Spectral imaging for measurement of nuclear pathology features in cancer cells prepared for in situ analysis

An image acquisition method for simultaneously detecting in a single field of view both a genetic rearrangement marker and nuclear morphology in a single cell or single sample containing a population of cells from a tissue, said method comprising:

• a. subjecting the single cell or single sample to a fluorescent nuclear stain for making visible sizes of nuclei, shapes of nuclei, and amount of stain contained in nuclei, and subjecting the single cell or single sample to fluorescence in situ hybridization (FISH) for detecting the genetic rearrangement marker using labeled nucleic acids;
• b. in a single field of view of the single cell or single sample, with a stabilized light source capable of repeating illumination at a plane of the single cell or single sample with less than 1% variation in absolute illumination level so that variability in brightness levels reflects true stain distribution and with a calibrated quantitative detection (CCD) spectral detection system, acquiring a raw spectral data cube using a rapid series of exposures for a set of wavelengths across a wavelength range from 400 nm to 800 nm, the raw spectral data cube comprises intensities for each wavelength at each pixel;
• c. using linear unmixing to deconvolve the raw spectral data cube into specific wavelength channels representing a relative distribution of signal at each pixel, generating individual images for each channel of interest, then generating an image representing nuclear morphology and an image representing the genetic rearrangement marker;
• d. calculating for each nucleus except non-glandular nuclei or non-cancer nuclei (i) nuclear roundness;
• (ii) relative chromatin content;
• and (iii) status of the genetic rearrangement marker;
• and
• e. comparing the nuclear roundness, relative chromatin content, and status of the genetic rearrangement marker of each cell to nuclear roundness, relative chromatin content, and genetic rearrangement marker data from a library of reference cancer cell populations to determine a cancer sub-type.