This is quite similar to a previous post, however I’m pretty new to ImageJ so I was hoping to get some guidance on the best way to do colocalization with three fluorescent markers in an object-based manner.
Basically, I have two yeast proteins (1 and 2) tagged with RFP and GFP. I know that 1-RFP recruits 2-GFP creating larger GFP puncta. But I’d like to see if these puncta then co-localize with the mitochondria, which I have stained using DAPI. Unfortunately, all puncta are pretty dim so I lose a lot of information if I just threshold the image. But I can easily pick out the puncta using Find Maxima. Ideally the question I’m trying to answer is: when protein 2 colocalizes with 1, is it at the mitochondria?
For reference, my previous experience with colocalization has been using the Metamorph software and combining the granularity app (for identification of dots) with the colocalization app (to look a colocalization of granule area in the two channels).
Sorry if this is a super simple question! Any help would be greatly appreciated!!