SPECTRALLY RESOLVED MORPHOMETRY OF THE NUCLEUS IN HEPATOCYTES STAINEDBY 4 HISTOLOGICAL METHODS

A novel concept of spectrally resolved morphometry for histological sp ecimens 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-S chiff and Masson's trichrome were assessed. Light intensity in the ran ge 450-850 nm was recorded from 10 000 pixels of nuclear domains of ea ch stained cell and represented as light transmittance spectra and opt ical density. in order to identify spectral shifts caused by stain-mac romolecule interactions, we compared the spectra of individual stain c omponents with those of DNA and bovine serum albumin. Chromatin and in terchromatin areas were classified spectrally using a chosen spectral library followed by morphometric calculations of nuclear domains for e ach staining method. The spectral fingerprints of Masson's trichrome s tain distinguished the nucleolus from the rest of the nuclear chromati n, enabling the demarcation and calculation of the nucleolar area. Spe ctrally resolved imaging of human hepatocytes stained by Masson's tric hrome stain revealed marked differences between the nucleolar area in normal human hepatocytes compared with hepatocellular carcinoma. Masso n's trichrome stain also distinguished the nucleolar area in human bre ast carcinoma cells and keratinocytes. (C) 1998 Chapman & Hall.