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A CHECKLIST FOR EVALUATING IMAGE QUALITY      315

                       otherwise, a too-high black-level setting may produce distinct segments of separate col-
                       ors that under conditions of lower black-level setting and reduced contrast are observed
                       to be contained in the same structures. For critical applications, ratio imaging methods
                       can be used to distinguish between signals that are colocalized vs. signals that are only
                       partially overlapping.



                       A CHECKLIST FOR EVALUATING IMAGE QUALITY

                       The following are criteria we might use in evaluating the quality of a scientific image.
                       They contain the principles we have described along the way for specimen preparation,
                       optical adjustments, and image acquisition, processing, and presentation.

                          Fixation and labeling
                            absence of fixation artifacts
                            adequate fluorochrome intensities
                            absence of bleed-through and nonspecific signals
                            supporting control experiments
                          The acquired image
                            proper optical zoom or magnification to preserve the required degree of optical
                             resolution
                            full range of gray-scale values utilized
                            appropriate camera gain (and offset)
                            notes on microscope optics and camera acquisition parameters
                          Image processing
                            image studied to match pixel values with locations on the image histogram
                            histogram white and black sets adjusted to include all of the data in the image
                            no or minimal black and white saturated areas in the image
                            conservative use of gamma ( ) scaling and sharpening filters
                            insets and explanations made separately for emphasized features requiring
                             extreme image processing
                          Use of color
                            single fluorochromes shown in gray-scale format
                            for colocalization of different fluorochromes, minimum and maximum values of
                             color channels properly adjusted on the image histogram
                            for display of multiple fluorochromes in a montage, gray scale used for single
                             channels, color used for combined multichannel image

                          The check list and guidelines presented in this chapter will allow new microscopists
                       to use image processing knowledgeably and appropriately. Although processing needs
                       vary depending on the application and the particular image, at a minimum, this chapter
                       will help direct discussion on what processing operations should be performed. When a
                       reader of your article can understand and reproduce an observation in his or her own lab-
                       oratory, you will know that you have mastered the essentials of image processing and
                       many fundamentals of light microscopy and electronic imaging.
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