Subtract red from green?

howdy,

I am having trouble figuring how to subtract background in some of my images. I have astrocytes in mCherry and oligos in GFP. Oligo’s sometimes “bleed through” the images and appear in my astrocyte channel and vice versa. Any guidance as to how I can completely remove the cross over?

@juturna

Doing this computationally after-the-fact is not ideal… so I’d advise you to re-evaluate your acquisition. Perhaps you can use different fluorophores that are more easily separated? Ideally - you’d have single-stained controls too… so you can properly measure this ‘cross over’. It might be worth posting this on the Microforum: