Flat-field correction AND background subtraction?


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!



Yes, what you suggest sounds reasonable. Yes, do this:

At this point, this first step is all you need to do. If your background now still shows great variation from image to image, then I would double- and triple-check that the scope isn’t auto-scaling the images in some way. It may not be, but you should be sure.

But yes, if you then want to get the absolute difference between foreground and background for each image, then do as you say:

And it is fine to uncheck “Rescale intensities”. It ought not to make any difference, because presumably all the images would be “corrected” the same amount, but it is possible that something might go awry with your metadata. This rescaling is most often useful for 12-bit cameras that save images in a 16-bit format, and thus look dark if the display is not scaled appropriately.

Good luck!