Spectral imaging for quantitative histology and cytogenetics.

Evaluation of cell morphology by bright field microscopy is the pillar of histopathological diagnosis. The need for quantitative and objective parameters for diagnosis gave rise to the development of morphometric methods. Morphometry combined with spectral imaging provides multi-pixel information from a specimen, which can be used for further image processing and quantitative analysis. The spectroscopic analysis is based on the ability of a stained histological specimen to absorb, reflect, or emit photons in ways characteristic to its interactions with specific dyes. Spectral information obtained from a histological specimen is stored in a cube whose appellate signifies the two spatial dimensions of a flat sample (x and y) and the third dimension, the spectrum, representing the light intensity for every wavelength. By mathematical analysis of the cube database, it is possible to perform the function of spectral-similarity mapping (SSM) which enables the demarcation of areas occupied by the same type of material. Spectral similarity mapping constructs new images of the specimen, revealing areas with similar stain-macromolecule characteristics and enhancing subcellular features. Spectral imaging combined with SSM reveals nuclear organization and identifies specifically the nucleoli domains. Therefore, differentiation stages as well as apoptotic and necrotic conditions are easily quantified. The commercial SpectraCube system was developed for the application of spectral imaging in biology, recording both transmitted light and fluorescence. The SKY technique utilizes the advantages of the SpectraCube for multi probe FISH and chromosome karyotyping, identifying marker chromosomes, detecting subtle chromosome translocations and clarifying complex karyotypes.