How to get number of co-localizing particles?

I’m trying to quantify coexpression of two genes with fluorescent in situ hybridization. I have a lot of sections so 'd love to speed up the process with automated counting by ImageJ. What I did until now is, split the channels, inverted the image, adjusted the threshold and used the analyze particles function. Now I have my ROIs for both the red and the green channel. Is there a plugin for overlaying these and telling how many of them overlap?


You can go back a step and once you have the threshold set - Create Mask - to get a binary mask for each channel. Then you can do simple Math with the two binary mask and ask where pixels are located only within both (AND). This assumes that your objects don’t overlap in multiple places (so would work well with circular objects)… then you would get a binary image showing only the pixels where there was overlap - so you could run Analyze Particles once again and count / measure those areas.

Perhaps you can share your original images - and we can better help you come up with a workflow to answer this? But the above should help get you started. :slight_smile: