inmotion while simultaneously clicking (image is super-magnified).

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Crop

Unlike a drawing, word processing, or graphing program, white space or the whitebackground 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, theextra area needs to be “cut out,” or cropped. This function does not add or reducethe number of pixels making up the part of the image that remains. For those whowish to also crop to match two different images in terms of size, or for those whowish to adjust image sizes to match their output (for a page, a slide, an on-screenpresentation, etc.), it is recommended that you do that after several other steps. Itis 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 dialoguebox 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 orbetween corners along the outline until arrows form ( ), thenclick and drag to reposition.

4. Press Enter or Return key, or click inside box to complete the crop (or clickon Crop tool in the toolbar and respond to message that will pop up).

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 thepart of the image you desire (middle of crosshair is the “writing” point), andmove 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 Trans-form. Under Image, select Crop.

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Crop tool outline - reposi-tion after outlining bymoving cursor overcorner boxes. Click anddrag to reposition aroundarea of interest.

Outline is drawn fromcenter of Crop tool icon.

Excess white space

Cropping Off White Space

Mode

The following functions in Photoshop will not work unless the image file has certaincharacteristics. 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 iscalled a “black and white” in photography: all gray tones including pure black andpure white), RGB (Red, Green, Blue, or color files containing the three primary colorsof light), and CMYK (Cyan, Magenta, Yellow, and Black, or color files containing thethree primary colors of pigment plus black). Color files should be worked on as RGBuntil the end of the editing process, since computer screens work on the principle ofmixing 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

Changing Mode to RGB (Color)

1. In Actions Window, Click on Mode RGB. Or, under Image, select Mode andhighlight 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 andhighlight 8-Bits/Channel (near bottom of drop down list).

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 onwith all functions available. Note that Indexed Color files (those files, typically fromthe web, which contain 256 colors versus millions) and Bitmap files (those fileswhich contain only pure black and pure white with nothing else) must be changedto RGB and Grayscale respectively. Other modes are rarely encountered.

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Photoshop will not open most 12-bit files. These need to beconverted into 8-bit or 16-bit in the program that created the 12-bit file.

Dust and Scratches

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

Smaller Specks of Dust

1. In Actions window, scroll down to gray Sharpen/Dust sequence ofbuttons

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 muchdithering.

Or, under Filter, select Noise, then Dust & Scratches. Use a radius of 1 or2. A larger radius eliminates more dust, but also introduces more blurring.

Larger Specks of Dust or Scratches

1. In toolbar, click on the Rubber Stamp tool. In menu under Windows selectShow Brushes for 4x & 5x(if Hide Brushes is shown then the Brushesdialogue box is already open); in 6x & 7x look for Brushes in the toolbarunder the menu.

2. Select a brush or footprint that is slightly larger than the speck or scratchyou wish to eliminate (by choosing Precise in Preferences—seepage 28—an proportionately sized circle will form as the toolbar ismoved onto the image).

3. Hold down the Alt/Option key and with the rubber stamp click on an areavery 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 fromvarious areas. The borrowed area “follows” the rubber stamp tool in thedefault setting.

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Dust & Scratches Dialogue Box

Before changes After changes

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

Rubber Stamp Tool

Before changes After changes

Place footprint overdust speck afterselecting brush andborrowing an areawith the RubberStamp tool.

Sharpening

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

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 sharpeningintroduces pixelation.

Or, under Filter select Sharpen and Unsharp Mask. Set the slider Amountby eye (make sure Preview is checked), and keep the Radius between 1 and2 and the Threshold at zero.

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

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

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Contrast & Color: A Primer

If images are grayscale, good scientific photographs often contain a full range oftones from black to white; if images are in color, they contain rich, deep hues. Eitherinstance requires judicious use of contrast and color controls to not only enhanceimages, but to produce images that reproduce as they appear on the computerscreen. Typically, images need to have shadow areas that do not contain blacksthat 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 sowhite as to be transparent. All other outputs need to be the lightest of grays so thatedges of images do not print at the same color as the white background. What isgenerally true about midtones (those tones between blacks and whites, or thosecolors 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 brightoverall versus too dark. The caveat for images destined for printing presses and theweb is that they “can never be too bright.” Midrange tones are also the best to alterwhen 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 theirmeanings:

Contrast (or Black Level): Contrast refers to how dark the black end, or the darkesttones in your image, appear. If your black end is very dark, everything else appearslighter in comparison. Thus, your image has an appearance of greater visual impactand greater contrast.

Saturation: Saturation, in terms of color, refers to how instances in which a hue isat its maximum color intensity. Another way of conceiving saturation is to think of ahue furthest away from grey. Almost all colors used in research are saturated colors,

which makes these images particularly unfit for reproduction. Overly saturatedhues usually create problems when viewing on the screen and even more so whenprinting to a printing press. Note that the meaning of saturation changes somewhatwhen the reference is to untextured highlights.

Brightness (or White Level): Brightness refers to how bright the white end, or thebrightest tones in your image, appear. The danger with brightness is to set thewhitest 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 otherhand, 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 iswhat most improves the reproduction of an image. It also changes the linearity ofimage tones and makes them logarithmic.

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

Do not change contrast on images in which density/inten-sity measurements will be taken.

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Contrast/Color Auto Actions

Correcting Color and Contrast for Typical Images (generally fromscanned negatives, slides, or digital cameras)

1. In Actions window, scroll down to blue Contrast/Color sequence ofbuttons.

2. Choose Contrast/Color Autobalance 1 to keep darkest shadow levels (butnot 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).

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

If Auto-Balance Doesn’t Auto-Determine Hues Correctly andWhite Areas Exist in Image File

1. In Actions window, scroll down to blue Contrast/Color sequence ofbuttons. Select Color-White Level. Or, under Image select Adjust, thenLevels.

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 (todistinguish the edge of the image from the white of the page your image willbe printed on).Under Edit, select Undo if you do not like the effect (or use keys Control/Command + z).

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White balance Eyedroppertool

Click on whitest area onimage.

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

Levels Dialogue Box

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 toeither 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 plentyof white and black values exist (a wide histogram), move grey triangle in middle to the left (to lightenshadows) 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 theblack triangle to at least 10 (to lighten deep blacks to printable levels).

Contrast Adjustment Using Levels

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

After adjusting top sliders, move black slider toright until Output Level reads at least 10 (sethigher if blacks diminish fine details).

Put white level on 254 for images (vs. drawingsor graphs or white area around images) bymoving Output Level slider to left (this makeswhite slightly greyer than its surrounding whitepage when printed).

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

Move grey (middle) slider to left to increasebrightness of shadows, to the right to increasecontrast in mid-tones.

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Manual Adjustment of Color

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

1. In Actions window, click on Contrast-Manual Adjust (or, in menu, underImage 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 beforeevaluating colors.

4. When done, if colors are still incorrect, go into problematic channel and backoff on sliders (typically the white slider). For example, if image is too redafter adjusting sliders in each channel, go to red channel and back off onthe white slider until image looks more correct (this may have to be done inmore than one channel).

OR... If that doesn’t work, cancel and start again. The problem could be asaturation in one or two hues (e.g., the red in an H&E stain can be neoncolored) 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 shouldbe maintained for measurement or evaluation.

Narrow histograms comprise a narrow brightness range. In thatinstance, white and black sliders need to be set by eye in eachchannel iteratively.

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Correcting Color Shift in Whites & Selected Areas

Cross curves. Cross curves comprises a situation in which a correction in onecolor 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 hueinto white backgrounds. In that instance, specific areas need to be selected beforemaking 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 oneshade 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 theexactness of selection (if you desire, for example, only the hue-shiftedwhites, click as many points as possible and you will be less likely tooverlap into other parts of your image that contain other colors).

3. Adjust Fuzziness to select no more or no less than desired parts of theimage.

4. If the selection is white, and it’s a photographic image, under Edit selectFill and Use: White. Go back into Levels to backoffOutput to 245 or so (tomake white somewhat gray). If the selection is a particular element of thesample (say, blue-stained cells), use Levels to adjust contrast by going intopertinent channels and moving the middle slider, and use Hue & Satura-tion (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 definedborders are softened best by using a higher Fuzziness value.

Color Range will put a scrolling selection around areas of interest. Thisselection can obstruct detail. Use Control/Command + h to hide scrollinglines. When finished with Contrast/Color tools, don’t forget to deselect byusing Control/Command + d or by clicking outside selection (make suremarquee tool is selected), or, in menu under Select, choose Deselect.

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Correcting for Hue-Shifted Whites

Use Eyedropper tool toclick on what should bewhite, then use PlusEyedropper to click onmore points.

Be sure Sampled Colors isselected and Selection ischecked underneath graphicimage.

Set Fuzziness after clicking onseveral points. Determinesetting visually.

After using Fill to fill with white, set outputslider in levels until white becomes lightgray.

Before (top) and after(bottom) selectingwith Color Range andthen correcting colorshift.

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 ablack background, most often it is the black background that encroaches upondetails. That may not be seen so clearly on the computer screen—you may see, forexample, fine neurites against deep black on the computer screen—but whenprinted some details are lost. The other problem has to do with the brightest

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

Fluorescence-brighter1 Brightens fluorescence and makes processesvisible in dark areas by lightening blacksslightly. Changes gamma. Continue clickingon this button to keep increasing detail inblack areas.

Fluorescence-brighter2 Slightly greater increase in brightness thanprevious option.

Fluorescence-up contrast1 Increases contrast by slightly darkeningshadow end of image and brightening whiteend. Changes gamma.

Fluorescence-up contrast2 Slightly more contrast than previous option.

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

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

1. In Actions, click on Contrast-Manual Adjust (or, in menu under Imagechoose 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. Ifyou overshoot, click Cancel and start again.

3. Keep black Output Level at 0 and white at 255.

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Adjusting For Grayed Whites on Bitmap Images

White Eyedropper tool

Contrast: Measuring Pixels & Matching Background Gray Levels

Contrast For Images with Gray or Neutral Backgrounds (such asgels 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 Imagechoose Adjust, then Levels.

5. Set white level by using white triangle slider. Measure level of backgroundgrey by moving cursor over representative background areas in your imageand looking at info window. (Grayscale values are set by percentages, 0 iswhite, 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 tomatch similar images to the one you are presently working on.

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 anyloss of image information.

3. Undo, if necessary.

4. Proceed to steps outlined above if you wish to further adjust brightness ofgray background or darkness of blacks. More information about this actioncan be found at www.quickphotoshop.com.

Extreme graininess, such as that found in images of scanned phosphorscreens can only be reduced using proprietary filters, such as thoseprovided by Bio-Rad with their analysis software.

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When moving cursor onto image, itturns into an Eyedropper tool. Inforeadout will give the values of gray(the K: value) at the dropper end ofthis icon. Find representativebackground and adjust slider to seebefore/after values. Check in a fewplaces to get a mean value.

K: Previous Gray valuepercentage / Gray valuepercentage after adjust-ing slider

Click on arrow-head and selectPalette Options.

Set First Color Read-

Set SecondColor Readoutto RGB Color.

Background Adjustment

Move slider to left toincrease brightness.

Slide black triangle to right forOutput Level if blacks appeartoo dark. Do this by eye.

Info Window

out to Grayscale.

Manual Color Controls/Subtle Colors

Variations (for Subtle Modifications of Color and Contrast)

1. Under Actions, select Color-Variations. Or, in menu under Image chooseAdjust, 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 Imagechoose Adjust then Hue & Saturation.

2. In dialogue box, choose the hue by clicking on the Edit arrowhead andchoosing from the drop down list.

3. Expand or contract the range of colors in your selection by moving theslider 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 orgaudy.

5. Change Hue if the color is incorrect, or change Lightness if the color istoo dark or too light.If you see no change, chances are you have chosen the wrong color tomanipulate. For example, you may have chosen red when it should havebeen magenta.

Changing the Color Blue

Scientific images often contain a handful of hues, or shades of only one. The blue-to purple-blue hue can be seen on the computer screen but is likely to print asalmost black. Thus, color changes are necessary for reformulating a blue with redin 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 methodand change hue to make greener (more cyan). Shift Hueslider 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, orMake-Blue (shifted to cyan).Manual method: The manual means for adding color is outlined atwww.quickphotoshop.com.

Adding Pseudocolor to Grayscale images (for visualization purposes)

Pseudocolor images resulting from this action follow the visible spectrum from theshort wavelength of violet (deepest black parts of your image) to the longestwavelength red (brightest parts of your image). Your image becomes an RGB colorimage. 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 theposterize function. Used as an aid in visualizing and segmenting images.

1. In Actions window, select either Pseudocolor-8 Grays or Pseudocolor-8Colors.

Manual method: see www.quickphotoshop.com

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Subsequent Steps for Single Image

Save ImageIf you haven’t saved your image file yet, you should do so now. Save the file, forthe meantime, as a Photoshop file. Go on to subsequent steps before saving asother file types.

In Actions window, click the Save As button (or, in menu under File selectSave As).

Subsequent steps for Single ImageTo 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 interms of how it is outputted to a printing device as pixels per inch or pixels percentimeter. The dimensions in inches or centimeters must also be determined alongwith output resolution (the two go hand in hand) when printing to digital photo-graphic printers and to publications. These kinds of output devices print only atdefined output resolutions.

The resolution is not so important, however, when printing to ink jets and laserprinters. That’s because no particular gain in resolution is had by arbitrarily addingmore pixels to your image (what you do when you increase the number of pixels, orresample) with these devices. Ink jets and laser printers reinterpret pixels to makedots, and, without going into an exhaustive explanation, it’s fair to say that dots, bytheir nature, provide less resolution than pixels. To state it another way, you’llalways get more resolution from a digital photographic printer which has, say, aresolution at 300 pixels per inch than you will from an ink jet that provides 1400 dotsper 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 aremeaningless, unless the number of pixels across and down is low: at less than 500pixels or so at one-half page.

35mm slide-making devices, on the other hand, use software that fits the image tothe slide, so neither the dimensions nor the resolution is important. These print atextremely high resolutions. In that instance, even poor resolution (72 ppi) can beprinted upon so many thousands more pixels to “add” resolution (though poorresolution from the get-go can never be adequately corrected: chippy or aliasedlettering is evident). Nothing is gained by arbitrarily adding pixels to the image fileitself (thus changing resolution), or by changing the image dimensions.

In any case, a target resolution can be set to fit nearly all output devices. Thatsetting is what prints best to publication. If the image then needs to be printed to aprinting 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 itswidth/height relationship, also known as aspect ratio).

3. Enter Resolution of 300.Uncheck Resample Image when subsequently printing to ink jet or laserprinter.

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

Set most importantdimension in inchesor cm.

Set Resolution in pixels/inch. This is OUTPUTresolution.

Check Constrain Propor-tions.

Check Resample Image unless going to ink jet or laser printer.

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Image Size Dialogue Box

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 isabsolutely necessary. The beauty and bane of Photoshop is in its ability to keepimages on separate layers. While layers keep images from interfering with eachother, as though on transparent sheets one above the other, the sheer multiplicity ofimages, along with text, can make Photoshop difficult to navigate. Action buttonshave been included to make it easier to keep the number of layers down to one ortwo, 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 isalready 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, ifnecessary.

Photoshop 6x & 7x Layers window looks quite similar.There are some added functions, and some things are indifferent places, but the basic window is the same.

Layers can be dragged to new positions byclicking and dragging. The higher layercovers the layer below.

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

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Layers Window

Click on arrowhead to reveal layering options.Normal is most often used (other options arediscussed elsewhere in this manual).

Eyes can be clicked on or off. When off, thelayer disappears.

Chains can lock one layer to another, so that ifone layer is moved, both move (this doesn’talways work).

Click on arrowhead to activate drop down list.Ability to flatten layers or to merge visible (onlythose with “eyes” on) is selectable from dropdown list and also from Layers in the menuselections (5x, 6x).

Transparency or opacity of layer is set byclicking here. Layers are made more translu-cent if opacity is reduced.

Capital “T” indicates that this layer is a non-rendered text layer (5x-7x). Thus lettering isable to be edited by double clicking on thelayer, but images cannot be added to it.

Adjustment Layers can be made only for con-trast/color adjustments. A new adjustmentlayer is found in the drop down list.

Black cutouts indicate a masked layer. Newimages/text cannot be added to black partof mask.

New layers can be made by clicking on paper icon.

Layers can be deleted by dragging them into the trash icon, or by simply selectingthe layer and clicking on the trash icon.

Layers: Actions & Flattening

Layers Action Buttons

Layer- Flatten: flattens all layers. This is necessary when saving to formatsother than the Photoshop (.psd) format, and when desiring to make the filesize smaller. Or, under Layers (or in the Layers drop down menu) chooseFlatten 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 filemore manageable.

Layer- Render Text: Makes text layer into “normal” layer. Text layer is nolonger editable by double clicking, but text can be easier to move around (thetext layer need not be double clicked to move text around on the image).Because limited text is typed in Photoshop, rendering text should be consid-ered a useful function. Also available under Layers when selecting Type, thenRender (5x) or in 6x & 7x under Layers, then Rasterize and select Type.

Merge Visible: Too many layers can make navigation difficult, and so it isadvisable to merge layers of one type into a single layer (such as all the text orall the images). This can be accomplished manually by clicking on eye iconsto 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 dropdown 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, acontrol 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 imagescropped in the same place. This can be done as long as both images are the samesize 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 setcropping with the “fixed image size” description below.

If your image files are different sizes in dimension and/or resolution, then you willneed 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 fromone 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 fromimage with largest dimension.

If your first attempt at drawing an outline over the region of interest failsto outline correct area, click outside the outline to eliminate “marchingants” (scrolling outline) and start again. Or, under Select choose TransformSelection (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 chooseimage from which you saved the selection. On a Mac, choose also AlphaOne from Channel.

6. Repeat steps 4 and 5 for remaining images.

7. In Actions window, click on Crop button (or, in menu under Image, selectCrop) 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 maynot open in other programs). In menu under Window, select Show Chan-nels. Drag alpha channels to the trash icon.

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Transferring selections from one image to another

Make selection with Marqueetool. Save to new channel.

Load selection from 1st file/alpha 1 channel.

Selection transfers to 2nd image in same location

Cropping Images that are the Same Resolution but Different Dimensions

Sometimes images are scanned at the same resolution, but are slightly different insize, or the locations of related images have shifted from one scan to the next. Inthat instance, an outlined area of interest (a selection) can be made on one image fileand then dragged to the related image file. The location of the selection can be eye-balled 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 fromimage with smallest dimension.If your first attempt at drawing an outline over the region of interest failsto outline correct area, click outside the outline to eliminate “marchingants” (scrolling outline) and start again. Or, under Select choose TransformSelection (not available in 4x). Selection can then be reshaped.

3. Crop that image. In Actions window, click on Crop button (or, in menuunder 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 useControl/Command + a).

5. Click and drag selection outline to the related image. Align by eye (hope-fully shared landmarks exist in the images), then crop. Repeat for additionalimages.

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Cropping Images of Different Sizes and Resolutions toa Specified Size

In some instances, you are aware of the final output size, and fitting all images tothe same dimensions and resolution guarantees that two or more images side byside will be identical. Again, it is best to shoot for a target resolution that will yieldthe largest file at the most ideal resolution. Other files can be made from this masterfile at smaller and smaller resolutions.

This method for determining dimensions requires a bit of simple math. If, forexample, you are using a U.S. letter page size as your output, and you are printingtwo 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 shorterdimension is 8.5 inches, but 1/2 inch is generally white margin). Thus, each of thetwo images will be about four inches wide with a small border between the two. Inthat instance, you would set your width to slightly less than four inches and theheight to whatever fits (one dimension is typically considered more important thanthe other). Your Resolution is at 300 ppi.

1. Click and hold on Marquee tool to reveal crop tool (4x, 5x) or, in 6x & 7xsimply 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, andset resolution to 300 pixels/inch. Note that one dimension is more impor-tant than the other, and the latter may have to be determined by clickingand dragging to make crop outline to see whether it falls short or long (bytrial and error). Note that when crop selection is enlarged or reduced, itmaintains its aspect ratio (ratio of width to height).

3. Double click inside crop outline to complete crop, or click again on Croptool. Return to Marquee tool when done.

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Merging ImagesMerging Images/Colocalization

In this instance, merging is considered to be the combination of two or more RGBcolor images, one on top of the other: an equal percentage of color from differentchannels. This technique is commonly used in fluorescence to show coexistenceor colocalization of two fluorophores labeling similar cell features. Red and greencolored labels merge best since the combination of the two produces the easilyrecognized 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 red-colored image.

3. You will then be prompted to open the second image, which needs to bethe green-colored image.

4. Optional. In some instances, the images can be slightly misalignedbecause of color shift from one wavelength to another. This action putsyour green image on a layer above the red image. Simply hold down theControl/Command key and move green image over red image or use Movetool. While holding down, use arrow keys for fine movements.

Manual methods can be found at www.quickphotoshop.com.

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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 thismanual.

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 estimateamount of extra space you’ll need to accommodate extra image(s). Giveplenty of room, since extra space can be cropped later.

3. In Canvas dialogue box, choose Anchor grid square to give direction foradditional space. Click okay.The image will expand beyond the boundaries of its window. You’ll haveto zoom out to see what you have done, or pull on corner of image filewindow 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 chooseCut; under File choose Close—don’t Save).

6. Now first image should be the active window (or you may have to selectthe 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 moveonly on the x or y plane (horizontally across or vertically down)

9. Repeat for additional images.


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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 menuunder Edit (Layer in 4x), select Transform then Scale).

2. While holding down Shift key (so that proportions don’t change), placecursor over a corner (to get double arrow). Click and drag until desired sizeis reached.

Make Final Contrast/Color Adjustments, Crop, and Flatten Layers

1. In Actions, you may need to go back to Contrast- Manual Adjust to matchcontrast 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 whitespace.

3. You will want to flatten your image when finished, since each image sits ona 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 drawingprogram like Canvas, CorelDraw or Illustrator), you may need to add more whiteimage area above, below, and/or to the sides of your image. It isn’t as easy assimply 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).

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 presenta-tion, especially important when images are downloaded from the web, orwhen items at <10cm have been scanned incorrectly.

Adding White Space and Resolution

1. In Actions window, select Combining- Add Space (Canvas). Or, in menuunder Image select Canvas Size.

2. In Canvas dialogue box, choose a unit of measurement and estimateamount of extra space you’ll need to accomodate lettering. Give plenty ofroom, since extra space can be cropped later.

3. In Canvas dialogue box, choose anchor grid square to give direction foradditional space. Click Okay.

The image will expand beyond the boundaries of its window. You’ll haveto zoom out to see what you have done, or pull on corner of image filewindow to expand its size.

4. In Actions window, select Image Size (or, in menu under Image, selectImage Size). Increase resolution to 300 pixels/inch if necessary (Note thatoriginal data will be changed. Make sure Resample Image is checked).

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

Adding White Space Using Canvas

Choose unit of measurement fromdrop down by clicking on arrow-head. Put in values for amplespace.

The chosen square on grid definesthe position of the image. Extracanvas is added in the directionof arrows.

Move cursor over corner,click and drag to revealmore image. You mayalso have to zoom outafter making canvaslarger.

Find pixels across and downhere (only visible whenResample Image is checked).

Put in resolution of 300 atdesired height or width whenworking with lower resolutionimages

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Image Size Dialogue Box

Manual Lettering of Images (4x, 5x)

Good lettering depends upon the resolution of the image. Because Photoshop mustplace lettering over so many pixels, a user should expect lettering to look on paperclose to how it appears on the screen (at a reasonable zoom: zoomed about as faraway as it would be when reading at a reasonable viewing distance). Photoshopputs a ghost image at the edges of lettering to fool the eye. That is called anti-aliasing (see page 15).

Users should be aware of a few features of the Type tool. Each time you type andthen click okay, you create another layer. When the Layers window shows a T, thatlayer can be double-clicked and you can edit the lettering (not in 4x, however). Youcannot edit the lettering after the layer has been rendered, which occurs whenlayers are flattened (5x - 7x). To prevent layer after layer from building from theaddition 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 inversion 6x - 7x by eliminating the dialogue box so that a user can type on the imageitself. Photoshop is useful for small amounts of lettering. Extensive lettering shouldbe done in Photoshop’s companion program, Illustrator, or in other page layoutprograms such as Canvas, CorelDraw, etc.

Text from other programs can be cut and pasted intodialogue box, but formatting may be lost.

Adding Text in 4x - 5x

1. Drag guidelines from ruler area for alignment of lettering.

2. Select the T from the toolbar, or type the letter “t.”

3. Click on place where you’d like to letter.

4. Dialogue box appears. Type into this box. Text appears on your image asyou type in 5x, but not in 4x.

Use Helvetica or Arial text for better readability after images are shrunk inpublication. Choose Regular, Bold, Italic, etc., and color (by clicking on thecolor 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 draggingover it) and type in new size. These changes can be previewed in 5x, but in4x the lettering must be placed on the page (by clicking Okay). In 4x, ifchanges need to be made to the type size, typeface, etc., it is best to simplyremove the text layer on which changes need to be made (drag layer intotrash icon in the Layers dialogue box), and start again.

6. Move text in place by dragging with cursor while in dialogue box (5x), orafterward 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 orHelvetica.

Set Size of Typehere.

Color is set to thesame color as theforeground; or, bydouble clicking onbox, you can set adifferent color.

Lettering must behighlighted beforechanges can bemade.

Be sure Fit in Windowis checked so thatyou can view alllettering.

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

Type Tool Dialogue Box (5x)

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 eitherclick at the point where you’d like to place lettering, or click and drag to make a textbox. In the latter instance—when making a text box—formatting can be changedby 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). Changescan 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 layerscan 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 unlessthe check mark or “T” box is clicked first.

When first using 6x or 7x (after being accustomed to 4x and 5x), new work habitsneed to be incorporated. These are indicated below.

1. Select T from the toolbar, or type the letter “t.” Click at the desired spot forplacing lettering or click and drag to make a text box.

2. Click on Palettes below the menu to reveal the Character Palette (if notalready visible). Choose Helvetica or Arial for the typeface, along withattributes such as Regular, Bold, Italic, etc. Choose color by double-clicking on the color window, or by changing the foreground color at thebottom 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 thetypeface, color, size, etc. in the Character Palette.

Re-positioning text. Before clicking on T in the Layers dialogue box (whenstarting at a click point only and not within a text box): move cursor awayfrom the text area. The text symbol will change to a move symbol. Clickand drag to move.

After clicking on T in Layers box: Hold down the Control/Command key oruse the Move tool.

When finished with applying text: Click on the check mark below the menuon the right (or click on the Marquee tool). Clicking on the X eliminatestext.Returns at the ends of lines must be included with “click point” text, butnot with text boxes.

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Click check markwhen done; clickX to eliminatetext.

Text in 6x, 7x

Set strength of anti-aliasing(Strong is recommended)

Set size of typeChoose Arial or Helvetica

Choose attributes here

Click to openCharacterPalette

Indicate color byclicking on colorbox

Set paragraphstyle

Character Palette“T” box in Layers

LeadingTrackingKerning

Click on “T” box before makingchanges in the Character Palette

Enhancements to LetteringOutlining Black Lettering in White, or White Lettering in Black

1. Select your text layer(s). In Actions window, click Show Layers then clickon 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 textinto 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 theforeground 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 youdo 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 uponwhich images are placed (should be below text layers).

2. Make a new layer: in Actions window, select Layer- Make New (or, inLayers dialogue box, click on paper icon).

3. Select Marquee tool from toolbox. Click and drag behind text to the size ofbox desired.

If you are working on more than one image, drag guidelines down from rulerarea (or turn on grid under View: 5x - 7x only) for alignment purposes.

4. In Actions, select Fill- With White or Fill-With Black (or, inmenu under Edit, select Fill and click on arrowhead at top to get dropdown list. Choose white, black, foreground or background).

5. Click inside “marching ants” selection and move selection to next positionby clicking inside the marquee and dragging (hold down the shift key whiledragging to move in horizontal or vertical direction). Repeat step four.

If you accidently lose the “marching ants” outline, it can be retrieved byfinding that step in the History window (5x - 7x only), or by clicking Undounder Edit.

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Alignment and Orientation of TypeAlignment (5x - 7x)

Already much has been said about using either guidelines or a grid for aids whenaligning text and images. To allow for yet another choice, Photoshop includedanother method for alignment of several image or text layers in versions 5x - 7x. Totake advantage of this method, text and images need to be on their own separatelayers versus placing all text or all images into one layer.

1. In Layers dialogue box, select a desired text or image layer. Choose layersto which chosen layer is associated, and place lock symbols by clicking inthe boxes to the right of the eye symbol.

2. In menu under Layer choose Align Linked if you wish to align text orimages from several layer along an axis. If you wish to make spacingconsistent between text or images from several layers, choose DistributeLinked.

Rotating Text 90 Degrees

Labeling in research can often mean setting type at an angle. This can be done inPhotoshop, but it requires a few steps:

1. Click on T icon on toolbar. On image, click on approximate place whereyou’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 6xor 7x, move cursoroutside box area and wait until the cursor becomes arounded 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 onMarquee 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 clickingat the top point, holding down shift key, then clicking at the bottom pointat 45 degrees to the right of the top. A line will form that istransparent 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 withthe 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 atupper right to get drop down list. Select Delete Path (6x. 7x) or Turn offPath (5x). Select Marquee tool!

7. In Actions window, click Transform- Rotate button (also located under Editin menu).

8. Move cursor to corner (arrow becomes rounded), click and drag to rotateinto horizontal position above image area. Use guidelines to aid position-ing.

9. Double-click inside outlined area to complete the rotation, or click on theMarquee tool and click Apply.

10. If the spacing between the entries are not great enough, double click ontext 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 yourown values.

11. Rotate text/numeric entriesagain to align horizontally. Fine tune alignmentby 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 theentire column 45 degrees. One by one, outline entries with the lasso tool (underthe Marquee) and move into place above image area. Line up by eye or by usingguidelines.

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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 atemplate. Actions to complete templates can be downloaded fromwww.quickphotoshop.com. These templates fall into two categories: templates forsimilarly acquired images from a digital camera or PMT/laser system at fixedresolutions and everything else.

Everything else. “Everything else” includes what has been scanned, drawn in otherprograms, or gathered together from different sources. These are images in whichseveral different image resolutions are put onto the same page. In this instance, itbehooves the user to choose a page size that matches a target size. While noparticular target size fits every circumstance, the most universal size would be onethat complies with the needs of publishers AND satisfies the need to make a print or35mm 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 andbottom for printer margins. In this instance, the resolution is still defined by pixelsacross and down, but it is more commonly understood in terms of how many pixelsare crammed into an inch or centimeter, since the image eventually gets printed topaper. Three hundred pixels per inch is ideal, for it translates well to publisherguidelines, it provides enough pixels so that images look clean and non-pixelatedwithout 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 largefiles (files that take up a lot of memory and hard disk space) can be made moremanageable (see “Saving Files,” page 83).

Digital Imaging Systems. Digital camera systems and PMT/laser systems operateby using a sensor with certain number of photon collection points going across anddown, as in 640 x 480. This correlates to the number of pixels across and down inyour image. The number is fixed--the system can take no more or no less than thenumber across and the number down. This defines the resolution of the camera.

Every newly manufactured camera improves upon the old by multiplying thisnumber, and new cameras are available just about every month, so the templatescurrently available cannot possibly cover every camera system made. For updatedtemplates, check www.quickphotoshop.com,or contact the author with the numberof sensors across and down for your digital device, and you will be provided with atemplate for your use.

Choose your template by the way in which you acquired your images. Find outhow 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 youhave completed work on your single images, you are now ready to put all yourimages 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. Thetemplates for a publication page fit most journals. A slight difference in column sizecan 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 ofdifficulties in estimating heights from various sources. You can use the templatesfor U.S. Letter and then re-size to “publication” dimensions using Image Size underImage (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 grayscaleor RGB (color) and USA Letter Size or A4 (European, etc.).

3. You will be prompted to open images. The 1st image is the templateimage, 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 rightor up to down.

Some images may look pixelated if image was from web (less than 500pixels in both dimensions), or if it was scanned at low resolution (say, at72ppi). You will need to re-acquire the image, or locate the original.

single image1/4 page

single image1/2 page

single imagefull page

single image1 column

single image2 column

single image full“publication”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 pixels-across-by-pixels-down size that matches the camerasystem you used.

3. You will be prompted to open images. The first image is the templateimage, and the following image files will start with the topleft image, then either the image to the right or the image below, and so onleft to right or up to down.

All of the following templates assume six images total. If you have less thansix images, simply cancel the action mid-stream, and then crop to size (seeCrop, page 38).

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

Some Templates for Image Files at Fixed Resolutions

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Auto-Layout Corrections For Lettering

Lettering is in the wrong position. Lettering is normally placed in the corner inwhich the least amount of image information exists.

1. Select the Text Layer in Layers dialogue box (in Actions window, useShow Layers button, then select text layer).

2. While holding down the control/command key, click and drag lettering tore-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 cannotbe edited by double-clicking on its layer in the Layers window (in otherwords, if you wish to change type later, you will need to trash text layerand start fresh).

Lettering Needs to be Outlined in Contrasting Black or White (to separate letterfrom 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 fromvarious sources and resolutions (the images fit into their respective positionsusing a “best fit” function). Your images may be either wider or taller than theheight and width of each “panel” these do not conform to what is called the aspectratio (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 theimage 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 twolayers can also be linked (by selecting the image layer in the Layersdialogue box and clicking grey box next to the text layer to create locksymbol), 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 ContactSheet II function (if you have Photoshop 5.0, you can upgrade to 5.5 by visiting theAdobe web site for the upgrade software). This is an automated function that

1. Under File, select Automate, then Contact Sheet II.

2. Click Choose button and find directory in which your image files arelocated.

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 surroundingimages will not be consistent. Use technique on previous page to reposi-tion images. You will have to apply lettering on your own.

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

Contact Sheet II dialogue box

Click Choose to find directory inwhich your files are located.

Set Width, Height and Resolu-tion.

Indicate how many images youwish to place across (Rows)and down (Columns ).

Uncheck this box if checked.

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You cannot specify width ofborders between images.

Manual Layout of Multiple Images to Make a Figure or Plate

For ease, you may use templates that are provided, already replete with guidelinesfor 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 thebasis of a fixed size in which magnification often needs to be maintained.

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 pagesize (see table on page 8 for commonly used sizes). Set Resolutionto 300 pixels/inch.

3. Set Mode to Grayscale or RGB.

Indicate Width andHeight in inches orcentimeters.

Set Mode to Grayscaleor RGB.

New Image Dialogue Box

Templates Based Upon Fixed-Resolution Images (e.g., from digitalcamera systems)

1. In toolbox, be sure to make background color white (or the color youprefer for the background color).

2. In Menu under Image, select Canvas Size. You will be adding extra spaceto your existing image to give yourself more “canvas,” as in a painting.

3. In Canvas Size dialogue box, enter in height and width the appropriatenumber of pixels to allow for remaining images plus extra pixels forborders. 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 fourimages in your figure, you'll need 2 x 640—1280—for the width and 2 x480—960—for the height PLUS room for borders between pictures—20pixels. 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.

Enter Width and Heightin Pixels.

Click on desired box.White space is addedin direction of arrows.In this instance, imageis on top, left: canvasis added to the rightand below image.

Canvas Size Dialogue Box

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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 thetemplate or page. Photoshop allows for images to be dragged from one window toanother 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 twowindows are open on your screen is to select all of one image, then to cut and closethe 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 buttonis provided to shorten the process.

1. Open image file that will become the top, right image. In Actions, click onSelectAll/Cut/Close, then click on file to which canvas space has beenadded (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 Movetool.

If images are too large, too small, or at an odd aspect ratio, re-size the imageusing Transform- Scale button (or, in menu under Edit, select Transformand then Scale). Hold down Shift key (to keep proportions) when pullingon 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 whatseems to be a good border in between. Place guidelines at the bottom andsides of the images for lining up images and for making borders by drag-ging guidelines from ruler area at the side of the image (if rulers aren’tvisible, in menu under View, select Show Rulers). Hold down Control/Command key when dragging guidelines. Alternatively, You may preferusing a grid overlay for layouts, thus avoiding the use of guidelines. Inmenu under View, select Show Grid (4x, 5x); in 6x & 7x under View chooseShow then select Grid.

3. Fine adjust your images so that they line up along guidelines by movingthem one-by-one until they “magnetize” into place. Make sure Snap toGuides is checked in menu under View.

These will be on different layers, and so you will have to select theappropriate layer before moving in the Layers dialogue box.

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Dragging 2nd Image onto Template to WhichCanvas Has Been Added

2nd image is moved and eye-balledinto place on template after usingSelect All/Cut/Close then usingPaste to paste onto template.

1st image

Drag guidelines to defineedges and borders.

Eliminate extra image area byoutlining with Marquee tool andpressing delete button (make surebackground color in toolbar iswhite).

Zoom in to point at which ruler readings are interpretable (you may haveto change units (keyboard command: Control/Command + k, choose

Determine distance and make new borders for subsequent images at thesame distance apart.

Zoom back and remove images to align to guidelines, if necessary.


Units and Rulers).

Manually Adding Images to a Page, Continued.

4. If you are laying out more than two images, zoom in and use the ruler todetermine the width of your border between the first two images. Dragguidelines into place for additional images along with borders, matchingthe width of your first border.

5. Repeat steps to add additional images.

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.

Add new images.

Crop entire image to removeextraneous image area.

Final Image

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Symbols

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

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 findsymbol desired.

3. Each symbol is on a different layer. Open Layers window (in menu, underWindow, select Show Layers. If you see Hide Layers, then Layers windowis open). Select layer and symbol of interest.

4. Hold down Control/Command key and drag symbol from the downloadedsymbols file to your image file.

5. If size is too large or too small, in Actions window, select Transform- scalebutton (or, in menu under Edit, select Transform then Scale). Place cursorover corner of outline, hold down shift key, click and drag. Some symbolsmay fit your intention better by NOT holding down shift key and scalingfile in one dimension (by width only or height only). You may wish also torotate 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 whileholding Control/Command key to make selection around feature of interest.

2. Click on Make White (Layer) button in the Actions window. Or, in menuunder Edit choose Fill: choose White from the Use: drop down box.

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

Symbols and patterns sizes are matched to an image size that is approximately300ppi. Symbols can be scaled down; patterns, however, may not work as well.

Dragging Symbol from Downloaded Symbols File

Symbol is dragged fromdownloaded symbols file.

In Actions click on Transform-Scale. Re-scale image to desiredsize while holding down Shift key.In Actions click on Transform-Rotate if you wish to rotatesymbol. Copy and Paste (underEdit ) to make additional symbolsat same orientation and size.

To make symbol white select layersymbol lies on and click asecond time on that layer whileholding down Control/Commandkey. In Actions window, click onMake- White.

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 byclicking on Eyedropper tool and then clicking on desired contrasting colorin your image).

3. In Actions window, click on Fill- foreground color button. Or, in menuunder Edit choose Fill: choose Foreground Color from the Use: dropdown box.

Making Symbols Manually

If the symbol you are looking for does not exist in the “symbols” file, you will haveto make the symbol manually.

Arrows and Dashes

1. Make a new layer in Layers Window. (click on top, right arrowhead toreveal drop down list. Select New Layer or use “page” icon at bottom ofdialogue box).

2. In toolbar, set foreground to the color desired for the arrow or dash. Thenfind 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 andarrows (Undo and repeat, if necessary until correct width of line is obtained). For arrows, click on Start or End next to Arrowheads.

In Arrowheads dialogue box, set the Width, Length, and Concavity (orleave concavity at 0) to make arrowhead longer or shorter. An equal widthand length would make an isosceles triangle as an arrowhead.

5. Hold Shift key down, click and drag to draw arrow or dash. Do not holddown 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. Copyand Paste existing arrows and lines to duplicate (remember that each will beon its own layer).

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

Making Arrows Using the Line Tool

In Photoshop 5x, Line toolis beside Pencil tool.

In Photoshop 6x,the Line tool isunder the Texttool.

Set thickness of the lineby placing value inWeight, usually be-tween 4 and 8.

Check Start or End forArrowheads, then clickShape to set values forarrowhead.

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Boxes and Circles

1. Make a new layer in Layers Window. (Click on top, right arrowhead toreveal drop down list. Select New Layer or use “page” icon at bottom ofdialogue 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 orellipse.

3. Make outline (selection) on desired part of image. Hold down Shift keywhile 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 alongyour outline.

5. In Stroke dialogue box, set width to desired size. (2 - 8 is typical). Do notset too small or line may disappear when published or reproduced.

6. Undo and repeat, if necessary until correct width of line is obtained.

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

Photoshop 6x & 7x SymbolsA 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 touse until figuring out how to get rid of the automatic fill (that can be done byclicking on check mark at far right of menu). Working with the Marquee tool, asdescribed in this section, may be the better option, but you can decide for yourself.

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Making Square Outlines

In menu under Edit,select Stroke.

Set width of line youwish to trace alongsquare or circle. (2 -8 is typical).

Outline areayou wish tomake box orcircle around.

Stroke followsoutline.

Filling Image Areas with PatternsPatterns are normally placed into a shape or into an existing feature in your imagefile. In order to do that, you must first select a region of interest. This can eitherbe done manually by using the marquee or lasso tool, or, more than likely you canautomatically select that region of interest with the wand tool. The wand toolselects an area radiating out from where you click on the feature. The selectable

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

1. Open any one of several patterns files downloaded fromwww.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 orgrayscale values, use the Marquee tool or the Lasso tool to make or alter aselection. If you wish to make several selections, hold down Shift keywhile 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 downand use Pattern).

Wand Tool and Dialogue Box

Start with Tolerance at32. Increase ordecrease dependingon efficacy of Wandtool to outline desiredarea of interest.

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Magnification and Scale Bars

In the world in which the size of objects is important when presenting data, somemeans must be employed to provide scale. When a ruler or recognizable objectcannot be included in images, a scale bar provides the best way to give a referenceto 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 isto multiply the lens power by the eyepiece, so that a 10X lens is multiplied by a 10Xobjective to give a magnification of 100X. This way of determining magnification isincorrect because what is imaged by a digital camera is not always at the samemagnification as the objective multiplied by the eyepiece, and magnification is never

exact unless the final publication size is known and the original view of the imagecan 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 electronmicroscopy devices. Calibration slides can be obtained in the microscopy worldthrough microscope sales personnel. Once a ruler of some sort exists, the means formaking a scale bar is fairly straightforward.

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 seeHide 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 onemeasurement mark to another. The Info box will show how many pixelsequal your ruler units.

If you are also using the calibration for subsequent calibration inquantitation software, be sure to measure in both the x and y axis in theevent that your camera system does not produce square pixels.

5. Draw a scale bar using the Line tool while holding down shift key andlooking at the Info box (see page 27 for location of Line tool). Draw thepixel length which equals the length of the ruler unit.

For example, you may draw a line across two measurement marks that equal10 centimeters. The info box may show that you have drawn across 250pixels. If you wish to make a scale bar on an image taken under the sameconditions, you can draw a 250 pixel line and label it as 10 centimeters.

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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 AdobeAcrobat format. Adobe Acrobat files (.pdf) have several intrinsic advantages: thesefiles are compressed, so they don't take up so much hard disk space; the fonts aresaved in such a way that various computers do not need to have the same font foropening the file; and publishing agencies are quickly moving to use this format foroutput 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 one-fourth 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. Itdoes not contain layers. Photoshop gives the option of LZW compression,but that should not be used because then the file can only be opened inPhotoshop and a few other programs.

3. Adobe Acrobat file (.pdf). This file format is now being requested byeditorial boards of journals for paperless reviews of submissions. This fileformat can also be e-mailed, put on a web page, and, increasingly, it can befor the submission of documents, such as grants. This file format is savedwith ZIP compression, because it is non-Lossy (no image details aresacrificed). Choosing ZIP compression does not always result in file sizes

Saving by Using Action Buttons

In Actions, choose buttons for several ways to save (Save asPhotoshop, TIFF, JPEG button is recommended): Save as Photoshop, TIFF,JPEG (this saves in all current formats for several outputs, starting withPhotoshop so that layering is preserved for subsequent editing). Otherbuttons include, Save as- Photoshop; Save as- TIFF, Save as- CMYK TIFF,or Save as-CMYK EPS; Save as- JPEG; and Save as- PDF (Adobe Acrobatfile). You choose the compression for JPEG files: suggested compression ismedium to maximum.

The reality is, however, more sobering. At this printing, it is best to save files notonly in several different formats, but also in more than one mode (ideally in CMYKas well as RGB). Because hard disk space is currently cheap, and because CDrecordable disks and the concomitant devices are also inexpensive, the sufferinglevel 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 ownfolder: One folder is for Original files, a second for Photoshop files, a third for TIFFfiles, a fourth for CMYK files and a fifth for PDF and JPEG files. Thus, actionbuttons are included to save as follows:

that are significantly smaller. Files can also be saved with JPEG compres-sion at several settings. Remember that some image information may becompromised. Check for yourself against the original image.

4. JPEG (Joint Photography Experts Group) file. This file format is ideallysuited for web publication and for PowerPoint. Various levels of compres-sion 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 topublications. Its range of colors (colors it can print) is far less than thevisible range that we can see by eye and the range we can see on ourcomputer screen.

Note: If you wish to save manually, remember that only the Photoshopformat contains layers. To save in other formats, layers need to beflattened (in drop down list in Actions dialogue box, select Flatten Lay-ers. Also found under the Layers Menu in 5x - 7x). If you have openeda PDF, Illustrator file, or from some other formats, you will need to flattenlayers though you may have made none (Photoshop, frustratingly, in-troduces 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 thathave to do with output resolution, or how many pixels per inch/centimeter. Outputresolution values may be changed or left alone, depending on the printer. If youused 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,

Overview of Printers

Setting dimensions and resolution by the kind of printer. Printers fall into fivebroad categories: laser printers (most of which are postscript printers); ink jetprinters and their larger version: poster printers; and pre-press printers. A fourthcategory defines slide writers—those devices which expose digital files to 35mmfilm—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 highresolution printers (defined by dots per inch), images do not suffer much at all interms of clarity. In any case, the detail in the shadow areas and in the whitesusually is not preserved and blacks aren’t deep.

Photoshop typically does not print well to laser printers. Lettering can look fuzzywhen it is re-formed into dots. These printers do not print well with large files, soreduce the size or make JPEGs so that the printer does not “hang up.” Unlessprinters require CMYK, you can print RGB files to these printers.

Ink jet printers. These printers have shown the most promise as versatile, highresolution 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 experimentwith printer settings to obtain the kind of output that matches your computerscreen. For example, on a PC, the Epson® printer makes the best images fromPhotoshop when using the Advanced Setting, and then choosing ICM (InternalCalibration of the Monitor). Choose paper stock carefully to obtain the bestreproduction. Paper stock should be thick and somewhat shiny (coated) for bestresults.

Large files can be printed to these printers with a fairly good tonal range. Somecolors, however, do not have good range at all, such as shades of red. As time goes

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

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

RGB files can be printed to ink jets. Many of these use drivers that eitherconvert files to CMYK transparently, or require CMYK files for output. In eithercase, it is best to make CMYK files yourself so that you can predict the outcome.

Pre-press printers. Several companies make CMYK printers. These includedye-sublimation printers, wax transfer, and a plethora of ink-based systems. Thedye-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 theseto appear in publication, but don’t expect exact color matching unless you havegone through a great deal of work calibrating colors.

These printers also anticipate that you have set both resolution and dimensionsbefore printing: 300 pixels per inch at your desired dimensions (or you may beable to choose the option Fit to Page).

Slide writers. Unlike Pre-press printers, these devices anticipate that files areRGB, not CMYK! Lettering, graphs and drawings are transparently made intoimage files, using a software process known as rasterizing or RIPping (RastorImage Processing). See page 18 for more information on RIPping.

In this scenario, lettering will print as it appears on the computer screen. Main-taining a high resolution of the file is only important insofar as lettering does notbecome pixelated. Resolution that is too high (higher than 400 pixels per inch atpage size) creates resolution that exceeds the device: empty resolution. Filesthat are higher than 15 megabytes exceed the resolution of film, and thus do notprovide 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 paperusing lasers for exposure. The print quality typically exceeds what can be obtainedfrom other printers. Resolution is set to match printer resolutions, and thendimensions are set.

These printers typically take output from Photoshop, and not vector programs,unless RIPping software is also purchased (results are not always expected: somefiles lose information after rasterizing. See other methods on page 16). Expectresolution, 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 OutputWhen desiring that your image prints to an exact dimension—as opposed tomaking a print that simply fills the page (by checking the Fit to Page option in theprinter’s dialogue box)—you will need to reset dimensions and, possibly, theresolution setting, using the Image Size dialogue box. How this box is set dependson the output device. See below for instructions on the output device you use.

For Pre-press printers, and Photographic Printers

1. Set Height and Width to desired dimensions.2. Set Resolution (this is really the output resolution) to 300ppi or to resolu-tion settings of printer. In the latter instance, match to printer’s resolution sothat file size remains about the same (check with manual that came with theprinter).

3. Make sure Resample Image is checked.

Laser Printers and Ink Jets

1. Type in desired Height and Width dimensions. Ignore Resolution values.

2. Uncheck Resample Image

For Publication

1. Set Height and Width to desired dimensions. Normally these sizes areindicated in the publisher’s guidelines. If unsure about the ultimatedimensions, make image two times the likely size of reproduction.

Set Resolution as follows:

If scanned images or text started as low resolution files (<800 or so pixels),these will need to be re-scanned.

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

Images 300ppi

Images & Text 600ppiText/Drawings 1200ppi

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Laser Printers and Ink Jets

Type in desiredHeight and Width

Uncheck ResampleImage

Pre-Press Printers, PhotographicPrinters

Set Height and Width todesired size.

Set Resolution (this is reallythe output resolution) to300ppi or to resolutionsettings of printer.

Make sure Resample Image ischecked

Resolution Values from Digital Systems and Scanned Films

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

For example, if Resolution shows a value of 72 ppi, but the width and heightdimensions show 24 by 24 inches, simply uncheck Resample Image and change theresolution 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 ResampleImage and type 8 inches for width or 10.5 inches for height (the largest printpossible on an 8.5 X 11 inch sheet, allowing for one-quarter inch on each side for aborder).

In like manner, files that were acquired with a slide scanner show a high outputresolution value (e.g., 1200 ppi) and small height and width dimensions (e.g., 1.3inches 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 isto go from RGB files—those files that look good on a computer screen—toCMYK—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, colorscan shift once more from what you see on your computer screen to what is seen onthe screen at the publishing agency and then to what is published.

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

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

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

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

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

Check To See How Much Color Shifts

1. Under View, select New View. A second image will appear.Arrange image windows so that both, or important parts of both, can beviewed (image can also be duplicated).

2. Under View, select Preview then CMYK. Evaluate whethersignificant color shifts have occurred.

3. If colors look okay, select one of the two images. Under Image, selectMode then CMYK.

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If colors are unacceptable, start with the following steps:

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.

Method 1. Color and Contrast Restoration on CMYK Images withSmall Color Shifts

1. Select CMYK image.

2. In Actions window, click on Color- hue & saturation (or, under Image, selectAdjust then Hue & Saturation). Work on colors one by one.

3. In dialogue box, click drop down box next to Edit. Choose a color that hasshifted.Or... Use Eyedropper tool to select offending color.

4. Slide Saturation slider to the right to increase saturation, to the left todecrease. You may also want to lighten or darken with the Lightness slider.

These tools, by themselves, may bring color into acceptable range. Ifthese do not solve the problem with color shift, restore color and con-trast with Method 2.

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

Hue & Saturation Dialogue Box

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

You can edit your selection further by using the Eyedropper tool to clickon the features in your image that contain the colors you’re interested inchanging. Click on the first feature with the unmarked Eyedropper tool,and then on additional features with the plus (+) Eyedropper tool. If youaccidently choose an unwanted color, use the minus (-) Eyedropper toolto 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 (availablewhen a color is selected).

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Most problems withCMYK have to dowith saturation ofhue. Move slider toright to brighten, toleft to affect contrast.Subtle adjustmentsin Hue and Lightnessmay also help.

Method 2. Color and Contrast Restoration on RGB Images withLarge or Problematic Color Shifts

1. Select RGB image. Duplicate RGB image and align on screen so thatimportant 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. Selectcolors by using eyedropper tools as described in the previous method.

Keep gamut warning overlay on, especially when selecting bright objectsagainst a dark background (as in darkfield microscopy). Be sure to use plus(+) Eyedropper tool to select to edges of objects, much easier seen whengamut warning is active.

4. Adjust the Saturation slider first. Keep in mind that all colors will print ifsaturation is set low enough (to the left). Adjust slider until a fair amountof the gray overlay disappears.

5. Adjust Lightness slider by increasing (moving to right) shy of eliminatingtoo much contrast. Now increase Saturation as much as possible (moveslider toward center) without increasing gray overlay. The idea is to workbetween the two, lightening as much as possible without losing contrast,and keeping saturation as close to the center as possible. Don’t be sur-prised if the color you are working on needs dramatic desaturation.

6. Adjust Hue carefully to determine if a shift in one direction or the othermight increase chances of eliminating gray overlay. Play with all three toget close to your desired color and contrast.

You may need to turn gamut warning off and on. That is best done with thekeyboard 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. Thegray overlay dissapears when colors are adjusted correctly. That may nothappen with the saturatedcolors used in research. Expect to remove 90 - 95% ofgray overlay for any hue: the remaining unbalanced color will be at edges ofbright or dark samples. These small areas of unbalanced color are imperceivablewhen printed.

Use Gamut Warning to Preview Out-of Gamut Colors

Everything covered in gray repre-sents what will not transfer well toCMYK.

Gray overlay shows edges where toodark to be seen by eye. Afterclicking on the first point with theEyedropper tool in the Hue &Saturation dialogue box, be sure touse the plus (+) Eyedropper tool toclick on multiple points, includingdark peripheries.

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Image on left is howRGB image appearsas uncorrectedCMYK; on right isthe corrected CMYKimage in green hueonly.

Desaturation using the Satura-tion slider eliminates most ofthe gray overlay.

CMYK Correction for Blue

Shades of blue appear bright on a computer display, but tend to print dark andwithout contrast, especially after being changed to CMYK mode. This means forcorrecting blue tends to print close to what is seen on the screen, but, for exactmatches of hue, you’ll need to use the method on the following page.

1. Move the Hue slider to the left to add green (make morecyan). Move to about -30.

2. Adjust Saturation and Lightness by eye.

Evaluate the color after printing to a hard copy, as opposed to evaluatingby looking at the display on the computer screen.

3. When satisfied with the result, in menu under Image choose Mode, thenCMYK Color.

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

Only in certain colors, such as cyan-blue, does an increase insaturation help.

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 CMYKcolor space, then use the steps that follow. These use Pantone Inks® as colorguides. Because colors contain a range of hues from dark to light, and becauseonly one particular ink color is chosen, color changes may have to take place in

1. In menu under Window, select Show Color. Click on arrowheadat top right and select CMYK Sliders. Leave this window open.

2. Click on representative area of image with eyedropper tool containing thecolor you’d like to match.

3. Double click on foreground image at bottom of toolbar icon. In ColorPicker dialogue box, click on Custom. Click Book drop down list andchoose Pantone Coated (journals are typically printed on what’s calledcoated 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 thena mid-green, and so on. These steps require trial and error, and fiddling is a require-ment.

Click on arrowheadto select CMYKColor.


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Show Color Window

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 Fuzzinesshigh (50 -100). For bright objects against a dark background, keep gamutwarning overlay on to be sure you are selecting to the edge of yourobjects.

You may wish to hide the moving outline (selection) while makingchanges. To do so, press Command/Control key + h. It’s a lot easier to seeyour corrections, but the downside is that you can forget that you’ve hidthe selection later.


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Color Range Dialogue BoxSet to Sampled Colors.

Fuzziness should be set50-100 or so.

White areas indicatewhat has been se-lected.

Use leftmost Eyedropper tool toclick on first point in desired color.Use plus (+) Eyedropper to selectadditional shades of the samecolor.

CheckSelection.

Adjust Colors Using Curves

8. In Actions window, click on Contrast Adjust- Curves (or in menu underImage, select Adjust then Curves).

9. Click on representative color in image with Eyedropper tool (identical orclose to the position chosen when determining ink color).

10. By clicking and pulling line in Curves dialogue box to the left across thetop, or down along the right, adjust Red and/or Green and/or Blue Chan-nels individually while looking at the Show Color Dialogue box to matchCMYK values to those you have written down (see table on the followingpage). Turn View Gamut off and on using Command/Control + Shift + ykeys to test the efficacy of your changes. If the hue doesn’t look right, useCurves line across and down to fine tune by eye while ignoring CMYKvalues 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, adjustRed channel and Blue channel to achieve a similar reading. Values willchange on the Color Palette as adjustments are made in the Curvesdialogue box. Readjust by eye.

Do not expect to see a perfect match of hues! Some colors are outside thegamut (breadth of color available) of what can be printed by a printingpress. Do expect, however, to improve upon the RGB image with a slightlydifferent hue.

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Final Steps

11. Once satisfied with adjustments while in RGB color space, change Modeto CMYK Color: in menu under Image choose Mode, then CMYKColor. Flatten image if prompted to do so (in menu under Layers chooseFlatten Image). Be sure to have saved the RGB image before doing so.

12. For final adjustments and fine tuning, use Hue & Saturation or Curvesto 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, andonce you have corrected the color in CMYK color space so that it matches the RGBhues fairly well, the image may yet be devoid of contrast Often contrast can berevived by simply moving the bottom of the line in the Curves dialogue box to the

Overall Contrast

1. Use Color Range to select areas of interest according to proceduresoultined earlier.

2. In Curves dialogue box, click Channel drop down box. Select Black. Dragline 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 proceduresoultined earlier.

2. In Curves dialogue box, determine where values of interest lie along line. Todo that, click and hold with eyedropper tool on areas of interest. A circleshould appear along the line.

3. Make 3 points at that location along the line. Bend line so that hills andvalleys create shadows and highlights within narrow color range.

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 beintroduced into ranges of tones, whether these are shadow areas, mid-tones orhighlights.

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Reviving Contrast In Highlights Using Curves

Original RGB Image CMYK image after color corrections

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

Green and purple after Curvesadjustments

Working with PowerPointTo Make JPEG Images for Importing into PowerPoint

PowerPoint generally requires small file sizes, best accomplished by making filesinto the JPEG (Joint Photographic Experts Group) format. This format com-presses the file using what is called Lossy Compression. That means someimage information is inevitably lost with this format. The amount depends

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

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

To Correct for Darkened Images in PowerPoint

Images tend to become more contrasty and darker in Powerpoint. You can remedythis problem one of two ways:

If you are printing to 35mm slides, you can ask your service agency changethe gamma (typically the gamma is increased). Software made to write digitalfiles to 35mm slides often provides that. You simply need a willing technicianwho will experiment until an ideal gamma is found for your images.Otherwise, increase brightness and reduce black levels in Photoshop beforeimporting 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 wishto make a “live” presentation from your laptop.

The 35mm aspect ratio must be chosen under File/Page Setup when desiring tomake PowerPoint presentations to 35mm slides. That aspect ratio fits the width andheight of a 35mm slide, more a rectangle than a square. Both aspects are given inchdimensions (in the American versions) without resolution settings. PowerPoint actslike a laser or ink jet printer insofar as the resolution is not important: only thedimensions of the image.

1. Set the dimensions of the image using Image Size dialogue box (in menuunder Image, select Image Size). For side-by-side images, re-size bycalculating how much space you’ll need given the dimensions below whileallowing for room at the top for titles and at the sides (allow extra room atsides when making 35mm slides to allow for space taken by the slidemount). The cropping steps can also be used (see page 38). Be sure touncheck Resample Image. Fit both dimensions or either the width or heightto desired aspect ratio:

35mm Slides

On-Screen

11.25 X 7.5 inches (landscape)

10 X 7.5 inches (landscape)

PowerPoint Files to Photoshop

Early versions of PowerPoint saved to low resolution, pixelatedfiles. The latestversions allow for exporting to TIFF at higher resolution. The latter should bechosen when desiring to open a PowerPoint file in Photoshop. If that option doesnot exist on your version, see page 15 to determine how to get high resolution filesinto 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 agraduated blue background with yellow lettering. The use of these colors came outof 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 blackon clear high contrast negatives (Kodaliths) onto blueprint film. The film was thendeveloped by placing it in a jar with a concentrated solution of Ammonium Hydrox-ide. The result was a deep blue-purple background with white lettering. Thesewere typically mounted on 35mm glass slides. Diazos made sharp edges andprojected better than any other color combination even in rooms that were notcompletely dark. The disadvantage was that diazos faded over time.

Gradients. Those colors have been borrowed for modern computer graphics, withthe exception that a complementary color (yellow) comprises the lettering. Theability to place a darker blue-black at the top of the slide graduating to a lighterblue causes the viewer to look at the area of highest contrast (the top of the slidewhere 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 blueimage file. In Actions, click on any of the following buttons: White on Blue35mm, Yellow on Blue 35mm, White on Blue for On-Screen, Yellow on Bluefor On-Screen. These actions auto-select what is black and paste it over thebackground. 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 Re-size or Transform. (Or, under Edit select Transform, then Scale). Holddown Shift key while dragging the corners of the outline to re-size theimage.

To Make a “PowerPoint” Type Background:

1. Make a solid blue or blue gradient image file by clicking the Blue Gradient35mm Slide, Blue Without Gradient 35mm Slide, Blue Gradient On-Screen,and Blue Without Gradient On-Screen buttons in the Actions dialogue box.

Slides in Photoshop. In that spirit, the following procedures will create White-on-Blue or Yellow-on-Blue graphics for 35mm slide format or for on-screen presenta-tions (at 1024 x 768 pixels) in the landscape dimension. These are meant for black-on-white graphs and drawings with text. You can try to select only the black partsof 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 imagesversus text or graphs on a blue, graduated background, choose the action buttonsin the first step below. In any case, remember that the shade of blue from thetemplate/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 atwww.quickphotoshop.com.

Yellow Lettering on Blue Background from aBlack on White Illustration

Use appropriate Actionsbuttons to auto-createyellow-on-blue imagefrom a white-on-blackimage.

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Color Shifts with Grayscale Photographs

The digital revolution has left us primarily with color digital printers. These tend toshift in color, and mostly toward blue, in contrast to photographic prints from thedarkroom, which often shift toward a brown color. This shift in color is not to beregarded as trivial. The overall color can affect our perception of resolution anddetail. Once, as an experiment, I made several slides with different color casts,

1. Make a typical print in the darkroom using the photographic paper ofchoice.

2. Scan negative from which typical print was made or choose similar nega-tive.

3. Make print without any color adjustments to your digital printer.

4. Compare digital print with print made from darkroom and determine whichway the gray color has shifted.

5. Change mode of grayscale image to RGB color (or CMYK Color if you havea 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, Greenor Blue channels.

7. Change color of each channel in degrees, by 5-10 points, in the direction ofthe color you hope to achieve.

For example, if the color of an RGB image has shifted to blue on the digitalprinter, and you hope to achieve a browner tone, you will want todecrease blue, therefore adding yellow and increase red (to get brown,which is a combination of red and yellow). The 1st print could be -10yellow, the 2nd -10 yellow +5 red, the 3rd -10 yellow and +10 red, and soon. This is called a ring-around print. You may need to do more than onering-around print to arrive at a satisfactory result.

8. Evaluate prints using typical lighting in the room. Even better, evaluateprints under 5500 degree kelvin lighting (available as fluorescent tubes atprofessional photographic and graphic arts supply stores).

9. Save the result by clicking on Save in the Curves dialogue box and apply tofuture images (using Load).

This may not be foolproof, since factory runs of digital print paper mayshift differently. This procedure may have to be repeated after receivingnew 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 thecolor. A radiologist to whom these were shown told me that the bluish-gray imageswere best because these were highest in resolution. The caveat is that the colorshifts inherent to grayscale images printed on a color printer should be seamlesslymaintained from what was printed in the darkroom to what is now done on a digitalprinter. Here’s a method:

Matching Grayscale Colors to Photographic Prints

Photographic Print Digital RGB Print

Select Channel ofinterest

Click arrowhead so thatdark gradient is on the leftbottom.

Slide top of Curvesline to the left ordown to increase ordecrease channel’scolor.

Record Input andOutput Values; typethese on ringaround print.

Determine whichCurves correctionworks best. Clickon Save to save theCurves correctionand apply tosubsequent imagesusing Load.

Organizing & Archiving FilesArchiving. As of this printing, many experts agree that film is yet the cheapest andsafest way to archive material. Of course, that also depends on how the film isstored (in moist conditions mold can form) and in how well the image is fixed to thefilm (if fresh fixer is not used when processing the film, it can yellow and fade after20 - 30 years). If images begin as digital files, then it is wise to use current technol-ogy and update as technology changes. At this time, that would mean burning filesto CD-ROM (do not use smelly inks to write on the CD itself) and, in the nextcouple years, transferring those files to DVD-ROM. When the next technologyarrives, transfer again.

Organizing. Photoshop provides two ways to organize files: as hard copy onpaper 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 theweb as both a one-page contact sheet for low resolution, and as high resolution byclicking on the file name. In this scenario, your files can be reviewed via a webbrowser, such as Netscape or Microsoft Explorer. These functions are not availablewith 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, andnumber of images across and down. Size of lettering, unfortunately, cannotbe made small enough for long file names, except in 7x.

3. Click Okay. This function will continue processing images and makingnew images until it finishes with all files in the directory you chose.

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 filesmade 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 ina banner above your images.

4. Unless you have specific sizes you wish to make, leave Thumbnails andRe-size Gallery Images on default settings (where they are).

5. Click Okay. After all files are processed, your default web program willopen and display the file.

6. Rename file to better describe nature of image files, and leave theextension .htm or .html (Photoshop will only make a file namedIndex.htm. If a file with that name is uploaded to your website, it willbecome your home page. Renaming the index.htm file will solve thatproblem).Because Photoshop only makes a web page named “index.htm,” any newimage files must be included with existing files when making additions.

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Example of Image Files madeinto a Contact Sheet

Summary of Steps

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Index

12-bit 3816-bit 393200 degrees kelvin 935mm slides 843D objects 75500 degrees kelvin 972 dots per inch 158-bit 39. See also Bit-Depth

A4 70A4 size 69Accumulate 14Actions 30Adding color 51

to grayscale images 51Adding white space. See LetteringAlignment 67Anti-aliased lettering. See LetteringAnti-aliasing 64Archiving 101Arrows. See SymbolsArtifact 40Auto-balance 43Automate 74Autorads 8

Brightfield 9Brightness 5, 12, 42

Calibrating monitor. See MonitorsCalibration tool 22Canvas 61

anchor 61CD 20Character palette 65Circles. See SymbolsCMYK 2, 83. See also Mode

color/contrast restoration 89reviving contrast 96

CMYK color 88color shifts 88correcting blue 91difficult corrections 92large corrections 90

Colocalization 60Color picking 92Color range 46, 92

fuzziness 46, 92Color shifts. See CMYK colorColor temperature 9Color wheel 13Combining images 61

two or more into one 61Complementary filtering 13Computer screens. See MonitorsContact sheet 74Contrast 5, 12, 42Coomasie blue gels 8Correcting color 43

changing certain colors 50changing the color blue 50manual corrections 45selected areas 46subtle colors 49

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

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

104

Symbols

A

C

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

B

D

whites 46Correcting contrast 43

manual correction 44Corrupted files 33Crop 38

different dimensions 58fixed target size 59several to same size 57

Crop tool 59Cropping white space 38CRT (Cathode Ray Tube) 21Cursor icons 26Curves 95

adjusting colors 94

E

Index

FDA approval 20Figures 70. See also TemplatesFile formats

Acrobat (pdf) 16BMP 15EPS 16GIF 33JPEG 33PICT 15, 33PostScript 16RAW 33TIFF 15, 33

Film scanner 6Flatbed scanner

filter 2scanning from books 7

Flatfield image 10Flatten layers 83Flip 35Fluorescence 47Frame average 12Frame-average 9Fuji Pictrography 18Fujichrome 6

IDE controller 21Illumination, Kohler 10Illumination, uneven 9Illustrator 16, 18Image files 17Image size 53

resample image. See Image sizeImage type

CMYK 2Image types. See Film scannerImage window 26Indexed color. See also ModeInk jet printer 18Ink jet printers 18Integration 14

Keyboard symbols 80Kodak Gold 6

Laserjet printer 18Layers 55

adjustment layers 55flatten 56

merge visible 56render text 56

LCD (Liquid Crystal Displays) 22Lettering

45 degree rotation 68add white space 63alignment and orientation 67anti-aliased lettering 15corrections for auto-layout 72manual (4x, 5x) 64Manual (6x, 7x) 65outlining 66rotating 67

Levels 44channel 45Levels dialogue box 43output levels 44

Line tool 29, 79Lines disappear 7Lines per inch 5LUT (Look Up Table) overlay 10, 12

105

F H

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

I

G

Gamma 5, 12, 23, 42monitor's gamma 24

Gamut 18Gamut warning 90Glare 13Grayscale. See Mode. See also Mode

color shifts 100Guidelines 32

J

L

JPEG 83

K

M

Magnification 82Marquee tool 32Matching gray levels 48Median filter 5Memory

setting for Macintosh 25Menu 26Merging images 60Microscope slides 7Microsoft Word 17Mode 39Moire patterns. See also Laserjet printerMonitors 22

self-calibrating 22

Index

Open 33Open As 33Open as 33Optical Character Recognition (OCR) 2Optical resolution. See ResolutionOrganizing 101Orientation 67Outlining. See StrokeOutput levels. See LevelsOutput resolution 53. See also ResolutionOutput size 3Over-sampled. See SamplingOver-saturation. See Saturation

Pagemaker 18Palette 26Patterns 81pdf 83Phosphors 23Photo printer 18Photons 69Pixelated Image 15Pixelated lettering 7Pixels

measuring 11, 48Plates 70. See also TemplatesPolarizing 13Postscript interpreter 17

PowerPoint 17, 53, 97correct for darkness 97make yellow on blue 99making JPEGs 97setting dimensions 98

Pre-press printers 86Preferences 28Print to file 16Printers 84

features 85inkjet 84, 86laser 84, 86poster 84pre-press 84, 86slide writers 84

Prints 84Processor speed 21Profile. See also MonitorsProfiles 28Pseudocolor 51Publication 86

RGB color. See Flatbed Scanners; ModeRIP (Raster Image Processed) 18Rotate 35Rotation

Arbitrary Rotation 34Rubber stamp tool 40Ruler units 82Rulers. See Preferences

Saturation 42over-saturation 11

Scale bars 82Scaling 3Scratch disk space. See Disk spaceScratch disks 29. See PreferencesScratches. See Dust and scratchesSCSI controller 21Shading image 10Sharpening 5, 41. See also Flatbed scannersShow color 93Stroke 66Summary 102Symbols 78

manually making 79

Templates 69, 71manual assembly 75

Textarial 64helvetica 64

The Image Processing Handbook 5TIFF 83Toolbar 27Transform 35Troubleshooting 29True color. See Flatbed Scanners

106

N

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

O

P

S

T

Q

R

Quark 18

RAM (random access memory) 21Reducing graininess 48Refresh rate. See Video cardResolution 6, 15, 17

adding 63by file size 6for output 86from digital systems 87setting by file size 3setting for images 53setting resolution 3

RGB 83

Index

Variations 48Vector 17Video card 21, 22

White balance. See Digital cameraWhite Level 42Word Processing Programs 15Working With Color CMYK Files (for Publication and88Working with PowerPoint 97

35mm slide aspect ratio 97on-screen aspect ratio 97PowerPoint files to Photoshop 99rasterizing 18setting dimensions of images to fit PowerPoint 97to make JPEG images 97

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

107

Type tool 64

U

Undo 36Unsharp mask 41USA letter size 70

V

W

Z

quick photoshop for research a guide to digital imaging for photoshop 4x 5x 6x 7x

Documents