How do I outline/identify a stain for co-localisation in images with “background” stain?
I am new to CellProfiler and I would like to use it to measure signals from fluorescent stainings in some human tissue. I have some problems when it comes to identifying/outlining my primary objects and I hope someone in this forum can be of help.
I have two images; one of a green stain and one of a DAPI stain which I would like to use to identify co-localisation so I can use that to measure the green fluorescent stain that co-localises to the nuclei. The green stain is basically everywhere in my tissue, including in nuclei, with varying intensities. The DAPI stain I can identify and outline without any major problems:
I also have the following (green) stain:
I am only interested in measuring the green fluorescent stain that overlap/localises with the nuclei stain. When I try to outline the green stain it is, probably understandable, difficult to identify only the nuclei/round shape of the green stain. I have tried to make a pipeline that identifies the nuclei stain and all of the green stain, and then used the DAPI stain to identify were the DAPI and green stain overlap:
But the outline for this is not perfect so when I measure the signal intensity, I don’t measure enough of the positive stain that co-localises to the nuclei:
For identifying primary objects for the green stain, I have tried different thresholding methods and I have tried different ways of distinguishing and dividing clumped objects. The last image is the closest I have gotten to outlining the co-localisation (distinguished by intensity, divided by propagate).
So, my question is if anyone knows if it is possible to optimise this co-localisation further? I have attached my pipeline if that is needed:
HDAC2Co-locNuclei_pipeline.cppipe (13.7 KB)