Determination of correction factors of 3H-?-self-absorption for quantitative evaluation of grain number in autoradiographic studies

Abstract

Deparaffinized and Feulgen-stained sagittal sections of the mouse brain were studied interferometrically in order to measure optical path differences of euchromatin and heterochromatin of various cell types. Furthermore, the ratio eu-: heterochromatin of each cell type was determined. From these data mass densities of karyoplasm and, finally, correction factors of ³H-β-self-absorption were calculated for comparing grain numbers of different cell types in quantitative autoradiographic studies after application of tritium-labelled substances. Remarkable differences of correction factors up to a factor of 2.18 were found. Furthermore, the actual section thickness was determined interferometrically. A reduction to about 0.60 of the microtome setting was measured in two different areas of the brain. Using mass densities together with actual section thickness correction factors for a thickness of 1 μm were calculated. This was done also for cell types outside the brain the data of which were taken from literature. Thus, differences in correction factors up to about a factor of 4 were found pointing out the importance of considering ³H-β-self-absorption in quantitative autoradiographic studies.