I’m something of a novice user and have recently run into a problem related to variable background illumination in different wells. The basic principle behind my algorithm is that objects with pixel intensities above a pre-determined threshold are classified as positive, and those below it are classified as negative. The majority of wells have a similar range of pixel intensities for positive and negative cells, but a handful of wells (10-15% of wells in a 384-well plate) have elevated background intensities, leading to classification of non expressing cells as positive.
I’m curious if there is a way to standardize the pixel intensities for each respective image such that the range of intensities between a normal and high background image would be roughly the same.
Thanks in advance for any assistance!