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Techniques
Analyzing Electrophoretic GelsThe following is one possible procedure for using Image to analyze a one-dimensional electrophoretic gel. It also demonstrates some of the less obvious features in Image, and also a few shortcuts. Note that this technique cannot be used to compare bands on different gels unless the gels are calibrated to known standards. Any results obtained using this procedure should not be trusted without testing using standards with known concentrations or by comparing with results obtained using a densitometer.A 23 page manual ("gel_macros_doc.hqx") that describes this procedure in much greater detail is available by anonymous FTP from zippy.nimh.nih.gov, in the /pub/nih-image/documents directory. 
Note that this macro package changes several of the settings in the Analysis/Options dialog box. It enables Wand Auto-Measure so that the area is automatically measured when you click with the wand tool under a peak. It disables Label Particles so that peaks are not automatically numbered by the wand tool. It enables Include Interior Holes . If this were not done, the wand tool would measure zero area. It enables Adjust Areas to compensates for the tendency of the wand tool to underestimate the size of small peaks. The size of small peaks is underestimated because some of the actual peak area is represented on the screen by the pixels which define the boundary, and, on small peaks, the ratio of boundary pixels to interior pixels is higher. Using SelectionsSelections are user defined regions or lines within an image that can be measured, filtered or edited. Region selections are created using the rectangular, oval, polygonal or freehand selection tools. Line selections are created using the line selection tool, which has three forms selected from a pop-up menu. Line selections can be straight, freehand or segmented. Selections are outlined by a moving marquee, sometimes referred to as the "marching ants". Only one selection can be active at a time. Moving a Selection. Selections can be moved by clicking
inside them and dragging. The Info window displays the coordinates of
the upper left corner of the selection (or the bounding rectangle for
non-rectangular selections) as it is being moved. Notice that the cursor
changes to an arrow when it is within the selection. Straight line selections
can be moved using the handle (small black box) in the center of the line.
If you want to move the contents of a selection, rather than
the selection itself, do a Copy (command-c), a Paste (command-v), then
click within the selection and drag. Use the shift key to constrain movement
to be horizontal or vertical. Use the arrow keys to nudge the selection
one pixel at a time in any direction. Using Image with Flatbed ScannersThe Acquire command provides direct support for most scanners that have Photoshop plug-ins. Image supports three scanning modes: 8-bit grayscale, 8-bit indexed color and 24-bit color. The plug-in that comes with the La Cie Silver scanner is one that works well with Image .You also have to be careful not to generate images that are too large for Image to handle. Image was optimized for the 640 x 480 (300K) images produced by frame grabber cards, whereas an 8 x 10 inch page scanned at 300 DPI is 2400 x 3000 pixels (7.2MB), much larger than Image was designed to handle. The following table gives suggested maximum scan areas for different scanning resolutions and monitor sizes. Image can handle scans somewhat larger than these, but you will probably run into problems if you greatly exceed these recommended sizes. Monitor 75 DPI 150 DPI 300 DPI 14" (640x480) 8" x 6" 4" x 3" 2" x 1.5" 17" (832x624) 11" x 8" 5.5" x 4" 3" x 2" 20" (1152x870) 14" x 11" 7" x 5.5" 3.5" x 2.75" Use the Preferences command to increase the size of the Undo and Clipboard buffers to 600K when working with scans of the size suggested for 17" monitors, and to 1000K for 20" monitors. Note that making the Undo and Clipboard buffers larger then necessary can require a lot of extra memory. For example, increasing the buffer sizes from 1000K to 1500K increases Image's memory requirements by 1MB. You should probably stick to 75 DPI unless scanning small selections. 75 DPI also has the advantage of producing images that are near actual size when displayed or printed, since nominal screen and printer resolution on the Macintosh is 72 DPI. Recovering Data from Line PlotsImage can be used to recover numeric coordinate data from printed line plots using the following procedure. 2) Edit the plot to remove the x-axis, y-axis and labels. 3) Rotate the plot 90 clock-wise. 4) Create a line mask similar to the one above using the line drawing tool and repeated use of Copy and Paste. 5) Convert the line plot to a scatter plot by ANDing the plot with the mask using the Image Math command. 6) Select X-Y Center in the Analyze/Options dialog box. 7) Deselect Invert Y-Coordinates in the Preferences dialog box. 8) Use the Analyze Particles command to record the X-Y coordinates. Alternately, and much easier, use the macros in the file "Line Plots->Data", which automates most of these steps. Use the wand tool to outline the plot (click to the left of the curve), then use the Clear Outside macro to erase everything except the plot. Next, use Convert Line Plot to Points, which will perform steps 3-8 above. It assumes the active window contains a binary image and you have selected, using the rectangular selection tool, a single isolated line plot drawn on a white background. Use the Plot Points macro to display the resulting coordinate data. Using the Option KeyHolding the option key down when using many of the commands and tools in Image causes alternative functions to be performed. When using menu commands, you only need to hold the option key down when you first select the command.Starting Image - Use QuickCapture card instead of Scion card, assuming both are installed Menu Command Options Close - Changes to Close All Magnifying Glass - Zoom out instead of zooming in
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