I’m having a problem measuring fluorescent intensity on tissue arrays. What I have been doing is subtracting the background and selecting 3 random ROI’s within the tissue and measuring the mean pixel intensity. However, I’m not sure this is the best route. Autofluorescence of the tissue seems to be a big issue here and I’m not sure if I’m measuring autofluorescence or the actual fluorescent marker. I have tried comparing the control (with no fluorescent marker) to the tissue with the marker but in all of these cases the autofluorescence seems to be brighter than the tissue with the fluorescent marker. The mean intensity and the max is always higher in the control. I’m not sure if there is a way to discriminate the two? Does anyone know of a way I can separate the two or measure the marker only?