Hyperspectral imaging has become more widely used in biomedical appplications. One of the methods used is the evaluation of KI-67 labeled cells in tissue sections. KI-67 is an antigen that is associated with the high rate of cell division typical of several types of tumors. Calculating the KI67 labeling index involves discriminating the nuclei containing the antigen, labeled with a brown colored immunohistochemical end product (containing the end product of an enzymatic reaction between the antigen and a discloser of its presence called DiaminoBenzidine or DAB).

Cell nuclei are then stained a blue color with hematoxylin. These two dyes or disclosers reveal the proliferating cells (DAB positive) from the total nuclear population (hematoxylin positive). The two populations can be discriminated from one another using spectral unmixing of stacks of images using an AOTF based system. In the case shown below images were acquired from 450-650 nm in 10 nm bands then processed to determine DAB positive cells (containing KI-67 antibody) and hematoxylin stained nuclei discriminating them from the weakly or unstained background tissue in the section.

Hyerspectral Images Stacked

Hyperspectral images as acquired from 450nm-650nm.

Thresholded DAB KI-67 positive (left) and the total nuclear population (right) which allows the user to discriminate between positive (proliferating) cells and the total cell population and calculate KI-67 labeling index.

Positive stained nuclei in red, negative stained nuclei in blue.