Appropriate Choice of Threshold

[ABGane]

Dear all,

I am using Image J to compare the level of two channels to each other. The Green channel is a specific component of the Red channel (a mitochondrial subunit stain and a mitochondrial stain).

The images requires thresholding before analysis. I think I will autothreshold the Red channel for each image (Default) but for the thresholding of the Green channel should I autothreshold that too or should I set the green threshold to the same as was automatically determined for the Red channel?

What I'm concerened about is the computer trying to 'equalise' the levels of green detected by a more all-encompassing threshold. I'm assessing the deficiency of green within red so I need the method to be constant across mutliple images too (Which autothreshold at different levels).

Background noise is present in both channels.

Thanks in advance for any help you can provide! This is very important for the validity of my experiment and I just can't get my head round the iterative reconstruction techniques involved so approaching the problem mathematically isn't working for me!

Thanks again!

Angus