Card-carrying bioinformaticist here....
Are you positive you can't segment using GenePix or an equivalent? Their spot-segmentation algorithms are really very nicely done, and modern versions handle one, two, and four-colour arrays. You don't have to scan on an Axon scanning to use GenePix -- it should handle your Typhoon data no problem. The reason I suggest this is that segmentation is pretty tricky, and when you suggest "corresponding pixels in two spots" type comparisons, that means that even a one-pixel segmentation error will corrupt the comparison. If I reviewering a paper doing this I'd be extremely concerned, and would probably ask for: a direct comparison to an established quanting program on a spot-by-spot basis, direct comparison to an established quanting program on an overall results basis, and public release of the code used for your program. Just my two cents....
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