QUANTITATIVE ANALYSIS OF HISTOCHEMICAL REACTIONS: IMAGE ANALYSIS OF LIGHT AND ELECTRON MICROSCOPIC RADIOAUTOGRAMS
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In order to quantify histochemical reactions, light and electron microscopic radioautograms obtained from mouse organs were analyzed by means of various kinds of available image analyzers. Cultured cells in vitro or epithelial cells of the stomachs, small and large intestines, the livers and the pancreases of mice aged from fetal day 19 to postnatal 2 years were labeled with RI-labeled precursors for macromolecules, e. g. 3H-thymidine for DNA, 3H-uridine for RNA, 3H-amino acid for proteins, 3H-glucosamines and 35SO4 for mucosubstances, 3H-glycerol for lipids and all the cells and tissues were fixed in glutaraldehyde and osmium tetroxide, embedded in Epon, sectioned either thick (2μm) or thin (0.2μm), coated with either Konica NR-M2 or-H2 radioautographic emulsions, exposed for several weeks or months, developed in either SDX-1 or GL-phenidon developers and stained with either toluidine blue or lead citrate for either light or electron microscopy.Light microscopic radioautograms (LMRAG) were observed with Olympus Vanox AHB-LB or Nikon Optiphot-2 and analyzed quantitatively with IBAS-II (Carl-Zeiss) or Quadra 900 (Macintosh) for labeling indices and grain counts. Electron microscopic radioautograms (EMRAG) were observed in Hitachi H-700 or JEOL JEM-4000EX electron microscopes at accelerating voltages of 200 or 400kV, and enlarged photographs were analyzed with several kinds of image analyzers, PPA-250 (Rhesca), MOP (Kontron), Digigramer-G (Mutoh Kogyo) and Quadra 900 (Macintosh) for grain counting or measuring the sizes and numbers of cell organelles. Some of EMRAG were analyzed in analytical electron microscopes, combined with Hitachi H-700 Horiba EMAX-1800E, JEOL JEM-200CX Kevex 7000-77, and JEM-4000EX Tracor Northern TN-5400.From the results, it was demonstrated that correct data can, to some extent, be obtained by controlling both full automatic and semi-automatic analyzers by operating them manually.
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