I was wondering a) if I should perform flat-field correction and background subtraction sequentially and b) if so, how do I go about about doing this? The foreground is comprised mostly of cells, which are evenly distributed across the image, and as with all microscope-based images there is heterogeneity in illumination due to the imaging system. Would I first use the images (~1,000 per channel) from each plate to generate an illumination function using CorrectIlluminationCalculate (with the “regular” and “all” options) then flat-field correct each image using CorrectIlluminationApply (with the “divide” option)? Since I want to subtract the background fluorescence (i.e. the fluorescence from the non-cell regions) from these flat-field corrected images would I next use the CorrectIlluminationCalculate module (with the “background” and “all” options) to estimate the background fluorescence and finally subtract this from each image using CorrectIlluminationApply (with the “subtract” option)? Also, since I am most interested in measuring the fluorescence intensities of the cells I should not “rescale intensities” in either case correct? Thanks for your help and I look forward to hearing your thoughts!