Counting foci in large tile scans

Hi everyone,

I’m looking for any help on setting up gH2AX foci counting from my images. I have some tilescans with over 100 images in them and some lone images. Preferably I would like to stitch the images of each biological replicate together, make a composite image (if necessary) and then apply the counting plugin. My images are all .lif files.

Is it necessary for me to make composite images for this purpose? I did gH2AX staining with Alexa Fluor 488 and DAPI, so I have DAPI in one channel and AF488 in another. Or do I only need to do the counting in one channel?

I was also going to stitch the images in my tilescans together using this

But I also have this lone acquisitions which don’t overlap at their borders, can I stitch those images together too for counting?

I was going to count the foci by using this method:

Many thanks for any pointers.