The next step in the process is background substraction, which automatically applies a threshold across the entire image to compensate for any ununiform staining. Because there are a number of modes available for doing this, the user can view and compare the results of each approach on the screen. Image matching Although there are many approaches to matching images from different electrophoresis samples, the most common starting point is to select one gel to be used as a base reference or control. Phoretix compiles an index for all of the spots on this control image, which is then used to automatically correlate the spots on each subsequent image. The algorithms that perform this matching first identify a common focal point in the two images and then work outward until the images are fully correlated.
Background subtraction is one of the most crucial steps in accurately quantifying spot material.
Phoretix includes three automatic methods and one manual method, all of which treat each spot independently to account for variations in background across the gel. The software can be set to perform this image matching automatically, or the user can assist the software by manually identifying the same spot on two images as a common reference point. The user overlays the reference gel spots and links various identifiable spots on the distorted gel to similar spots on the reference gel. Once satisfied with the positioning of the seeds, the warping is removed and the pattern-matching process proceeds. The process doesn't alter either the reference or the image being matched to it.