Simultaneous Feulgen densitometry and autoradiographic grain counting with the Quantimet 720D image-analysis system. I. Estimation of nuclear DNA content in 3H-thymidine-labeled cells.

Open Access
Publisher Website
Google Scholar
Abstract
A Quantimet 720D computer-based television image-analysis system was configured and programmed to determine simultaneously the DNA content and nuclear grain count of 3H-thymidine-labeled cells in Feulgen-stained autoradiographs. This is the 1st time the dual measurements were made in individual nuclei with a single instrument. Using a light pen, .apprx. 800 cells may be measured/h. The integrated optical density of Feulgen-stained DNA is determined in labeled nuclei from the product of the mean optical density of the grain-free nucleus and the projected area of the entire nucleus. In defining the grain-free nucleus for measurements, the grain phase is masked by new thresholding procedures. The methodology was validated in diploid G-1 rat kidney cells labeled with 3H-thymidine. Densitometry bias due to glare and halo effects around grains was systematized, using signal autodelineation, and then measured as a function of grain coverage. A model of the nuclear-grain phase edge-function was developed for evaluating the error components at the boundary. Algorithms were developed which minimize densitometry bias so that the accuracy and reproducibility of the Feulgen-stained DNA estimates is independent of grain coverage in well-flattened nuclei. A procedure is described for establishing the nuclear sampling requirement for the proper use of the algorithm in various cell types. The simultaneous grain counting methodology with the Quantimet is detailed in a companion paper. The methodology provides a rapid means of determining 3H-thymidine labeling throughout the DNA histogram on a cellular basis and for estimating relative specific activity of labeled nuclei. Critical applications are suggested for studies of DNA synthesis and cell cycle kinetics.