Patent ID: 8335360

Claim:
A computer implemented method for localizing and quantitating a particular biomarker within a first marker-defined biological compartment relative to a second marker-defined biological compartment present in a biological sample comprising: a) incubating the tissue sample with a first imaging agent that specifically labels the first marker-defined compartment, a second imaging agent that specifically labels a second marker-defined sub-cellular compartment, and a third imaging agent that specifically labels the biomarker; b) obtaining a first high-resolution image of the first imaging agent labeled sample, a second high-resolution image of the second imaging agent labeled sample, and a third high-resolution image of the third imaging agent labeled sample; c) determining the first and second imaging agent pixel intensity in each of the pixel locations in the first and the second image; d) performing an automated clustering analysis on the pixels to identify three data centroids to provide automated differentiation of signal from background and differentiation of the first and the second marker-defined sub-cellular compartment associated pixels; and e) analyzing in the third image the pixel locations assigned to the compartments so as to identify those pixel locations with an intensity value indicative of the third imaging agent, and determining the total intensity value of the third imaging at the pixel locations assigned to each of the first and second compartments, so as to thereby localize and quantitate the biomarker in the first compartment relative to the second compartment.