Spectrally resolved morphometry of the nucleus in hepatocytes stained by four histological methods

A novel concept of spectrally resolved morphometry for histological specimens was developed using light microscopy combined with spectrally resolved imaging. The spectroscopic characteristics of rat hepatocytes stained by Haematoxylin and Eosin, Romanowsky-Giemsa, periodic acid-Schiff and Masson's trichrome were assessed. Light intensity in the range 450-850 nm was recorded from 10000 pixels of nuclear domains of each stained cell and represented as light transmittance spectra and optical density. In order to identify spectral shifts caused by stain-macromolecule interactions, we compared the spectra of individual stain components with those of DNA and bovine serum albumin. Chromatin and interchromatin areas were classified spectrally using a chosen spectral library followed by morphometric calculations of nuclear domains for each staining method. The spectral fingerprints of Masson's trichrome stain distinguished the nucleolus from the rest of the nuclear chromatin, enabling the demarcation and calculation of the nucleolar area. Spectrally resolved imaging of human hepatocytes stained by Masson's trichrome stain revealed marked differences between the nucleolar area in normal human hepatocytes compared with hepatocellular carcinoma. Masson's trichrome stain also distinguished the nucleolar area in human breast carcinoma cells and keratinocytes.