COMMENTS RELATED TO SCIENTIFIC IMAGING

"CONFOCAL" COLORS ARE WRONG. The red, green and blue colors commonly used for confocal images simply do not appear with equal brightness on the computer screen, nor do these reproduce. These color tables need to be re-created in order to have colors fit for output (see pdf of Microscopy Today article). Automated actions for creating "output" colors can be obtained with actions provided at seminars given by Jerry.

EVENLY ILLUMINATED SAMPLES A MUST FOR IMAGE ANALYSIS. Especially when thresholding to separate features of interest from background, an evenly illuminated field is critical. This requires flatfield (shading) correction when acquiring images, and non-koehler illumination (condensor aperature all the way open when acquiring in brightfield) to eliminate excess detail within features.

PHOTOSHOP CAN CORRECT UNEVEN ILLUMINATION FOR EM, GELS, LIGHT MICROSCOPY. You won't find this in a Photoshop manual, but methods exist within Photoshop for correcting uneven illumination. See pdf for more info.

PHOTOSHOP CAN SEPARATE FEATURES OF INTEREST FROM BACKGROUND (SEGMENT). Several means to segment images can be found within Photohoshop. These include grayscale erode & dilate (minimum/maximum), separation of features by color, several color modes from which a grayscale channel can reveal an easier means for separation, median filtering to remove noise, high pass filtering to flatten background, etc.

H&E IMAGES ARE OFTEN SATURATED IN THE PINK RANGE. Many digital cameras make eosin neon-pink or neon-fuschia. These colors need to be desaturated by using the Hue & Saturation tool (Image > Adust(ments) > Hue & Saturation) or by correctly applying auto-color. Both methods are covered in Jerry's seminars.

THE "IMAGE SIZE" DIALOGUE BOX IS THE MOST DANGEROUS BOX IN PHOTOSHOP. Resample should be UNCHECKED unless a specific output resolution is required (e.g., when going to publication). Otherwise the inherent resolution of the image can change!

YOU CAN'T ALWAYS RELY UPON 16- TO 8-BIT CONVERSION UNDER MODE IN PHOTOSHOP. Software included with some scientific cameras include exporting or saving to 16-bit TIFF files. These files open in pre-CS versions of Photoshop as 16-bit, but the values are truncated to the 8-bit range (this can be seen by looking at the histogram: under Image > Histogram). Adjustments in grayscale levels must be made BEFORE converting image to 8-bits/channel.\

IMAGE STACKS FROM CONFOCAL SOFTWARE CAN BE OPENED AS LAYERS IN PHOTOSHOP... but only by using GIMP first. GIMP is a program that contains many functions that also exist in Photoshop, but the interface is unfamiliar. It is worth downloading, however, for purposes of reading TIFF stacks and placing these in layers so that the stack can be saved in the Photoshop format (make sure, however, that each layer uses Lighten, which is similar to the maximum setting in confocal software applications). The website for downloading GIMP is http://gimp-win.sourceforge.net/stable.html for Windows and http://www.gimp.org/macintosh/ for Macintosh OSX. For Windows, both the GIMP program AND the GTK software needs to be downloaded. For the Mac, it appears that both FINK and GIMP.APP need to be downloaded.

SHARPENING METHODS CAN BE USED TO SHOW BONE DENSITY IN X-RAYS, AND TO EMPHASIZE FEATURES FOR PRESENTATION PURPOSES. The use of unsharp mask and a layering method employing the high pass filter can emphasize details within features.

PSUEDOCOLOR TABLES CAN BE APPLIED TO GRAYSCALE IMAGES IN PHOTOSHOP. Color tables using the visible spectrum is the most useful psuedocolor option in Photoshop. See the psuedocoloring article for more details.

WHITE AND BLACK VALUES ARE NOT APPLIED APPROPRIATELY IN SCIENTIFIC IMAGES. The deepest black values for images should NOT be set to 0: instead, this value should be set for output devices. The generic output values are given in seminars given by Jerry, or these can be found in ...