How do I outline/identify a stain for co-localisation in images with "background" stain?

How do I outline/identify a stain for co-localisation in images with “background” stain?
Hello,
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:
HDAC2StainOutlines_correct

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:
DAPIandHDAC2Identified_correct

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)

Best,
Camilla

Hi @CamillaTErichsen,

I am only interested in measuring the green fluorescent stain that overlap/localises with the nuclei stain.

If I understand your question correctly, it seems like the MaskObjects module could be helpful. Using this module would allow you to mask your HDAC2 objects and keep only the regions that overlap with DAPI. You could then continue to use the RelateObjects module in order to know which HDAC2 region belongs to which nucleus. You can make also then make intensity measurements on only your new HDAC2 objects that are for the regions that overlap w/ DAPI.

I hope this helps. Feel free to post more description of your challenge, if not, and a sample image set. Good luck!
Pearl

1 Like

Hi @pearl-ryder,
Thank you very much for your suggestion. My problem has now been solved!

Camilla