Simultaneous Feulgen densitometry and autoradiographic grain counting with the Quantimet 720D image-analysis system. III. Improvements in Feulgen densitometry.
A method has been developed for densitometric estimation of the Feulgen-stained DNA content of 3H-labeled nuclei in autoradiographs in conjunction with automated grain counting using a Quantimet Imaging System. Refinements in the methodology are reported which include 1) the incorporation of an Image-Editor Module into the Quantimet module configuration; 2) the optimization of incident illumination based upon evaluation of various light sources; 3) changes in the optical configuration which reduce glare and minimize the level of monitor shading correction; 4) the optimization of scanner sensitivity; and 5) the evaluation of cell-flattening and staining with respect to densitometry resolution and sensitivity. These refinements resulted in a CV of less than 6.4% in the G-1 and G-2 DNA peaks of rat kidney cells in autoradiographs compared to the previous CV of 10.5%, and a G-2 to G-1 ratio of 2.025. For a fixed field position the CV was 5.1% and the replication error less than 1.0%.
D000818:Animals; D001345:Autoradiography; D002478:Cells, Cultured; D004396:Coloring Agents; D004247:DNA / Q000032:analysis*; D003720:Densitometry / Q000295:instrumentation / Q000379:methods*; D006651:Histocytochemistry / Q000295:instrumentation*; D007399:Interphase; D007668:Kidney / Q000166:cytology; D008029:Lighting; D055095:Optics and Photonics; D051381:Rats; D012394:Rosaniline Dyes; D013690:Television / Q000295:instrumentation* / Q000379:methods
D004396:Coloring Agents; C007722:Feulgen stain; D012394:Rosaniline Dyes; D004247:DNA