Uneven illumination occurs when the light does not evenly illuminate your sample across the field of view. This results in darker areas and more brightly illuminated areas, as you can see below.
Often you’ll see places (usually in the corners) that are darker than the rest of the image. It makes quantitation very tricky, if not impossible.
Figure 1. Cells stained with a nucleic acid dye are not evenly illuminated, most likely due to a misaligned light path in the microscope.
If you notice uneven illumination, that means the microscope light path needs to be aligned.
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