substraction two images at a roi

Hello,

I have two videos (40x40 pixels each with around 300 images each) from fluorescence imaging with some bleed through from one channel (channel 2 in the image) to the other (channel 1 in the image) only in the center of the image (picture attached). I tried substracting, but the problem is that the channel causing the bleed through has a lot of background noise, so it also substracts pixels at other region. Is it possible to do a program with matlab where I would give him the coordinates at which the substraction is to be done?

Thank you very much in advance.