I want to quantify the nucleoplasmic signal of my protein of interest but excluding the staining in the nucleoli (structures inside the nucleus). I have experience in generating pipelines to identify objects inside the nucleus and measure properties and relate it to the parental nuclei. However I have never used those objects inside the nucleus to create a “negative mask”, meaning a nuclear mask that excludes them. I have sets of 3 pictures: channel 1=DAPI, channel 2= my protein of interest, channel 3= a protein that stains nucleoli in order to identify these structures. Is it possible to generate this kind of nuclear mask without the nucleoli? Or is it better to quantify the signal in the whole nucleus but then subtract the signal from the nucleoli? if this is the case, how can I subtract the signal of all the nucleoli to the same parental nucleus? Also, I would like to keep the data per individual nucleus.
Please, can you help me?