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2002 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow Print 2002 Kluwer Academic/Plenum Publishers New York All rights reserved No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: and Kluwer's eBookstore at: http://kluweronline.com http://ebooks.kluweronline.com
Preface
Quick Photoshop for Research: A Guide to Digital Imaging contains essential information for the use of Photoshop specific to researchers. This step-by-step guide is the only book published for users whose needs are not for the purpose of graphic or web design: instead, this book only addresses the tools and functions necessary for the ethical enhancement of scientific images, and subsequent layout of these images into figures or plates. The aim is to provide information about digital imaging in an easy-to-follow guide from the beginning of the imaging process to its end. Additional information about scanning and acquiring images via a digital camera or laser/PMT system is also covered, as well as information about printers and PowerPoint. This book is intended for occasional users, as well as beginning and intermediate users primarily in the life sciences, though it can be applied to forensics, astronomy and engineering. It can also be distributed from core imaging facilities from within universities and corporations for those who use microscopes, scanners, confocal systems, and other imaging devices. Actions can be downloaded from quickphotoshop.com to automate functions covered in this book. Part 1 of the book covers what users should know about imaging, ethics, computer requirements, differences between vector and imaging programs, setting up preferences in Photoshop, and calibrating computer screens before using Photoshop. Part 2 explains Photoshop functions for imaging: open, duplicate, rotate, flip, undo, history, zoom, crop, mode; along with corrections for dust and scratches, color and contrast, and for adding color. Part 3 addresses what functions are used when working with several images to make a plate. You'll learn about layering, cropping several images to the same dimensions, combining images, adding white space for lettering, using the text tool, and changing the orientation of text. Part 4 covers the lay-out of multi-image figures or plates; the addition of symbols such as boxes, arrows, and circles; how to fill with patterns; the construction of scale bars; what file formats need to be saved; how to set resolution for output; the steps for converting RGB files to CMYK maintaining color saturation and contrast; ways to solve hue shifts in grayscale photographs; methods for organizing and archiving files; and specific ways to integrate Photoshop with PowerPoint. This book finally addresses what every researcher needs to know about when working with images with the most popular and widely-used imaging program. It speaks to what researchers know intrinsically in a world that has become far more visual: images often convey more than words; and, like it or not, images leave an impression in recipients' minds, often making the difference between a grant that receives more points rather than less, or between a paper that is accepted in a prestigious journal versus one that is less desirous. Like it or not, images can make or break careers.
Acknowledgments
Thanks to the following individuals
Layout and design: Jerry Sedgewick, Laurel Connett, and Linda Peterson Cover design: Nam Pham Editing: Laurel Connett, Reena Maheshwari, Aria Williams, and Jenny White Templates: Jill Plumb-Smith Cover photos: Paul Letourneau, Ph.D. (fluorescent growth cone), Stan Erlandsen, Ph.D. (EM of mosquito) Inside photo credits: Martin Wessendorf, Ph.D. (fluorescent neurites); Stan Erlandsen, Ph.D. (EM photos) Special thanks to my wife, Jan Shafer, and to my colleagues, John Oja and Jim McCabe, without whom this book would never have been completed.
Contents
Part 1 Before Getting Started In Photoshop
Flatbed Scanners
Typical Choices When Scanning Image Type Resolution/Output Size (or Scaling) Resolution: Setting by File Size Filter Options Contrast, Gamma, Brightness
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Making Non-pixelated, High Resolution Images from Graphing, Drawing & Word Processing Programs Differences Between a Drawing Program (Vector Graphics) and a Paint Program (Image Files) Ethics and Photoshop Computer Requirements for Running Photoshop Computer Screens (Monitors)
Calibrating Your Monitor
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Setting Memory for Photoshop on a Macintosh Visual Definitions Overview of Toolbar Setting up The Photoshop Workspace Troubleshooting Load Actions in Photoshop
Part 2
Mode
Changing Mode To RGB (color) Changing Mode to Grayscale (or 16-bit to 8-bit)
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Rotate/Flip
For Small Degrees of Rotation For Interactive and High Degree of Rotation (useful for rotating parts of an image)
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Dust and Scratches Sharpening Contrast & Color: A Primer Contrast/Color Auto Actions
Correcting Color and Contrast for Typical Images (generally from scanned negatives, slides, or digital cameras) If Auto-Balance Doesn't Auto-determine Hues Correctly and White Areas Exist in Image File
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Crop
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Adding Color
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Adding Color to Grayscale Images (Darkfield) Adding Pseudocolor to Grayscale images (for visualization purposes) Minimizing Grayscale/Color Values (for visualization purposes)
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Subsequent Steps for Single Image Final Resolution and Dimensions for Single Images
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Adding White Space For Lettering in Photoshop Manual 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
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Part 4
Finalizing Figures
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Working With Color CMYK Files (for Publication and Pre-Press Printers)
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Check To See How Much Color Shifts Method 1. Color and Contrast Restoration on CMYK Images with Small Color Shifts Method 2. Color and Contrast Restoration on RGB Images with Large or Problematic Color Shifts CMYK Correction for Blue Steps to Follow When Color Matching is Near-Impossible Reviving Contrast in the CMYK file
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Symbols
Symbols from Symbol.psd file
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Filling Image Areas with Patterns Magnification and Scale Bars Saving Files Prints and 35mm Slides From Digital Files
Overview of Printers
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Color Shifts with Grayscale Photographs Organizing & Archiving Files Summary of Steps Index
Part 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 grayscale graduations, especially in pastel versus primary colors. Also, be aware that the advertised resolution of a scanner is normally twice the true optical resolution. The advertised resolution is interpolated (pixels are duplicated to double resolution).
Image Type
Color materials. Set at True Color or RGB color (whether 16.8 million, 32-bit, 24bit, etc.) for scanning color materials. Photographic type materials (with a range of grays). Set at Grayscale. If a choice of bit-depth is shown (8-bit, 10-bit, 12-bit, and so on), use default setting or 8-bit unless the image density or intensity will be measured (while a higher bit-depth sees a greater number of gray levels, it normally becomes 8-bit again once printed or made into a 35mm slide). Text or line drawings in black/white. Set at Bitmap, Black-and-White, or Binary. At this setting, only pure blacks and pure whites (black/white) will be scanned.
Indexed Color (256 colors) or CMYK. Do not use either of these settings. Indexed Color only records 256 colors and is intended only for the web. You should let Photoshop convert RGB to Indexed Color after scanning your document. CMYK records color intended only for printing presses and is used primarily by graphic designers. Your computer screen shows colors as a mixture of red, green, and blue, thus it makes more sense to scan as RGB. Convert the file later in Photoshop.
Some scanner software makes image type choices for you. To avoid having your choice changed by automation, either change the automatic image type option in a preferences or options menu that may have come with your software; or, after previewing and outlining your area of interest and before your final scan select the image type.
Output Resolution
300 dots per inch (dpi) 600 dots per inch (dpi) 800-1200 dots per inch (dpi) 1200 dots per inch (dpi)
*Dimensions indicated only apply to some journals and are shown here only as ballpark or safe settings.
Output size: If Output Size is not available on your scanner software, use the file size method on the following page. Also use the file size method for a less detailed way to estimate a starting point for resolution settings. Text & black/white images. Smooth edges do not exist when zooming in on text, graphs, and drawings, even at 1200 dpi. The problem is that we can zoom in far
more than at a normal viewing distance (a comfortable reading distance for 12 point type). Print out on an ink jet before re-scanning to make a judgement about quality, or scan at 600 dpi as grayscale. Whites can be whitened and blacks blackened using contrast adjustments in Photoshop (see Page 48), and the image will contain smooth edges.
Be sure to save the original as a separate file in Photoshop. Files scanned at these resolutions can be made much larger in file size and dimensions without losing detail. If only a part of a scanned image is used, as in an EM negative, but the part has not yet been determined, scan at a higher file size.
Filter Options
Often scanner software includes several options for filtering your image. The most common include descreen (often median filtering) and sharpening functions. Sharpen. Sharpen functions work far better in Photoshop, so it is best to turn off any sharpening in the scanning software. Descreen (or median). This filter reduces or nearly eliminates the dot pattern in pictures from magazines and journals. These dots are typically at a density of 133 dots per inch(dpi), also called lines per inch (1pi). Newspapers are less dense at 80 dpi. Scanner software can do a great job of nearly eliminating the dot pattern to solutions that end up simply blurring the scanned image using a median filter (median filters really don't work well; these are made to retain edges and blur everything else). The best descreen functions use a fourier transform in their process. More information about fourier space can be found in The Image Processing Handbook by John Russ, or in the software manual that accompanies the Image Processing Toolkit, (a software program that provides Photoshop plug-ins) by Chris Russ (members.aol.com/ImagProcTK/). However, most descreen functions use a median filter, so be aware that descreen may not always give you the results you want.
Film Scanners
The same adjustments are made with film scanners as with flatbed scanners, except that film scanners allow for a far greater selection of image types, and the method for setting resolution is different. Image types. Film scanners allow for making a selection of image type on the basis of the kind of film used. For the most part, slide film is Ektachrome or Fujichrome and color negative film, as of this printing, is typically Kodak Gold. Generic settings that may be entitled raw negative and raw slid e exist if you are unaware of the film type. Resolution. The resolution of film is based upon pixels across and down. Use the file size approach.
Film which contains lettering will come out fuzzy. The image will need to be relettered, or you can sharpen in Photoshop (but dont expect great results).
Scanning Tips
Moire patterns. When scanning pictures from books and magazines, often you will see a moire pattern on the computer monitor, which looks a lot like two screens that are positioned at cross axis. This is NOT a resolution problem, it is simply the phenomenon of the dot pattern that makes up the computer screen crossed against the dot pattern on the magazine picture. Zoom in to make your evaluation. However, you may also notice a moire if you print out on a laserjet printer. This is because the laserjet also screens another dot pattern over your image to print. The easiest way to get around this is to print out on an inkjet. Disappearing lines. Also, when images are zoomed out, horizontal lines can disappear. Always zoom in to check to see that everything was scanned to your satisfaction. Pictures and text from books and journals. Whenever scanning from books and journals, you need to place a black piece of paper behind the page you are scanning to avoid any bleed-through from printed material on the other side of the page. Paper is usually thin enough that text bleeds through from the backside. You may want to Descreen to eliminate dot pattern if it is a picture (drawings usually don't get printed with a dot pattern). 3D objects. Do not hesitate to scan three-dimensional objects on a flatbed scanner. You'd be surprised at how focused the image can be, depending on the quality of the scanner. I have seen good results at up to 40mm depth through plexiglass. Microscope slides and like materials. Low power images can be taken with a flatbed scanner, useful up to about 2x - 4x. You will need to scan at ~1800ppi or better at true optical resolution. Certain slide scanners can be adapted to scan microscope slides. If you can adapt, these scanners give even more resolution, with the advantage of perfectly even illumination in background Pixelated lettering. Lettering that is zoomed up too far will look pixelated. Zoom out to a normal viewing distance to evaluate the quality of your scan; remember that horizontal lines may disappear. Print out results to evaluate on an ink jet printer, if you wish to be sure.
Coomasie blue gels. These gels can be scanned dry or wet (as long as care is taken to keep water from running to the edge and seeping into the electronic equipment). Because the blue color of coomasie is so light, the gel either needs to lay on a sheet of darkened glass, or the contrast needs to be high while brightness is turned down. If the gel is wet, be sure to wet the glass with a squirt of water before transferring. Smooth out the bubbles that form underneath. Scan as quickly as possible to avoid the accumulation of more bubbles due to the heat from the scanner. Autorads. Autorads, or x-ray films, are fairly straightforward. Scanner software can read these easily with its internal auto settings. If the film is exceptionally grainy, however, the graininess may be removed by placing the film on a sheet of glass, and then raising that glass off the surface by using coins or consistently sized spacers. Gels can suffer from slight out-of-focus detail and still look good. Don't be timid about de-focusing bands to eliminate grain. Electron microscopy films. Beware of Moisture rings if placing on glass. Do not press on glass. I havent found a good way to prevent these rings from occuring when in contact with glass, except to raise the film slightly off the glass using strips of tape. Nitrocellulose. Any material is easier to scan than nitrocellulose. The stain is so dim that every last wrinkle in the paper must be brought out to see bands. Place the nitrocellulose blot on a piece of darkened glass and raise the glass off the surface with coins or spacers and then raise contrast and lower brightness. Note that nitrocellulose is notorious for moisture rings (see above). Newer scanners typically do not use glass for large format films.
Scanning of Gels
Moisture Rings
White balancing is done with brightfield images (dark objects on a white background) NOT with darkfield, such as fluorescence (bright objects on a black background). With darkfield, not only do pure hues exist, making color interpretation unmistakeable; but the lamps and lasers do not provide color temperature references.
Color Temperature is a reference to light on the basis of whether the light is warmer or cooler. Measurement values are given for light in degrees kelvin. A warm light would be incandescent light: a yellow light. A cool light, on the other hand, would be sunlight on a blue sky day, which tends toward blue. If you think of an iron pot being heated by a fire, a cooler pot would be yellow, where a much hotter pot would be bluish. Its precisely that conception which gave rise to this way of thinking about and referring to light.
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Avoid Over-Saturation
Avoid saturating white- and black-end of images (especially important with confocal images). Avoid increasing brightness, or lowering black level so that image details are lost. Nothing is more unsightly than blobs of gleaming, flat, fluorescent-stained cells on a puddle of pure black. Pixels can be measured. By placing the cursor over representative pixels on your computer screen, pixel values often display at bottom of the screen in your acquisition sofware. In Photoshop, the values are indicated in the Info Box. In menu under Windows, select Show Info (see page 48) A pixel value of 0 indicates over-saturation in the blacks; a pixel value of 255 (8-bit), 4096 (12bit) and 65,536 (16-bit) indicates over-saturation on the white end. It may be opposite on a Macintosh.
Rule of Thumb: In Photoshop you can always INCREASE contrast (make blacks blacker and whites whiter), but you cannot create detail where it never existed (where pixels are over-saturated). Choose highest bit-depth available. Your camera system may include a higher bitdepth than the standard 8-bit-per-channel. Choose a higher bit-depth for greater resolution of gray values and somewhat more ability to make out details in the shadow and bright areas. Your software should also have the option of saving as 16-bit. Photoshop can only open 8- and 16-bit image files. Human eyesight doesnt see all grays. More detail exists than we are able to detect on the monitor. Human eyesight is limited to the ~100 gray level. Detail is unseen, especially in the shadows or in the black region. Thus it's better to keep detail in blacks in case you need it than it is to throw it away by over- saturating.
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How to Set Levels When Acquiring Images to Avoid Oversaturating Pixels. Using LUT overlay. Confocal and CCD software often includes a pseudocolor overlay to show when blacks or whites are over-saturated. On Bio-Rad software, for example, a LUT (Look Up Table: a graphic overlay that is not part of the image) named SetCol is used to show green wherever the image is over-saturated in the black region, and red where the image is over-saturated in the white region. By adjusting both the white level (alternately called brightness) and the black level (alternately called contrast), you can bring pixels in range. By providing this overlay, our limited vision, and the variances of how the computer screen is set in terms of brightness, no longer play a part in determining where to set the black and white levels (this is especially important when subsequent intensity or density measurements will be made). Look for pseudocolor overlays, often called LUTs, in your image acquisition program. Change gamma when whites/blacks are out of range. Some instances occur in which whites are so intense in parts of your image, and dark values so dark, that your only option for capturing both within a single image is to change the gamma value of the camera (not always possible on a CCD camera). Gamma settings bend the midtone grays, typically increasing the brightness of the darkest pixels in your image. If a gamma setting does not exist, take two separate pictures: one underexposed to capture the light part of the image, and the other overexposed to capture the dark parts. Recombine in Photoshop or Image J (see www.quickphotoshop.com). Please note that density/intensity measurements cannot be taken when the gamma deviates from a linear slope of one. Any bending of the gamma results in a log scale, and makes subsequent measurements incorrect. Also note that background should be subtracted when subsequently measuring density/intensity to correct for dead pixels on the CCD chip. The background image is acquired when light does not illuminate sample. This image can be saved, then subtracted later, or included in automated protocol that may have come with acquisition software.
LUT Overlay
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Glare. Problems with glare can be solved three ways: If possible, scan on a flatbed scanner. Try unlikely samples, including those that are 3-dimensional. If possible, submerge in a bath of water. Polarize the light. Purchase polarizing filters for BOTH the light sources AND a rotatable filter for your imaging system. Place filter in front of your light source on its own stand. If more than one light source exists, set polarizing filters at the same axis (axis should be marked). Then turn filter in front of camera lens while viewing through camera. Glare should disappear when turned to the point at which you obtain cross-polarization. Continued on next page >
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Functions For Brightening Dim Samples Accumulate (for dim signals). Accumulate can be chosen to brighten an exceptionally dim signal. Accumulate adds one frame to another when acquiring an image. Thus, brightness builds as images are acquired. Electronic noise also increases, but this can be used with frame averaging to improve signal to noise. Integration (a cheaper alternative to buying a cooled camera: cooling is to reduce dark current noise as a result of heated sensors on the camera's chip). Another way to increase signal from dim samples is to integrate on the chip. This is really no different than increasing shutter speed on a 35mm camera. Integrating allows the chip to collect photons over longer periods of time, but it, too, increases noise and requires frame averaging.
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Making Non-Pixelated, High Resolution Images from Graphing, Drawing, & Word Processing Programs
Many drawing, graphing, UNIX, and word processing programs do not provide the option for making high resolution image or text & drawing files. With the exception of a few notable programs like CorelDraw, Canvas, Illustrator, and the newest version of PowerPoint, options for saving or exporting files to TIFF, BMP (bitmap: in this instance, a bitmap is simply an image file, NOT a pure black and pure white drawing), or PICT can only be done at 72 dpi. The problem with a resolution that spreads text and lines across only 72 dots or pixels per inch is twofold: 72 dots per inch comprises too few pixels and text looks chunky and pixelated. No provision is made for creating text with a ghost image behind it for confusing the eye (what is called anti-aliased lettering). Only programs that allow you to save or export files at higher than 72dpi, and also allow you to check "anti-aliased lettering," will produce high enough resolution. Images saved as JPEG can look good, but these are compressed files, and some image information is thrown away (judge for yourself). Please note that the indication of an output resolution of 72 dpi does not always indicate low resolution of the image itself. If the source of the image is a digital camera or Photomultiplier Tube (PMT)/laser system, then Photoshop is guessing the output resolution incorrectly.
Setting Save As, or Export Options in Programs that Allow High Resolution Images and Text: 1. Set image size in height and width to desired size, or choose from below. Set at these resolutions, depending on image type: Image Type Resolution Output Size: 1 column/2 column Color or Grayscale images without text 300 dots per inch (dpi) 3.3 inches wide (8.5cm)/6.85 inches wide (17.4cm) Color/Grayscale with text 600 dots per inch (dpi) 3.3 inches wide (8.5cm)/6.85 inches wide (17.4cm) Text and Black/White Drawings (binary) 1200 dots per inch (dpi) 3.3 inches wide (8.5cm)/6.85 inches wide (17.4cm) 2. If option exists, check anti-aliasing.
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If you can only produce low resolution, aliased files from your drawing/graphing/word processing program, try one of the following methods:
Method One (Works 100%) Print the File/Scan Result 1. Print the file to an ink jet or laser printer. 2. Scan resulting page on a flatbed scanner (see Flatbed Scanners for details). 3. Save to an image file (such as TIFF).
Method Two (Works 99%) Save Big/Make Small in Photoshop. 1. Quadruple the size of your graph or drawing and its page size. 2. Export to a TIFF, PICT or BMP file at 72 dpi. 3. Open in Photoshop and scale down. Rough edges will disappear,
Method Three (Mostly works: postscript files work intermittently) Save as Illustrator File, EPS (Encapsulated PostScript), PostScript, or Adobe Acrobat 1. Look for some way to either Save, Export, or print the file to an Adobe Illustrator file, available on some software programs. Better yet, buy the full version of Adobe Acrobat* and print to an Acrobat file (PC), or drag to into the Acrobat printer icon (Macintosh). These files open in Photoshop. Be sure to set resolution/size accorting to chart (p. 18) if prompted in Photoshop. 2. If no option exists, then open your print dialogue box by printing your file to a laser printer (postscript printer) or, alternatively, to Laser Writer 8 (on a Mac). 3. Search for an option to Print to File versus printing to the printer. 4. You may have to name the file and select from postscript choices. If available, check EPS. If not, go with default settings. 5. Click OK. Postscript file will be generated. Open Postscript file in Photoshop** and set parameters for dimensions and resolution (300 dpi). Photoshop does not always open file. CorelDraw is far more universally accepting. * See quickphotoshop.com/making_tiffs.pdf for more information on Adobe Acrobat. * * You may only obtain lettering without image files.
Choose postscript printer. Check Write or Print to file and EPS (if possible). Rename file with .eps or .ps (for postscript file).
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Differences Between a Drawing Program (Vector Graphics) and a Paint Program (Image Files)
If you have been accustomed to using such programs as PowerPoint and Microsoft Word, you have been using what are called vector programs in programmers parlance. These programs are made with the intent of going to a laser printer, or at least with the intent of going through a postscript interpreter. The postscript interpreter, in the simplest terms, provides a way for the computer to talk with the printer and to assign it font descriptions. VECTOR FILES Vector is meant to be printed to a laser printer. Vector uses pixellated fonts and lines (on the computer screen) to make smooth fonts and lines in printouts. Vector needs fonts (which may or may not exist on another computer). 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). It also interprets so that the contents of your document can be scaled up or down in size without a loss in detail. Image files, on the other hand, are intended for photographic printers, ink jets, and 35mm slides. These are comprised of pixels, and the number of pixels is fixed by the files resolution. Some differences are listed below: IMAGE FILES 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% to 120% 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 an image comes closest to coexisting in Photoshop 6x and 7x. Most the time, either you have representations of letters and lines NOT meant to look good on your screen, but meant to print out well on a laser printer (vector), or it is made up of pixels (image files). Software programs are intended for specific output devices. In the broadest view, vector files are meant to be printed to laser printers. Image files are meant to be printed on photo printers or slide writers. When image files are printed to laser printers, results are unpredictable and not representative. Conversely, vector files usually cannot be printed as images unless RIPped (Raster Image Processed: made from vector to imageor rasterfiles). The best solution is had with ink jet printers, though these are slower than laser printers. Ink jets do a good job of interpreting text, lines and images. Remember, however, that these printers depend upon the quality of the paper to which files are printed, and these devices cannot print out as broad a range of color (gamut) as photographic printers (see page 84). Rasterizing, When vector programs like Powerpoint are used for making slides, each file must be made into a bit image) file before it can be written to a slide Again, the file needs to be RIPped.
Pagemaker, Quark, Framemaker, and Illustrator (all page layout programs, all vector) are meant to deal with both text and image files. In each of these programs, the image is not opened or imported into the program, it is PLACED: the image, in this instance, is simply linked or referenced so that it can be found when printed (thus, image files must be included with these page layout program files when submitting these to a publisher, along with fonts). What you see on the computer screen in these programs (with the exception of Quark) is a low resolution representation of the PLACED (or linked) image.
Dots vs. Pixels when printing. Note that a laser printer must form images from dots, not pixels. Ink jet printers use irregular dots, and these generally print photo quality. Photo printers, on the other hand, melt dye onto paper (thermal or dye sublimation printers) or expose a silver emulsion with lasers (such as the Fuji Pictrography printer). In both cases images are not separated into so many dots. Because images are not formed into dotswhich are typically larger than individual pixels even the highest resolution laser printers, which now print at 1440dpi, do not match the resolution of 400 pixel per inch photo printers.
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picture was taken and the nature of the material photographed, all of which constitutes good laboratory practices, and all of which can be defensible and legal for such matters as FDA approval. Notes in the form of annotations to the image itself can be included, if the acquisition software allows for it (Photoshop 6.0 gives the option for annotation). That image, or a second copy, can be watermarked so that it cannot be altered. These can be saved to a CD for permanence (at least the degree of permanence necessary in most of research: 10 or so years, or until the next technology takes over, at which time these images are transferred to the newest archival material). More details on this can be found on page 83, Saving Files.
Let those who view your images know when images have been enhanced in Photoshop. A simple, Images Enhanced in Adobe Photoshop will do that.
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1. Using controls on your monitor itself, adjust brightness so that it is at the halfway point, and contrast so that it is all the way up (as contrasty as possible). 2. If not done already, set up computer screen for color and contrast using Adobe Gamma: Windows: 1. Click on Start. 2. Select Settings/Control Panel. 3. Double click Adobe Gamma. 4. Choose Step by Step Wizard (See steps at right for details). 5. When setting gamma, choose 2.2 (Europe: 1.8); this is the author's suggestion.
Step 1: Description or Profile. Your Profile is found by Adobe Gamma. Change to another setting if you are certain of the brand (many Mac monitors are Trinitron monitors, and should be set, therefore, to Trinitron) by choosing Load. In your monitor manual, the manufacturer may suggest appropriate icminternal calibration for monitorsas a starting point. Otherwise, use the default description. Step 2: Brightness and Contrast. The contrast and brightness controls refer to the controls that come with your monitor. Older monitors have dials, newer ones have buttons usually situated below the screen. Once adjusted correctly, you will see a dark grey box against black box(es). Typically, the contrast dial or button is turned to its most contrast setting and the brightness dial or button is up about halfway. Step 3: Phosphors. Again, use the manufacturers recommendations if you wish to change Phosphors settings. Otherwise, go with Phosphors that are indicated.
Mac: 1. Click on Apple symbol (upper left). 2. Highlight Control Panels/Adobe Gamma. 3. Choose Assistant. 4. When setting gamma, choose 2.2 (Europe 1.8).
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Step 4: Gamma. Uncheck View Single Gamma Only. You will be able to set the three color channels, red, green and blue. The gamma setting determines how bright the values between white and black are (the midtones). Adjust sliders until the box inside matches, as best you can, the brightness of the inner box to the brightness of the patterned frame. Desired Gamma. Set to Windows Default so that your gamma is 2.2. At this more contrasty setting, you will be best situated to judge the output from 35mm slide making devices, from the web, and from many digital printing devices. Ink jet devices (as of this printing) tend to print less contrasty. If you consistently print to a less contrasty output device, you may want to set this to 1.8. The European standard is also 1.8 (Macintosh). Note: this option is not available on Windows systems that cannot control the monitor. Step 5: White Point. Use manufacturers recommendation, or click on measure and follow directions. You will be asked to choose the most neutral square. A cool square tends toward blue or green, a warm square toward red or yellow.
Note: If, at 5000k, the calibration looks dull, dark or yellow, you may need to replace monitor. Use 5000k specifically if you wish to print or make slides. Use 7500k to 9300k when working on images for the web. Various settings can be saved and then loaded depending on how you plan to ouput the image.
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Visual Definitions
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Overview of Toolbar
Toolbars. Image programs use tools for functions. Somebut not alltools are duplicated in the menu. Not all tools are shown. Click and hold to see more tools on those tools with small arrowhead at bottom right. Click and drag on solid bar to move. Move Tool. To save time going back and forth between the Marquee tool and the Move tool, simply remain on the Marquee tool. When you want to use the Move tool, hold down the control/command key and the Marquee tool will become the Move tool. Photoshop 6x, 7x: The Photoshop 6.0 and 7 Toolbar contain all of the same tools as well as some more, and they are arranged slightly differently.
Marquee Tool. The default tool is the Marquee tool in the uppermost left corner. When not using another tool, click on this.
Line Tool. If you were used to using Photoshop 4x, the Line tool was found at another position. It now existshere for 5x. More than likely, you ll have to click and hold on the Pencil tool to choose it.
Hand Tool for navigation Foreground/Background. The bottom of the toolbar contains two squares; the one in front is foreground, behind is background. All tools use the foreground color. If you click on two smaller boxes under the two squares, you will make the foreground pure black and the background pure white.
Eyedropper Tool. The Eyedropper is good for finding a color in your sample and clicking to transfer that color to the foreground. The Eyedropper automatically activates in dialogue boxes for other functions, like when adjusting contrast. Thus, the foreground color can potentially change often when using Photoshop. If you click on the "two arrowhead" curved line, the foreground/ background will change position.
Make Invisible/Visible. You can make the toolbar invisible by pressing the tab key. To make it visible, press tab key again.
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In Menu Under View highlight the Following Show Rulers so that rulers appear alongside your image Show Guides so that guidelines will be visible (6x & 7x find Show, then Guides)
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Troubleshooting
Cannot complete request because the file layer is locked or Cannot complete because area is empty. 6x & 7x auto-locks the background or 1st layer. Duplicate layer by dragging background layer into page icon at bottom of dialogue box and make changes on the duplicated image. Or, choose the layer you intended to work on. Computer crashes often in Photoshop. Usually that means not enough space exists on the hard disk. Free up space by eliminating files, or set scratch disk space to take advantage of a second or third hard disk (see page 28). You may also want to defragment your hard disk. Crop tool magnetizes to the edge. See page 38. Crossed line over circle icon appears. You are on a text layer. Choose another layer in the Layers window. Illustrator and Acrobat files cannot be saved as anything but the Photoshop (psd) format. An invisible layer is introduced into illustrator and acrobat files. Flatten the layers by choosing that option in the Layers dialogue box drop down list, or by choosing that option under Layers in the menu (5x, 6x & 7x), then save the file to the format of choice. Function is chosen, but nothing happens. A selection has been accidentally drawn. Turn off selection by using Control/Command + d on the keyboard, or under Select, choose Deselect. Or, a selection has been hidden. Reveal the selection by using Control/Command + h; or, under View on the menu choose Show Guides in 4x and 5x, Show and then Selection Edges in 6x and 7x. Line tool doesnt make a line. Be sure that the foreground color on the toolbox is not the same color as the line you are drawing (such as a white foreground color and thus a white line - on a white background). If that is not the case, make a new layer (if it isnt 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 (its a Bitmap, Indexed Color or a 16-bit image) or a text layer is selected (in Photoshop 5x, 6x & 7x). Merge Visible is grayed out in Layers Menu when merging visible layers (layers with eye symbol). Click on a layer in which the eye symbol is visible, then Merge Visible from the Layers drop down list. No pixels are more than 50% selected. The selection edges will not be visible. When Feathering, this warning indicates that you will not be able to see the marching ants selection. Usually, you will need to make the selected area larger, or simply ignore the message (click Okay). Scratch disks are full. Photoshop requires at least three times the hard disk space than the size of your file. If you have more than one hard disk, make the least filled hard disk your primary scratch disk. Find Preferences (Control/Command + k) and then select Plug-Ins & Scratch Disks. Otherwise, you will need to delete or move files from your hard disk to make more room. You may want to reduce the size or resolution of your image using the Image Size dialogue box if the file is over 20 or so megabytes (under Image, choose Image Size); or you can Merge Layers or Flatten the image as layers add a great deal to the file size (use the Layers Window drop down box to find these). This action cannot be completed... Change the Mode of the file. The operating systems can also interfere with Actions. If its an action that came with this manual, check www.quickphotoshop.com for updates and/or to contact author.
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Marquee tool
1. Click on the Marquee tool at the top left of the toolbar, or type the letter m. 2. Hold down the Marquee tool to reveal other shapes you may want to use. 3. Click on a starting point for your selection, hold and drag to outline the area of interest, then release. 4. To get rid of a selection, you can click just outside the selection once, inside the selection twice, or in the menu choose Select and choose Deselect. Alternatively, use the keyboard: Control/Command + d.
The Marquee tool should always be selected after using other tools. In that manner, the Marquee tool functions as home.
To Use Guidelines
1. In the menu under View, make sure Show Guides and Show Rulers are
selected (4x, 5x). It should not read Hide Guides or Hide Rulers. In 6x & 7x, under View, Show Extras should be checked. 2. To include a guideline, simply click on the ruler and drag. A guideline will appear. Drag down or across to the desired location. 3. To remove a guideline, hold down the control/command key, or click on Move tool, and drag the guideline back to the ruler area. Guidelines are useful for setting borders for areas of interest. Once guidelines are in place, these serve as constraints for either the Marquee or Crop tools.
You can choose View in the menu and then Snap to Guides if you want to outline an area of interest using guidelines to constrain the selection.
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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 open Photoshop. First open Photoshop, then find files under File/Open or File/Open As.
Open As Window
1. On PC: Under Menu select File/Open As. On Mac: Under Menu select File/Open and check Show All Files (4x, 5x). On 6x & 7x All Files show without check mark. 2. Select a file type from the drop down list. Start with Photoshop file, then TIFF, GIF, JPEG, PICT, and so on. Often a thumbnail image will appear to let you know you found the correct file type.
Files are TIFF files, but the software that created them is relatively new (or odd). Try opening these files in programs with broader definitions of what a TIFF file looks like. NIH Image on the Mac is such a program (or Image J in the Windows platform); or CorelDraw on the PC. Re-save as TIFF and as another image format (such as bitmap.bmp) just to be sure.
RAW. You can also open as RAW. If you know the values, you can either enter values for pixels across in X and down in Y, indicate whether the image is 8-or 16-bit, leave count and header where Photoshop sets it; or you can click Guess.
Click on triangle to expand list. Choose the file type you think it is.
Corrupted File
Files have been corrupted. Corrupted files sometimes open, but when they do the image looks like it has been cut in strips and the strips have been re-arranged. If the file does not open at all, open as RAW (see above). Grayed out menu functions. Some files open, but only a limited number of functions can be used. See section on Mode on page 39.
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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 do not save over your original!
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Rotate/Flip
Especially when scanning artwork and negatives on a flatbed scanner, resulting images are tilted. These images can also be upside down or, in the case of negatives, scanned with a negative flipped in the wrong direction. The following functions will correct these problems. 1. In Actions window, click on Rotate 180, Rotate 90CW (clockwise), Rotate 90CCW (counter-clockwise), Flip Horizontal, or Flip Vertical (these functions are repeated in the menu: under Image, select Rotate Canvas).
For Interactive and High Degree of Rotation (Useful for Rotating Parts of an Image)
1. Select area you wish to rotate by clicking and dragging with the Marquee tool. 2. Under Edit (Layer in 4x), highlight Transform, select Rotate (or use Control/ Command key + t) 3. Double arrow forms instead of crosshair for cursor. Click, hold and rotate. Unclick when desired rotation is acheived, then double click inside selection to complete the rotation (or click on Marquee tool in the Toolbar; then click Okay).
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History Window
History
You can undo several steps back by opening the History window and clicking on the last step you wish to retain. This option is not available in Photoshop 4x.
1. Under Window, choose Show History (if you see Hide History, then History window is available on the screen). 2. Click on steps back to where youd like to be.
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Double click on hand tool to zoom back to normal magnification. This can be done if image is zoomed in accidentally when magnifying tool is in motion while simultaneously clicking (image is super-magnified).
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Crop
Unlike a drawing, word processing, or graphing program, white space or the white background is part of the image. Thats because both the white space the image sits on and the image are made up of pixels (picture elements). Extra white space, in this scenario, comprises extra unnecessary pixels that need to be eliminated. Furthermore, only part of the image itself may be important. In both instances, the extra area needs to be cut out, or cropped. This function does not add or reduce the number of pixels making up the part of the image that remains. For those who wish to also crop to match two different images in terms of size, or for those who wish to adjust image sizes to match their output (for a page, a slide, an on-screen presentation, etc.), it is recommended that you do that after several other steps. It is best to adjust image size and resolution as one of the last decisions.
1. Click and hold on Marquee tool (dotted square icon) at top of toolbar. Select Crop tool (if you mistakenly double click on Crop tool and a dialogue box appears, simply close or ignore it for now). 2. Outline area of interest by clicking at corner and dragging. 3. Fine tune selection by readjusting outline. Move cursor over corner or between corners along the outline until arrows form ( ), then click and drag to reposition. 4. Press Enter or Return key, or click inside box to complete the crop (or click on Crop tool in the toolbar and respond to message that will pop up).
If the cropping outline is near the edge of your image window, it tends to jump. In that instance, zoom in before adjusting. Or, click on marquee tool, outline the part of the image you desire (middle of crosshair is the writing point), and move the outline into position, if necessary, pixel by pixel with the arrow keys. Re-shape the outline in 5x - 7x, if necessary, by choosing Select, then Transform. Under Image, select Crop. Excess white space
Crop tool outline - reposition after outlining by moving cursor over corner boxes. Click and drag to reposition around area of interest. Outline is drawn from center of Crop tool icon.
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Mode
The following functions in Photoshop will not work unless the image file has certain characteristics. In Photoshop parlance, that is called the mode of the image file. Only three modes can be worked on with all functions available: Grayscale (what is called a black and white in photography: all gray tones including pure black and pure white), RGB (Red, Green, Blue, or color files containing the three primary colors of light), and CMYK (Cyan, Magenta, Yellow, and Black, or color files containing the three primary colors of pigment plus black). Color files should be worked on as RGB until the end of the editing process, since computer screens work on the principle of mixing varying degrees of red, green, and blue light versus pigments. Furthermore, files also contain varying levels of gray tones from black to white. Typical files contain 256 gray values; these are known in programming parlance as 8-bit files. In the world of research, however, files can contain 65,536 gray values, what is known in programming parlance as 16-bit. Only 8-bit files can be worked on with all functions available. Note that Indexed Color files (those files, typically from the web, which contain 256 colors versus millions) and Bitmap files (those files which contain only pure black and pure white with nothing else) must be changed to RGB and Grayscale respectively. Other modes are rarely encountered. Photoshop will not open most 12-bit files. These need to be converted into 8-bit or 16-bit in the program that created the 12bit file.
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Before changes
After changes
Before changes
After changes
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Sharpening
Several actions have been provided for varying degrees of sharpening. The automatic sharpening actions use the Filter>Sharpen>Unsharp Mask function, which gives greater control over the sharpening effect. Almost every image can improve with the use of sharpening filters, but look carefully at the image to determine whether the effect has led to a pixelated or grainy image. Do NOT sharpen images from which density/intensity measurements will be taken.
1. In Actions window, scroll down to gray Sharpen/Dust sequence of buttons. 2. Choose among several sharpening amounts, marked lowest to highest. 3. Under Edit, choose Undo (or use Control/Command + z) if sharpening introduces pixelation. Or, under Filter select Sharpen and Unsharp Mask. Set the slider Amount by eye (make sure Preview is checked), and keep the Radius between 1 and 2 and the Threshold at zero.
Higher resolution images can tolerate greater amounts of sharpening. JPEG images, on the other hand, can suffer from sharpening regardless of resolution.
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If colors are altered on an image in which hues relate to intensities or some other measurement, be sure to include standards in the image BEFORE altering color.
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If Auto-Balance Doesnt Auto-Determine Hues Correctly and White Areas Exist in Image File
1. In Actions window, scroll down to blue Contrast/Color sequence of buttons. Select Color-White Level. Or, under Image select Adjust, then Levels. 2. Click white balance eyedropper tool on whitest spot in your image. Photoshop will auto-balance from the point you clicked on. 3. Set Output Levels to 254 on the white end if you are working on an image. If you are working on a graph, or in the white area above a gel, keep at 255. An output of 254 will make the white in the whitest areas slightly gray (to distinguish the edge of the image from the white of the page your image will be printed on). Under Edit, select Undo if you do not like the effect (or use keys Control/ Command + z).
Set White Triangle Output Level to 254 by sliding triangle to the left (only when its an image vs. a drawing or graph).
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Move black slider to the right to increase the darkness of blacks in your image, typically to the end of the black hill of the histogram.
Move white slider to left to increase brightness of image, typically setting by eye (rule of thumb: digital images can never be too bright).
After adjusting top sliders, move black slider to right until Output Level reads at least 10 (set higher if blacks diminish fine details). Put white level on 254 for images (vs. drawings or graphs or white area around images) by moving Output Level slider to left (this makes white slightly greyer than its surrounding white page when printed).
Move grey (middle) slider to left to increase brightness of shadows, to the right to increase contrast in mid-tones.
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Do NOT set middle slider when adjusting gels, blots, autoradiographs, or any other kind of image in which linear dark to light values should be maintained for measurement or evaluation.
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Use Eyedropper tool to click on what should be white, then use Plus Eyedropper to click on more points. Be sure Sampled Colors is selected and Selection is checked underneath graphic image.
After using Fill to fill with white, set output slider in levels until white becomes light gray.
Before (top) and after (bottom) selecting with Color Range and then correcting color shift.
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Fluorescence-brighter2
Fluorescence-up contrast1 Increases contrast by slightly darkening shadow end of image and brightening white end. Changes gamma. Fluorescence-up contrast2 Slightly more contrast than previous option.
Contrast for images only containing pure black and white (called bitmap in Photoshop)
1. In Actions, click on Contrast-Manual Adjust (or, in menu under Image choose Adjust, then Levels). 2. Use white Eyedropper tool and click on part of image you wish to be white. Click on various parts of image until white background is evenly white. If you overshoot, click Cancel and start again. 3. Keep black Output Level at 0 and white at 255.
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Info Window
out to Grayscale.
K: Previous Gray value percentage / Gray value percentage after adjusting slider Set Second Color Readout to RGB Color.
Background Adjustment
Move slider to left to increase brightness. Slide black triangle to right for Output Level if blacks appear too dark. Do this by eye.
When moving cursor onto image, it turns into an Eyedropper tool. Info readout will give the values of gray (the K: value) at the dropper end of this icon. Find representative background and adjust slider to see before/after values. Check in a few places to get a mean value.
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Adding Color
Adding Color to Grayscale Images (Darkfield)
In Actions Window, select desired button: Make-Red, Make-Green, or Make-Blue (shifted to cyan). Manual method: The manual means for adding color is outlined at www.quickphotoshop.com.
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In Actions window, click the Save As button (or, in menu under File select Save As). Subsequent steps for Single Image To add Lettering go to page 61. To Save existing file in several formats for several purposes go to page 83. To insert file into PowerPoint go to page 96. To add symbols go to page 78. To combine or include with other images read through Part 3. To set final resolution see page 53
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Setting Image Dimensions and Resolution 1. In menu, under Image, select Image Size. 2. Enter Height or Width, depending on the most important dimension (Constrain Proportions should be checked so that image will retain its width/height relationship, also known as aspect ratio). 3. Enter Resolution of 300. Uncheck Resample Image when subsequently printing to ink jet or laser printer.
Set most important dimension in inches or cm. Set Resolution in pixels/ inch. This is OUTPUT resolution. Check Constrain Proportions. Check Resample Image unless going to ink jet or laser printer.
Go on to Making Multi-Image Plates (next section) or to Cropping Several Images to the Same Dimensions (page 57) if more than one image is to be included in a figure or plate.
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Part 3
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Layers
For the following steps, an understanding of the layers capability in Photoshop is absolutely necessary. The beauty and bane of Photoshop is in its ability to keep images on separate layers. While layers keep images from interfering with each other, as though on transparent sheets one above the other, the sheer multiplicity of images, along with text, can make Photoshop difficult to navigate. Action buttons have been included to make it easier to keep the number of layers down to one or two, depending on the action. For most purposes, only two layers need to exist: 1. In Actions window, click on Show Layers (or, under Windows in menu, select Show Layers. If Hide Layers is listed, then the Layers window is already open somewhere on your screen. one for the image (typically the background layer) and one for text and symbols (what is labeled text in the Photoshop for Research action buttons). Images that contain layers can only be saved in the Photoshop format (.psd). These cannot be opened by very many other programs, but that number is rising. Its useful to save your images with layers so that the image can be edited later, if necessary. Photoshop 6x & 7x Layers window looks quite similar. There are some added functions, and some things are in different places, but the basic window is the same.
Layers Window
Click on arrowhead to reveal layering options. Normal is most often used (other options are discussed elsewhere in this manual). Eyes can be clicked on or off. When off, the layer disappears. Chains can lock one layer to another, so that if one layer is moved, both move (this doesnt always work). Layers can be dragged to new positions by clicking and dragging. The higher layer covers the layer below. New layers can be made by clicking on paper icon. Layers can be deleted by dragging them into the trash icon, or by simply selecting the layer and clicking on the trash icon.
Click on arrowhead to activate drop down list. Ability to flatten layers or to merge visible (only those with eyes on) is selectable from drop down list and also from Layers in the menu selections (5x, 6x). Transparency or opacity of layer is set by clicking here. Layers are made more translucent if opacity is reduced. Capital T indicates that this layer is a nonrendered text layer (5x-7x). Thus lettering is able to be edited by double clicking on the layer, but images cannot be added to it.
Adjustment Layers can be made only for contrast/color adjustments. A new adjustment layer is found in the drop down list.
Black cutouts indicate a masked layer. New images/text cannot be added to black part of mask.
Clicking on layer while holding down the Control/Command key selects the objects on that layer. You will see selections, or what are called marching ants, surrounding your objects.
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Cropping Image Files that are at the Same Resolution and Size
1. Open all images you wish to crop to the same size. From these images, determine what would be the largest dimension for cropping. 2. Select the Marquee tool from the toolbox. Outline region of interest from image with largest dimension. If your first attempt at drawing an outline over the region of interest fails to outline correct area, click outside the outline to eliminate marching ants (scrolling outline) and start again. Or, under Select choose Transform Selection (not available in 4x). Selection can be reshaped. 3. Save the outline or selection. In Actions window, click on Selection- Save (or, in menu under Select, choose Save Selection). 4. Go to 2nd image and load the selection you saved. In Actions window, click on Selection- Load (or, in menu under Select, choose Load Selection). 5. In Load Selection dialogue box, click drop down arrowhead to choose image from which you saved the selection. On a Mac, choose also Alpha One from Channel. 6. Repeat steps 4 and 5 for remaining images. 7. In Actions window, click on Crop button (or, in menu under Image, select Crop) for all images. Be aware that saving selections results in what are called alpha channels. These will need to be trashed before saving file as a TIFF file (or it may not open in other programs). In menu under Window, select Show Channels. Drag alpha channels to the trash icon.
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Cropping Images that are the Same Resolution but Different Dimensions
Sometimes images are scanned at the same resolution, but are slightly different in size, or the locations of related images have shifted from one scan to the next. In that instance, an outlined area of interest (a selection) can be made on one image file and then dragged to the related image file. The location of the selection can be eyeballed by landmarks on the image itself. 1. Open all images you wish to crop to the same size. From these images, determine what would be the smallest dimension for cropping. 2. Select the marquee tool from the toolbox. Outline region of interest from image with smallest dimension. If your first attempt at drawing an outline over the region of interest fails to outline correct area, click outside the outline to eliminate marching ants (scrolling outline) and start again. Or, under Select choose Transform Selection (not available in 4x). Selection can then be reshaped. 3. Crop that image. In Actions window, click on Crop button (or, in menu under Image, select Crop). 4. Select the entire cropped image. In Actions window, click on Select All (or, in menu under Select, choose Select All. Alternatively, you can use Control/Command + a). 5. Click and drag selection outline to the related image. Align by eye (hopefully shared landmarks exist in the images), then crop. Repeat for additional images.
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Merging Images
Merging Images/Colocalization
In this instance, merging is considered to be the combination of two or more RGB color images, one on top of the other: an equal percentage of color from different channels. This technique is commonly used in fluorescence to show coexistence or colocalization of two fluorophores labeling similar cell features. Red and green colored labels merge best since the combination of the two produces the easily recognized color of yellow. 1. Close and save all images. In the Actions window, select Merge Red & Grn-Start on Red. 2. You will be prompted to open the first image, which needs to be the redcolored image. 3. You will then be prompted to open the second image, which needs to be the green-colored image. 4. Optional. In some instances, the images can be slightly misaligned because of color shift from one wavelength to another. This action puts your green image on a layer above the red image. Simply hold down the Control/Command key and move green image over red image or use Move tool. While holding down, use arrow keys for fine movements. Manual methods can be found at www.quickphotoshop.com.
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Cutting and Pasting Into First Image File 4. Open image(s) you plan to combine with first one. 5. In Actions window, click on Combining- Cut & Close (or, in menu choose from several functions: under Select choose Select All; under Edit choose Cut; under File choose Closedont Save). 6. Now first image should be the active window (or you may have to select the first window, the one to which canvas has been added). 7. In Actions window, click on Combining- Paste (or, in menu under Edit, select Paste). 8. Hold down Control/Command keycursor turns to 4-way arrowclick and drag pasted image into position. If, after you click, you also hold down the shift key, the image will move only on the x or y plane (horizontally across or vertically down) 9. Repeat for additional images.
Continued on next page >
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Resizing Image, if Necessary Images that are pasted in may need to be enlarged or reduced. 1. In Actions window, click on Combined- Transform button (or, in menu under Edit (Layer in 4x), select Transform then Scale). 2. While holding down Shift key (so that proportions dont change), place cursor over a corner (to get double arrow). Click and drag until desired size is reached.
Make Final Contrast/Color Adjustments, Crop, and Flatten Layers 1. In Actions, you may need to go back to Contrast- Manual Adjust to match contrast and brightness and color of your images (or, in menu under Image, select Adjust, then Levels). 2. If necessary, select area of interest with crop tool and crop out extra white space. 3. You will want to flatten your image when finished, since each image sits on a separate layer. In Actions, click Flatten Layers (or, in menu under Layer, select Flatten Image).
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Adding White Space and Resolution 1. In Actions window, select Combining- Add Space (Canvas). Or, in menu under Image select Canvas Size. 2. In Canvas dialogue box, choose a unit of measurement and estimate amount of extra space youll need to accomodate lettering. Give plenty of room, since extra space can be cropped later. 3. In Canvas dialogue box, choose anchor grid square to give direction for additional space. Click Okay. The image will expand beyond the boundaries of its window. Youll have to zoom out to see what you have done, or pull on corner of image file window to expand its size. 4. In Actions window, select Image Size (or, in menu under Image, select Image Size). Increase resolution to 300 pixels/inch if necessary (Note that original data will be changed. Make sure Resample Image is checked).
Move cursor over corner, click and drag to reveal more image. You may also have to zoom out after making canvas larger.
As a general rule, pixels should NOT be added when images were obtained using a digital camera or PMT/laser system at a fixed resolution. Otherwise, data is added to the original image.
Put in resolution of 300 at desired height or width when working with lower resolution images
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images can be re-lettered rather painlessly at a later time, if editing is necessary. The Text tool dialogue box is rather clunky. Photoshop has improved the text tool in version 6x - 7x by eliminating the dialogue box so that a user can type on the image itself. Photoshop is useful for small amounts of lettering. Extensive lettering should be done in Photoshops companion program, Illustrator, or in other page layout programs such as Canvas, CorelDraw, etc. Text from other programs can be cut and pasted into dialogue box, but formatting may be lost.
Set Size of Type here. Color is set to the same color as the foreground; or, by double clicking on box, you can set a different color. Lettering must be highlighted before changes can be made. Be sure Fit in Window is checked so that you can view all lettering.
(5x - 7x) Leading is the spacing between lines, Tracking is the width between letters, and Baseline is for setting superscript/subscript. Leave at default for the most part.
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Text in 6x, 7x
1. Select T from the toolbar, or type the letter t. Click at the desired spot for placing lettering or click and drag to make a text box. 2. Click on Palettes below the menu to reveal the Character Palette (if not already visible). Choose Helvetica or Arial for the typeface, along with attributes such as Regular, Bold, Italic, etc. Choose color by doubleclicking on the color window, or by changing the foreground color at the bottom of the toolbar. Type in text. 3. If changes need to be made, the text need not be highlighted. Instead, simply click on the T in the Layers dialogue box, then make changes to the typeface, color, size, etc. in the Character Palette. Re-positioning text. Before clicking on T in the Layers dialogue box (when starting at a click point only and not within a text box): move cursor away from the text area. The text symbol will change to a move symbol. Click and drag to move. After clicking on T in Layers box: Hold down the Control/Command key or use the Move tool. When finished with applying text: Click on the check mark below the menu on the right (or click on the Marquee tool). Clicking on the X eliminates text. Returns at the ends of lines must be included with click point text, but not with text boxes.
Set size of type Choose Arial or Helvetica Choose attributes here Set strength of anti-aliasing (Strong is recommended)
Character Palette
T box in Layers
Kerning
Leading Tracking
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Enhancements to Lettering
Outlining Black Lettering in White, or White Lettering in Black
1. Select your text layer(s). In Actions window, click Show Layers then click on text layer (if Layers box doesnt appear, it is already open). 2. In Actions Window, click on Stroke- Black (Thin), Stroke- Black (Thick), Stroke- White (Thin), or Stroke-White (Thick). This actions will make text into rendered and uneditable text. Or complete several steps for each layer (5x): under Layer, choose Type then Render Text; hold down control/ command key and click on text layer to make selection around text (4x); under Edit, choose Stroke, enter Width of 2 - 8 pixels. Stroke uses the foreground color. For 6x and 7x, in menu under Layer choose Layer Style, then Stroke. Enter Size of 2 - 8 pixels, click on Color: box to choose color. Stroke is when a line follows a selection or the edge of letters. 3. In menu under Edit choose Undo (or use Control/Command key + z) if you do not care for effect, and repeat with a thicker or thinner stroke.
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Part 4
Finalizing Figures
If you need to put two or more images on a page, you may want to choose a template. Actions to complete templates can be downloaded from www.quickphotoshop.com. These templates fall into two categories: templates for similarly acquired images from a digital camera or PMT/laser system at fixed resolutions and everything else. Everything else. Everything else includes what has been scanned, drawn in other programs, or gathered together from different sources. These are images in which several different image resolutions are put onto the same page. In this instance, it behooves the user to choose a page size that matches a target size. While no particular target size fits every circumstance, the most universal size would be one that complies with the needs of publishers AND satisfies the need to make a print or 35mm slide. Thus, you are given the choice of U.S. letter format (8x10.5 inches) and A4 size (210 x 297mm), in both instances allowing for room at the top and bottom for printer margins. In this instance, the resolution is still defined by pixels across and down, but it is more commonly understood in terms of how many pixels are crammed into an inch or centimeter, since the image eventually gets printed to paper. Three hundred pixels per inch is ideal, for it translates well to publisher guidelines, it provides enough pixels so that images look clean and non-pixelated without a loss of detail, and these images look stellar on 35mm slides. For the web, laptop presentations, and e-mail, these images can be compressed so that large files (files that take up a lot of memory and hard disk space) can be made more manageable (see Saving Files, page 83). Digital Imaging Systems. Digital camera systems and PMT/laser systems operate by using a sensor with certain number of photon collection points going across and down, as in 640 x 480. This correlates to the number of pixels across and down in your image. The number is fixed--the system can take no more or no less than the number across and the number down. This defines the resolution of the camera. Every newly manufactured camera improves upon the old by multiplying this number, and new cameras are available just about every month, so the templates currently available cannot possibly cover every camera system made. For updated templates, check www.quickphotoshop.com,or contact the author with the number of sensors across and down for your digital device, and you will be provided with a template for your use. Choose your template by the way in which you acquired your images. Find out how in the pages that follow.
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Some images may look pixelated if image was from web (less than 500 pixels in both dimensions), or if it was scanned at low resolution (say, at 72ppi). You will need to re-acquire the image, or locate the original.
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Guideline dividers will not look evenly spaced even though the spacing is perfect. That is both a screen resolution and a work-around bug in the action.
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New Position
Lettering Needs to be White. 1. In Actions, click on Lettering- Make White (Renders) button. This action renders the text into pixels, which means that the text cannot be edited by double-clicking on its layer in the Layers window (in other words, if you wish to change type later, you will need to trash text layer and start fresh).
Lettering Needs to be Outlined in Contrasting Black or White (to separate letter from its background) See outlining lettering on page 66.
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1. In Actions window, click Show Layers button (or, in menu under Window, select Show Layers) 2. In Actions window, click on Layers- Flatten (or, in Layers dialogue box, select from drop down list) This will place lettering on image so that the lettering becomes part of the image and is no longer editable. 3. Click on Marquee tool on toolbar. 4. Click and drag to outline area you wish to move on the image. 5. Hold down Control/Command key, click and hold mouse button, and hold down shift key to move images with lettering in a straight line. Alternatively, layers can remain separate. First the image can be moved, and then the text layer can be selected and it can be moved. These two layers can also be linked (by selecting the image layer in the Layers dialogue box and clicking grey box next to the text layer to create lock symbol), but both layers dont always link together when re-positioning.
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Contact Sheet II
In Photoshop 5.5 and above you can also make auto-layouts using the Contact Sheet II function (if you have Photoshop 5.0, you can upgrade to 5.5 by visiting the Adobe web site for the upgrade software). This is an automated function that 1. Under File, select Automate, then Contact Sheet II. 2. Click Choose button and find directory in which your image files are located. 3. Set width, height, and resolution to your specifications 4. Under Thumbnails, set columns and rows. You cannot set the width of the borders, so white space surrounding images will not be consistent. Use technique on previous page to reposition images. You will have to apply lettering on your own. requires you to place the image files you wish to include in the layout in their own folder. Your screen needs to be empty of images.
Click Choose to find directory in which your files are located. Set Width, Height and Resolution. You cannot specify width of borders between images. Indicate how many images you wish to place across (Rows) and down (Columns ).
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Templates Based Upon Fixed-Resolution Images (e.g., from digital camera systems)
1. In toolbox, be sure to make background color white (or the color you prefer for the background color). 2. In Menu under Image, select Canvas Size. You will be adding extra space to your existing image to give yourself more canvas, as in a painting. 3. In Canvas Size dialogue box, enter in height and width the appropriate number of pixels to allow for remaining images plus extra pixels for borders. It's best to estimate more than what you need. For example, if your image is 640 x 480 and you wish to have a total of four images in your figure, you'll need 2 x 6401280for the width and 2 x 480960for the height PLUS room for borders between pictures20 pixels. Final canvas is 1300 x 980, estimated at 1400 x 1100). 4. Select square of grid from which youd like extra space to be added. Enter Width and Height in Pixels.
Click on desired box. White space is added in direction of arrows. In this instance, image is on top, left: canvas is added to the right and below image.
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Dragging 2nd Image onto Template to Which Canvas Has Been Added
1st image 2nd image is moved and eye-balled into place on template after using Select All/Cut/Close then using Paste to paste onto template.
Eliminate extra image area by outlining with Marquee tool and pressing delete button (make sure background color in toolbar is white). Zoom in to point at which ruler readings are interpretable (you may have to change units (keyboard command: Control/Command + k, choose Units and Rulers). Determine distance and make new borders for subsequent images at the same distance apart. Zoom back and remove images to align to guidelines, if necessary.
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4. If you are laying out more than two images, zoom in and use the ruler to determine the width of your border between the first two images. Drag guidelines into place for additional images along with borders, matching the width of your first border. 5. Repeat steps to add additional images.
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Symbols
Symbols include the addition of anything other than text to the image, including dashes (or tick marks), arrows, asterisks, boxes, circles, etc. More complicated shapes and drawings can be done in Photoshop, but that kind of activity normally takes place in a drawing program (the program that most resembles the drawing protocol in Photoshop is its sister program, Illustrator. Drawings done in Illustrator Symbols from Symbol.psd file 1. Open the image file upon which you wish to put symbols. 2. Open the downloaded symbols file. Zoom in and scroll down to find symbol desired. 3. Each symbol is on a different layer. Open Layers window (in menu, under Window, select Show Layers. If you see Hide Layers, then Layers window is open). Select layer and symbol of interest. 4. Hold down Control/Command key and drag symbol from the downloaded symbols file to your image file. 5. If size is too large or too small, in Actions window, select Transform- scale button (or, in menu under Edit, select Transform then Scale). Place cursor over corner of outline, hold down shift key, click and drag. Some symbols may fit your intention better by NOT holding down shift key and scaling file in one dimension (by width only or height only). You may wish also to rotate the symbol. In Actions window, click on Transform-rotate button (or, in menu under Edit select Transform then Rotate). Make White or in Color 1. White. Select desired layer in Layers window. Click a second time while holding Control/Command key to make selection around feature of interest. 2. Click on Make White (Layer) button in the Actions window. Or, in menu under Edit choose Fill: choose White from the Use: drop down box. 1. Color. Start by double clicking on foreground window on the toolbar. 2. Select a color from dialogue box (or select color from your image itself by clicking on Eyedropper tool and then clicking on desired contrasting color in your image). 3. In Actions window, click on Fill- foreground color button. Or, in menu under Edit choose Fill: choose Foreground Color from the Use: drop down box. Symbols and patterns sizes are matched to an image size that is approximately 300ppi. Symbols can be scaled down; patterns, however, may not work as well. Dragging Symbol from Downloaded Symbols File can be opened in Photoshop. See page 16). For your convenience, a set of symbols and patterns has been included for downloading at quickphotoshop.com. You can simply drag symbols onto your image from these image files, or make these using manual Photoshop functions.
In Actions click on TransformScale. Re-scale image to desired size while holding down Shift key. In Actions click on TransformRotate if you wish to rotate symbol. Copy and Paste (under Edit ) to make additional symbols at same orientation and size. To make symbol white select layer symbol lies on and click a second time on that layer while holding down Control/Command key. In Actions window, click on Make- White.
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Set thickness of the line by placing value in Weight, usually between 4 and 8.
Check Start or End for Arrowheads, then click Shape to set values for arrowhead.
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In menu under Edit, select Stroke. Set width of line you wish to trace along square or circle. (2 8 is typical).
Keyboard Symbols
Symbols such as prime, double prime, degrees, copyright, and so on are best found in fonts. Many of these can be found in the Symbols and Verdana fonts. You can find these symbols in Windows 2000 by clicking on Start, then going to Programs > Accessories > System Tools > Character Map. Other Windows operating systems require that the application (charmap.exe) be installed from your Windows CD using Add/Remove Programs in the Control Panel. On a Macintosh, special characters can be found under the apple icon in Keyfinder or Keycaps.
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1. Make an image of a ruler or calibration grid using your fixed camera system. 2. In menu under Window, select Show Info (if already shown, you will see Hide Info). 3. In Info dialogue box, click on top, left arrowhead to reveal drop down list. Select Palette Options. In Ruler Units, choose Pixels. 4. Use the Marquee or Measure tool (not available in 4x) to stretch from one measurement mark to another. The Info box will show how many pixels equal your ruler units. If you are also using the calibration for subsequent calibration in quantitation software, be sure to measure in both the x and y axis in the event that your camera system does not produce square pixels. 5. Draw a scale bar using the Line tool while holding down shift key and looking at the Info box (see page 27 for location of Line tool). Draw the pixel length which equals the length of the ruler unit. For example, you may draw a line across two measurement marks that equal 10 centimeters. The info box may show that you have drawn across 250 pixels. If you wish to make a scale bar on an image taken under the same conditions, you can draw a 250 pixel line and label it as 10 centimeters.
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Saving Files
It would seem that files could be saved in one format to make everything simpler. To date, the closest format to just one is not the TIFF format, but the Adobe Acrobat format. Adobe Acrobat files (.pdf) have several intrinsic advantages: these files are compressed, so they don't take up so much hard disk space; the fonts are saved in such a way that various computers do not need to have the same font for opening the file; and publishing agencies are quickly moving to use this format for output to publications (these reportedly have the advantage of little color shift). The compression on these files can be so incredible that cutting file sizes by onefourth can result in absolutely no visible change in image data! 1. Photoshop file (.psd). This is the layered image which can be edited later. 2. TIFF (Tagged Information File Format) file (.tif). This file format is currently the most universal and it is normally requested by publishers. It does not contain layers. Photoshop gives the option of LZW compression, but that should not be used because then the file can only be opened in Photoshop and a few other programs. 3. Adobe Acrobat file (.pdf). This file format is now being requested by editorial boards of journals for paperless reviews of submissions. This file format can also be e-mailed, put on a web page, and, increasingly, it can be for the submission of documents, such as grants. This file format is saved with ZIP compression, because it is non-Lossy (no image details are sacrificed). Choosing ZIP compression does not always result in file sizes Saving by Using Action Buttons In Actions, choose buttons for several ways to save (Save as Photoshop, TIFF, JPEG button is recommended): Save as Photoshop, TIFF, JPEG (this saves in all current formats for several outputs, starting with Photoshop so that layering is preserved for subsequent editing). Other buttons include, Save as- Photoshop; Save as- TIFF, Save as- CMYK TIFF, or Save as-CMYK EPS; Save as- JPEG; and Save as- PDF (Adobe Acrobat file). You choose the compression for JPEG files: suggested compression is medium to maximum. Note: If you wish to save manually, remember that only the Photoshop format contains layers. To save in other formats, layers need to be flattened (in drop down list in Actions dialogue box, select Flatten Layers. Also found under the Layers Menu in 5x - 7x). If you have opened a PDF, Illustrator file, or from some other formats, you will need to flatten layers though you may have made none (Photoshop, frustratingly, introduces invisible layers in these file formats). The reality is, however, more sobering. At this printing, it is best to save files not only in several different formats, but also in more than one mode (ideally in CMYK as well as RGB). Because hard disk space is currently cheap, and because CD recordable disks and the concomitant devices are also inexpensive, the suffering level for having to save in so many ways is at least minimized on the economic level. It is recommended to organize files in such a manner that each file has its own folder: One folder is for Original files, a second for Photoshop files, a third for TIFF files, a fourth for CMYK files and a fifth for PDF and JPEG files. Thus, action buttons are included to save as follows: that are significantly smaller. Files can also be saved with JPEG compression at several settings. Remember that some image information may be compromised. Check for yourself against the original image. 4. JPEG (Joint Photography Experts Group) file. This file format is ideally suited for web publication and for PowerPoint. Various levels of compression and image quality loss are selectable by the user. 5. TIFF or EPS (Encapsulated PostScript) CMYK (Cyan, Magenta, Yellow, Black) files. This mode is necessary for submission of color photos to publications. Its range of colors (colors it can print) is far less than the visible range that we can see by eye and the range we can see on our computer screen.
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Overview of Printers
Setting dimensions and resolution by the kind of printer. Printers fall into five broad categories: laser printers (most of which are postscript printers); ink jet printers and their larger version: poster printers; and pre-press printers. A fourth category defines slide writersthose devices which expose digital files to 35mm filmand a fifth includes photographic printers. Laser printers. These printers do very well with drawing or vector files. Images, however, suffer from being re-formed into so many dots. Sometimes, as with high resolution printers (defined by dots per inch), images do not suffer much at all in terms of clarity. In any case, the detail in the shadow areas and in the whites usually is not preserved and blacks arent deep. Photoshop typically does not print well to laser printers. Lettering can look fuzzy when it is re-formed into dots. These printers do not print well with large files, so reduce the size or make JPEGs so that the printer does not hang up. Unless printers require CMYK, you can print RGB files to these printers. Ink jet printers. These printers have shown the most promise as versatile, high resolution printers with good tonal range. These can also print sharp lettering, depending on the printer and choice of paper. Their drawback is in how slowly prints are made. It is a good idea to experiment with printer settings to obtain the kind of output that matches your computer screen. For example, on a PC, the Epson printer makes the best images from Photoshop when using the Advanced Setting, and then choosing ICM (Internal Calibration of the Monitor). Choose paper stock carefully to obtain the best reproduction. Paper stock should be thick and somewhat shiny (coated) for best results. Large files can be printed to these printers with a fairly good tonal range. Some colors, however, do not have good range at all, such as shades of red. As time goes
on and more ink colors are added to ink jet cartridges (the higher end Epsons now have 6+ colors), reproduction of all colors should improve.
RGB files can be printed to ink jets. Many of these use drivers that either convert files to CMYK transparently, or require CMYK files for output. In either case, it is best to make CMYK files yourself so that you can predict the outcome. Pre-press printers. Several companies make CMYK printers. These include dye-sublimation printers, wax transfer, and a plethora of ink-based systems. The dye-sublimation prints, such as those made by Codontics, require CMYK files. These printers are meant to give you a preview of how you should expect these to appear in publication, but dont expect exact color matching unless you have gone through a great deal of work calibrating colors. These printers also anticipate that you have set both resolution and dimensions before printing: 300 pixels per inch at your desired dimensions (or you may be able to choose the option Fit to Page). Slide writers. Unlike Pre-press printers, these devices anticipate that files are RGB, not CMYK! Lettering, graphs and drawings are transparently made into image files, using a software process known as rasterizing or RIPping (Rastor Image Processing). See page 18 for more information on RIPping. In this scenario, lettering will print as it appears on the computer screen. Maintaining a high resolution of the file is only important insofar as lettering does not become pixelated. Resolution that is too high (higher than 400 pixels per inch at page size) creates resolution that exceeds the device: empty resolution. Files that are higher than 15 megabytes exceed the resolution of film, and thus do not provide any more information than slides can resolve.
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Photographic printers. These include the Fuji Pictography and Durst printers, both of which are RGB printers. These print to continuous tone photographic paper using lasers for exposure. The print quality typically exceeds what can be obtained from other printers. Resolution is set to match printer resolutions, and then dimensions are set.
These printers typically take output from Photoshop, and not vector programs, unless RIPping software is also purchased (results are not always expected: some files lose information after rasterizing. See other methods on page 16). Expect resolution, detail, color and contrast range to be extraordinary on these devices.
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For Publication
1. Set Height and Width to desired dimensions. Normally these sizes are indicated in the publishers guidelines. If unsure about the ultimate dimensions, make image two times the likely size of reproduction. Set Resolution as follows: 300ppi Images Images & Text 600ppi Text/Drawings 1200ppi If scanned images or text started as low resolution files (<800 or so pixels), these will need to be re-scanned.
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Working With Color CMYK Files (for Publication and Pre-Press Printers)
From dynamic to dull. Perhaps the most frustrating process in the imaging world is to go from RGB filesthose files that look good on a computer screento CMYKthose image files that immediately lose pizazz to become lifeless and drab. Worse, contrast can decrease. And then, if these shifts werent bad enough, colors can shift once more from what you see on your computer screen to what is seen on the screen at the publishing agency and then to what is published. Saturated colors. In the former instance, the greatest contribution to this problem is the world of research itself: so many colors in research are saturated. They are completely green, completely red, completely blue, and so on. Research colors tend to be more primary versus colors in the real world which are more pastel and less concentrated. That phenomenon is especially true when coming from the world of Silicon Graphics computers and their screens, in which deep and saturated colors display richly, but the colors translate to no other output. From your monitor to publication. In the latter instanceregarding computer screens and how poorly one a computer screen matches the output at the publisherthe best advice is to use a high end hardware calibrated monitor (such as the Barco line of monitors). On the more affordable level, Macintosh computers with Apples 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 by Apple. Other solutions. Having said that, the next best solution is to make your best print from your RGB file (if possible), or make your best print on a CMYK printer. Send the print with the digital file and ask the publisher to match the colors. When viewing your own print, you might consider buying a viewing booth, or setting aside an area you can light with 5500 degree Kelvin fluorescent lights (available at professional photography retailers or graphic arts suppliers). These lights provide a standard color temperature for evaluating prints. If prints are evaluated under other lighting conditions, colors can shift to green (most fluorescent lighting), to red or yellow (under incandescent lighting), to blue (in shadows near window light), or a mixture of two or all three. Adjusting colors. You can adjust colors that have faded or lost contrast after changing from RGB to CMYK. Three methods are suggested, depending on the degree to which colors are out of gamut. In all situations, color changes are made using Hue & Saturation. Remember that each image has its own peculiarities, and the RGB to CMYK process requires trial, error and experimentation.
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Method 1. Color and Contrast Restoration on CMYK Images with Small Color Shifts
1. Select CMYK image. 2. In Actions window, click on Color- hue & saturation (or, under Image, select Adjust then Hue & Saturation). Work on colors one by one. 3. In dialogue box, click drop down box next to Edit. Choose a color that has shifted. Or... Use Eyedropper tool to select offending color. 4. Slide Saturation slider to the right to increase saturation, to the left to decrease. You may also want to lighten or darken with the Lightness slider. These tools, by themselves, may bring color into acceptable range. If these do not solve the problem with color shift, restore color and contrast with Method 2.
Remember that blues and purple-blues will print as dark blobs. Move Hue slider to left to add green.
You can edit your selection further by using the Eyedropper tool to click on the features in your image that contain the colors youre interested in changing. Click on the first feature with the unmarked Eyedropper tool, and then on additional features with the plus (+) Eyedropper tool. If you accidently choose an unwanted color, use the minus (-) Eyedropper tool to click on the same point, or start over again. Note: When using Eyedropper tool, be sure to select numerous points, including bright to dark values within each hue. Or... expand or contract range of colors by adjusting sliders (available when a color is selected).
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Method 2. Color and Contrast Restoration on RGB Images with Large or Problematic Color Shifts
1. Select RGB image. Duplicate RGB image and align on screen so that important areas are visible. 2. Under View, select Gamut Warning (or, better yet, use Control+Shift key + y). 3. In Actions window, click on Color- Hue & Saturation (or, under Image, select Adjust then Hue & Saturation). Work on colors one by one. Select colors by using eyedropper tools as described in the previous method. Keep gamut warning overlay on, especially when selecting bright objects against a dark background (as in darkfield microscopy). Be sure to use plus (+) Eyedropper tool to select to edges of objects, much easier seen when gamut warning is active. 4. Adjust the Saturation slider first. Keep in mind that all colors will print if saturation is set low enough (to the left). Adjust slider until a fair amount of the gray overlay disappears. 5. Adjust Lightness slider by increasing (moving to right) shy of eliminating too much contrast. Now increase Saturation as much as possible (move slider toward center) without increasing gray overlay. The idea is to work between the two, lightening as much as possible without losing contrast, and keeping saturation as close to the center as possible. Dont be surprised if the color you are working on needs dramatic desaturation. 6. Adjust Hue carefully to determine if a shift in one direction or the other might increase chances of eliminating gray overlay. Play with all three to get close to your desired color and contrast. You may need to turn gamut warning off and on. That is best done with the keyboard command while the Hue & Saturation dialogue box is open (Control/Command + Shift + y). You need not remove all the gray overlay, just 90 percent or so. 7. Make image CMYK: in menu under Image choose Mode, then CMYK Color. Use the gamut warning indicator to decide which colors need to be altered. The gray overlay dissapears when colors are adjusted correctly. That may not happen with the saturatedcolors used in research. Expect to remove 90 - 95% of gray overlay for any hue: the remaining unbalanced color will be at edges of bright or dark samples. These small areas of unbalanced color are imperceivable when printed.
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Example of RGB Image (on left) Corrected for CMYK in the Blue Hue (on right)
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6. In Actions window, click on Color Range button (or in menu under Select, choose Color Range). 7. In dialogue box, use eyedropper tools to select color you wish to work on. Use the leftmost eyedropper tool to select the first color, then use the plus (+) Eyedropper tool to add more shades of the same color. Set Fuzziness high (50 -100). For bright objects against a dark background, keep gamut warning overlay on to be sure you are selecting to the edge of your objects. You may wish to hide the moving outline (selection) while making changes. To do so, press Command/Control key + h. Its a lot easier to see your corrections, but the downside is that you can forget that youve hid the selection later.
Check Selection.
Use leftmost Eyedropper tool to click on first point in desired color. Use plus (+) Eyedropper to select additional shades of the same color.
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8. In Actions window, click on Contrast Adjust- Curves (or in menu under Image, select Adjust then Curves). 9. Click on representative color in image with Eyedropper tool (identical or close to the position chosen when determining ink color). 10. By clicking and pulling line in Curves dialogue box to the left across the top, or down along the right, adjust Red and/or Green and/or Blue Channels individually while looking at the Show Color Dialogue box to match CMYK values to those you have written down (see table on the following page). Turn View Gamut off and on using Command/Control + Shift + y keys to test the efficacy of your changes. If the hue doesnt look right, use Curves line across and down to fine tune by eye while ignoring CMYK values in Show Color. For example, if representative value reads 65% Cyan, 0% Magenta, 100% Yellow and 0% K (black) on the closest match of a Pantone ink value, adjust Red channel and Blue channel to achieve a similar reading. Values will change on the Color Palette as adjustments are made in the Curves dialogue box. Readjust by eye. Do not expect to see a perfect match of hues! Some colors are outside the gamut (breadth of color available) of what can be printed by a printing press. Do expect, however, to improve upon the RGB image with a slightly different hue.
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Final Steps
11. Once satisfied with adjustments while in RGB color space, change Mode to CMYK Color: in menu under Image choose Mode, then CMYK Color. Flatten image if prompted to do so (in menu under Layers choose Flatten Image). Be sure to have saved the RGB image before doing so. 12. For final adjustments and fine tuning, use Hue & Saturation or Curves to adjust colors and contrast.
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Adjustments Within Tonal Ranges (Shadows, Midtones, Highlights) 1. Use Color Range to select areas of interest according to procedures oultined earlier. 2. In Curves dialogue box, determine where values of interest lie along line. To do that, click and hold with eyedropper tool on areas of interest. A circle should appear along the line. 3. Make 3 points at that location along the line. Bend line so that hills and valleys create shadows and highlights within narrow color range.
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Gradients. Those colors have been borrowed for modern computer graphics, with the exception that a complementary color (yellow) comprises the lettering. The ability to place a darker blue-black at the top of the slide graduating to a lighter blue causes the viewer to look at the area of highest contrast (the top of the slide where the title exists) and then to naturally move down.
Slides in Photoshop. In that spirit, the following procedures will create White-onBlue or Yellow-on-Blue graphics for 35mm slide format or for on-screen presentations (at 1024 x 768 pixels) in the landscape dimension. These are meant for blackon-white graphs and drawings with text. You can try to select only the black parts of the image using Color Range (page 93), copy and then paste into PowerPoint, but the results are typically unsatisfactory (either the text is inadequately selected, or the white background is included with the selection). If you wish to place images versus text or graphs on a blue, graduated background, choose the action buttons in the first step below. In any case, remember that the shade of blue from the template/actions will not be an precise match to the dark blue color of PowerPoint. The manual method for making slides to match PowerPoint can be found at www.quickphotoshop.com.
1. Open or select drawing, text or graph file you wish to paste on desired blue image file. In Actions, click on any of the following buttons: White on Blue 35mm, Yellow on Blue 35mm, White on Blue for On-Screen, Yellow on Blue for On-Screen. These actions auto-select what is black and paste it over the background. Make sure these are high contrast (see page 44). 2. If the image is cut off at the top or bottom, re-size. In Actions, choose Resize or Transform. (Or, under Edit select Transform, then Scale). Hold down Shift key while dragging the corners of the outline to re-size the image.
To Make a PowerPoint Type Background:
Use appropriate Actions buttons to auto-create yellow-on-blue image from a white-on-black image.
1. Make a solid blue or blue gradient image file by clicking the Blue Gradient 35mm Slide, Blue Without Gradient 35mm Slide, Blue Gradient On-Screen, and Blue Without Gradient On-Screen buttons in the Actions dialogue box.
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Photographic Print
Select Channel of interest Click arrowhead so that dark gradient is on the left bottom.
Slide top of Curves line to the left or down to increase or decrease channels color. Record Input and Output Values; type these on ring around print. Determine which Curves correction works best. Click on Save to save the Curves correction and apply to subsequent images using Load.
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Organizing. Photoshop provides two ways to organize files: as hard copy on paper and electronically. On paper, what are called contact sheets can be made. These are pages of small pictures (called thumbnails) along with their file names. Electronically, you can have images printed as JPEGs (compressed images) to the web as both a one-page contact sheet for low resolution, and as high resolution by clicking on the file name. In this scenario, your files can be reviewed via a web browser, such as Netscape or Microsoft Explorer. These functions are not available with versions earlier than Photoshop 5x.
1. Under File, select Automate, then Contact Sheet II 2. Choose the directory from which you wish to make contact sheets. The dialogue box gives several options for page size, resolution, and number of images across and down. Size of lettering, unfortunately, cannot be made small enough for long file names, except in 7x. 3. Click Okay. This function will continue processing images and making new images until it finishes with all files in the directory you chose.
Electronic Images in Web Browser
1. Under File, select Automate, then Web Photo Gallery. 2. Choose a Source (the directory from which you wish to have image files made to a slide show) and choose a Destination (where web file is placed). 3. You can include a Site Name, Photographer and Date. These are placed in a banner above your images. 4. Unless you have specific sizes you wish to make, leave Thumbnails and Re-size Gallery Images on default settings (where they are). 5. Click Okay. After all files are processed, your default web program will open and display the file. 6. Rename file to better describe nature of image files, and leave the extension .htm or .html (Photoshop will only make a file named Index.htm. If a file with that name is uploaded to your website, it will become your home page. Renaming the index.htm file will solve that problem). Because Photoshop only makes a web page named index.htm, any new image files must be included with existing files when making additions.
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Summary of Steps
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Index
Symbols
12-bit 38 16-bit 39 3200 degrees kelvin 9 35mm slides 84 3D objects 7 5500 degrees kelvin 9 72 dots per inch 15 8-bit 39. See also Bit-Depth Brightfield 9 Brightness 5, 12, 42
C
Calibrating monitor. See Monitors Calibration tool 22 Canvas 61 anchor 61 CD 20 Character palette 65 Circles. See Symbols CMYK 2, 83. See also Mode color/contrast restoration 89 reviving contrast 96 CMYK color 88 color shifts 88 correcting blue 91 difficult corrections 92 large corrections 90 Colocalization 60 Color picking 92 Color range 46, 92 fuzziness 46, 92 Color shifts. See CMYK color Color temperature 9 Color wheel 13 Combining images 61 two or more into one 61 Complementary filtering 13 Computer screens. See Monitors Contact sheet 74 Contrast 5, 12, 42 Coomasie blue gels 8 Correcting color 43 changing certain colors 50 changing the color blue 50 manual corrections 45 selected areas 46 subtle colors 49
A
A4 70 A4 size 69 Accumulate 14 Actions 30 Adding color 51 to grayscale images 51 Adding white space. See Lettering Alignment 67 Anti-aliased lettering. See Lettering Anti-aliasing 64 Archiving 101 Arrows. See Symbols Artifact 40 Auto-balance 43 Automate 74 Autorads 8
whites 46 Correcting contrast 43 manual correction 44 Corrupted files 33 Crop 38 different dimensions 58 fixed target size 59 several to same size 57 Crop tool 59 Cropping white space 38 CRT (Cathode Ray Tube) 21 Cursor icons 26 Curves 95 adjusting colors 94
D
Darkfield 9, 47 Dashes. See Symbols De-interlace 13 Defragmenting 21 Descreen 5, 7 Digital camera 9 Dimension 53 Disk space 21 Display card. See Video card Dots per inch 5 Duplicate 33 Dust. See Dust and scratches Dust and scratches 40
B
Binary 2 Binning 12 Bit-depth 11 Bitmap 2, 15 bitmap 47 Black level 5, 12 Blank field 10 Box under text 66 Boxes. See Symbols
E
Ektachrome 6 Electron microscopy films 8 EPS 83 Ethics 19 Export options 15 Eyedropper tool 89
104
Index
F
FDA approval 20 Figures 70. See also Templates File formats Acrobat (pdf) 16 BMP 15 EPS 16 GIF 33 JPEG 33 PICT 15, 33 PostScript 16 RAW 33 TIFF 15, 33 Film scanner 6 Flatbed scanner filter 2 scanning from books 7 Flatfield image 10 Flatten layers 83 Flip 35 Fluorescence 47 Frame average 12 Frame-average 9 Fuji Pictrography 18 Fujichrome 6
H
Histograms 44 History 36 Hue 42 Hue & Saturation 45, 50 Hue & saturation 89
I
IDE controller 21 Illumination, Kohler 10 Illumination, uneven 9 Illustrator 16, 18 Image files 17 Image size 53 resample image. See Image size Image type CMYK 2 Image types. See Film scanner Image window 26 Indexed color. See also Mode Ink jet printer 18 Ink jet printers 18 Integration 14
merge visible 56 render text 56 LCD (Liquid Crystal Displays) 22 Lettering 45 degree rotation 68 add white space 63 alignment and orientation 67 anti-aliased lettering 15 corrections for auto-layout 72 manual (4x, 5x) 64 Manual (6x, 7x) 65 outlining 66 rotating 67 Levels 44 channel 45 Levels dialogue box 43 output levels 44 Line tool 29, 79 Lines disappear 7 Lines per inch 5 LUT (Look Up Table) overlay 10, 12
M
Magnification 82 Marquee tool 32 Matching gray levels 48 Median filter 5 Memory setting for Macintosh 25 Menu 26 Merging images 60 Microscope slides 7 Microsoft Word 17 Mode 39 Moire patterns. See also Laserjet printer Monitors 22 self-calibrating 22
J
JPEG 83
G
Gamma 5, 12, 23, 42 monitor's gamma 24 Gamut 18 Gamut warning 90 Glare 13 Grayscale. See Mode. See also Mode color shifts 100 Guidelines 32
K
Keyboard symbols 80 Kodak Gold 6
L
Laserjet printer 18 Layers 55 adjustment layers 55 flatten 56
105
Index
N
Navigating 37 Nitrocellulose 8 Noise 14 Noise, dark noise 12 Normal viewing distance 7 PowerPoint 17, 53, 97 correct for darkness 97 make yellow on blue 99 making JPEGs 97 setting dimensions 98 Pre-press printers 86 Preferences 28 Print to file 16 Printers 84 features 85 inkjet 84, 86 laser 84, 86 poster 84 pre-press 84, 86 slide writers 84 Prints 84 Processor speed 21 Profile. See also Monitors Profiles 28 Pseudocolor 51 Publication 86 RGB color. See Flatbed Scanners; Mode RIP (Raster Image Processed) 18 Rotate 35 Rotation Arbitrary Rotation 34 Rubber stamp tool 40 Ruler units 82 Rulers. See Preferences
O
Open 33 Open As 33 Open as 33 Optical Character Recognition (OCR) 2 Optical resolution. See Resolution Organizing 101 Orientation 67 Outlining. See Stroke Output levels. See Levels Output resolution 53. See also Resolution Output size 3 Over-sampled. See Sampling Over-saturation. See Saturation
S
Saturation 42 over-saturation 11 Scale bars 82 Scaling 3 Scratch disk space. See Disk space Scratch disks 29. See Preferences Scratches. See Dust and scratches SCSI controller 21 Shading image 10 Sharpening 5, 41. See also Flatbed scanners Show color 93 Stroke 66 Summary 102 Symbols 78 manually making 79
Q
Quark 18
P
Pagemaker 18 Palette 26 Patterns 81 pdf 83 Phosphors 23 Photo printer 18 Photons 69 Pixelated Image 15 Pixelated lettering 7 Pixels measuring 11, 48 Plates 70. See also Templates Polarizing 13 Postscript interpreter 17
R
RAM (random access memory) 21 Reducing graininess 48 Refresh rate. See Video card Resolution 6, 15, 17 adding 63 by file size 6 for output 86 from digital systems 87 setting by file size 3 setting for images 53 setting resolution 3 RGB 83
T
Templates 69, 71 manual assembly 75 Text arial 64 helvetica 64 The Image Processing Handbook 5 TIFF 83 Toolbar 27 Transform 35 Troubleshooting 29 True color. See Flatbed Scanners
106
Index
Type tool 64
U
Undo 36 Unsharp mask 41 USA letter size 70
V
Variations 48 Vector 17 Video card 21, 22
W
White balance. See Digital camera White Level 42 Word Processing Programs 15 Working With Color CMYK Files (for Publication and 88 Working with PowerPoint 97 35mm slide aspect ratio 97 on-screen aspect ratio 97 PowerPoint files to Photoshop 99 rasterizing 18 setting dimensions of images to fit PowerPoint 97 to make JPEG images 97
Z
Zoom Tool 37 moving image while zoomed in (navigating) 37 zooming in & out 37
107