First off, thank you for the very extensive answer, your help is much appreciated.

Now to clear up a few things. First, the sample image was only provided for those who have never seen a microarray slide to better understand what I mean by "slide", "spot", etc. the images I am working with are indeed jpegs are much larger and actually quite a bit cleaner (I wish I had a place to upload an actual sample... I'll see if I can find a way).

Most importantly - I am not doing any sort of analysis on the spots themselves. The analysis is done on the original TIFF files (which are much too large to reasonably store for this application, which is why I am using the jpegs, I am not sure how compressed they are, but they are still of very high quality) using software written by many people much smater than I over the course of many years :) All I need is to get the spot given the coordinates - it's just another visual clue for the user in the final report, so the spot colour doesn't need to be anywhere near as precise as what is used for the actual analysis.

It has not in fact occured to me that much easier than rotating the actual image would be to determine the distances to the border for each row/column and adjust the cropping accordingly, most likely this will be what I end up doing. In fact just doing this for each of the four cornet spots, which have a very high contrast with the background just for this purpose, should be sufficient to figure out the rest of them.

Incidentally, speed and memory requirements are not an issue at all here - I would most likely to this once on the image to pick out all the spots and store them individually, and we are only talking about a few hundred of these over the course of several years.