I need some of your expertise for analyzing GFP fluorescence of mammalian cells after CRISPR knockout of the stably expressed gfp gene. I am having some difficulties achieving this, and found great help from the solutions posted in similar topics, specifically to find the mean intensity of individual objects.
However, is this good to compare the intensities within one given image? Is the intensity given from a 0 to 1.0 scale an absolute intensity or is it a relative pixel intensity based on the image being processed? Given that the output of my KO is not an “on/off” type signal, I want to get the mean intensity of each individual object to be able to draw a histogram to compare the intensity distribution of WT vs. KO cells on ONE chart. I just want to make sure that the data I produce is not flawed.
I did my best to make this clear, let me know if my description was a bit fuzzy and I’ll try to clarify!