Glare-correction in DNA image cytometry.
We tested the reproducibility of a simple method for glare correction in a modern video-based optical densitometer (CYDOK) by performing repeated measurements of the DNA content on identical sets of different rat hepatocyte DNA-classes and lymphocytes. Using a computer-controlled scanning stage of the microscope we were able to re-localize each nucleus of air-dried Feulgen-stained preparations with different microscope settings. The proposed microscope-adjusted glare-correction algorithm is based on the measurement of the transmittance of opaque bars a standard micrometric glass. The procedure gives local glare errors and allows calculation of the mean glare error which is applicable for the entire view field. Subtraction of this mean glare transmittance from each object and normalization of the background allows the user to eliminate errors due to different nuclear Feulgen-staining intensities. The method eliminates the need to use cell-type-dependent constants to correct the known DNA disproportionalities in air-dried, Feulgen-stained preparations. An additional advantage of the glare correction algorithm is the low coefficients of variation (CV, < 3%) within each nuclear DNA-class which reflect the highly reproducible DNA measurements.
Kindermann
D
D
Institute of Pathology, University of Bonn, Germany.
Hilgers
C H
CH
D000465:Algorithms; D004247:DNA / Q000032:analysis*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000592:standards*
D004247:DNA