There are some good options out there.

Here's a good description of the concepts, and it lists a few plugins.

https://imagej.net/Colocalization_Analysis

I read this paper a while back but I haven't tried the plugin.

https://www.nature.com/articles/s41598-018-33592-8

You may need to do this with install: https://imagej.net/Following_an_update_site#Add_update_sites Giving the URL: http://sites.imagej.net/EzColocalization/

This ResearchGate thread is also pretty good: https://www.researchgate.net/post/How_to_quantify_colocalization_of_dual-color_immunofluorescence_image

There are also several videos on YouTube that take you step-by-step.

Have a very specific type of output you are looking for. For example, plugins like Coloc 2 don't give you information about how well your images/channels align with each other. Whereas, EZcolocalization may give you more of a per-cell-colocalization.

I hope that helps. Good luck!

- Melissa

Yes, one step is co-localization or registration of the images. FIJI has several useful plugins, look in "Plugins > Segmentation." Another consideration is segmentation of the cells from the images. That is, to convert your grayscale or intensity images to a binary representation (0's and 1's or 0's and 255's). As for the analysis, you can do math in FIJI with images, "Process > Image calculator." The intersection, or where both images are "1" or "255" will be found using the "AND" operation. One analysis of the intersection image could be to count the number of objects or blobs. There may be some automated tools for this, but you will inevitably need to do some of the pre- or post-processing of the data.