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I have been having issues with thresholding transmitted light microscope images of opaque particles. The thresholding works great when there are a lot of particles in the field of view. However, when there are only a couple to none, it tends to threshold the imperfection of the illumination from the microscope lamp. Then when the image is counted and measured the program finds particles that are not there due to the lighting. I have attached images to demonstrate my issue. The first way I tried to fix this was writing code to set a manual threshold limit, since these are grayscale images I set it to a certain value, but I am worried it may add bias to my data if not all images threshold at that value (causing some particles to be under or over measured). I have tried to use background correction, and it removes the uneven illumination, but then the image still does not threshold as clear (meaning an all black image). Am I missing something or is there something else that I can try?
Thanks in advance!
Grayscale image that contains no particles. It actually contains some particles but these particles are too small for my measurements.
Threshold of the grayscale image above.
Count and Measure of the image showing the program finding particles that are not there due to the illumination.