Patent ID: 7219016

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 compartments present in individual cells of interest contained in a tissue sample comprising: a) incubating the tissue sample with a first stain that specifically labels a first marker defined subcellular compartment, a second stain that specifically labels a second marker defined subcellular compartment, and a third stain that specifically labels the biomarker; b) obtaining a high resolution image of each of the first, the second, and the third stain in the tissue sample using an upright or inverted optical microscope 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, wherein each image comprises 1024×1024 pixel locations; c) (1) determining the first and second stain intensity in each of the pixel locations in the first and the second image and assigning those pixel locations having an intensity indicative of: i) the first stain only, to the first compartment; ii) the second stain only, to the second compartment; iii) both the first and the second stain, to the compartment for which the stain intensity is greater or to neither compartment if the stain intensity is substantially equal; (2) reiteratively analyzing the first and the second stain intensity in each of the pixel locations assigned to each of the first compartment and the second compartment and reassigning each pixel location based on a weighted ratio of the first stain intensity relative to the second stain intensity to reach a 95% degree of accuracy in the assignment of the pixel location; d) analyzing in the third image the pixel locations assigned to the first subcellular compartment or the second subcellular compartment in step (c) so as to identify those pixel locations having an intensity value indicative of the third stain, and determining the total intensity value of the third stain at the pixel locations assigned to each of the first and second subcellular compartment; so as to thereby localize and quantitate the biomarker in the first subcellular compartment relative to the second subcellular compartment.