Patent ID: 8639450

Claim:
A computer implemented method for localizing and quantitating a particular biomarker within a first marker defined subcellular compartment relative to a second marker defined subcellular compartment present in individual cells of interest contained in a tissue sample comprising: a) incubating the tissue sample with a first stain that specifically labels the first marker defined subcellular compartment, a second stain that specifically labels the second marker defined subcellular compartment, and a third stain that specifically labels the biomarker; b) obtaining an image, said image comprising pixel locations, of each of the first, the second, and the third stain in the tissue sample using an optical imaging device so as to obtain: i) a first image of the first marker defined subcellular compartment; ii) a second image of the second marker defined subcellular compartment; and iii) a third image of the biomarker; c) analyzing pixel locations in the first and the second image so as to assign the pixel locations to the first, the second or neither subcellular compartment based upon an intensity value of the first stain relative to the second stain at that pixel location; d) analyzing in the third image the pixel locations assigned to the first or the second subcellular compartment in step (c) so as to identify those pixel locations having an intensity value indicative of the presence of the third stain, and determining the total intensity value of the third stain at such pixel locations; so as to thereby localize and quantitate the biomarker in the first subcellular compartment relative to the second subcellular compartment.