Checking for Uneven Illumination in Microscope Images

How to check to see if microscope images have problems with uneven illumination: where images may be overall brighter in some areas and darker in others, often a problem with vignetting (a brighter center spotlight effect). Very important when measuring fluorescent intensities or densities from images; and absolutely essential when using tonal separation to isolate regions of interest (segment).

How to Check Uneven Illumination in GIMP

Colors > Threshold
Iteratively adjust threshold to see lighting pattern

How to Check Uneven Illumination in Image J

Image > Adjust > Threshold
Iteratively adjust threshold to see lighting pattern

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