Image adaptive physiologically plausible color separation

A method for separating colors in an image, the method comprising:

• receiving, at a processor coupled to a workstation, an assay information comprising an image data, the image data comprising a plurality of signals detected from an assay identified in the assay information;
• adjusting, by the processor, one or more reference vectors based on a correlation of one or more of the plurality of signals with one or more of a plurality of rules stored on a memory that is communicatively coupled to the processor;
• and
• unmixing, by the processor, the one or more of the plurality of signals using the adjusted one or more reference vectors;
• and
• determine presence or organization of one or more biomarkers by analyzing the image data unmixed using the one or more optimized vectors.

A non-transitory computer-readable medium for storing computer-executable instructions that are, when executed by a processor one or more processors of a computing system, cause the computing system to perform operations comprising:

• generating a histogram of color vectors within a reference vector space derived from an image data associated with an assay comprising a stain, the stain being associated with a plurality of markers and a corresponding plurality of reference vectors;
• comparing the histogram with a known histogram for the assay;
• and determining whether or not to adjust one of the plurality of reference vectors based on the comparison;
• wherein one or more adjusted reference vectors are used to unmix the image data;
• and
• determine presence or organization of one or more biomarkers by analyzing the image data unmixed using the one or more optimized vectors.