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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.