The confocal microscope collects high contrast 2-D images at different depths from thick (≥100 μm) tissues samples, thus providing data for quantitative measurements of 3-D objects. There are several factors that may limit quanititative analysis including photobleaching of fluors, attenuation of measured signal, mismatch in the various refractive indeces of the sample, and the width of the point spread function. We have addressed the first three of these limitations by using acriflavine-stained 100- [im thick sections of rat hippocampus and our 3-D automated analysis software. The laminar structure of the hippocampus provides tissue sections with areas of high and low nuclear density (pyramidal and extra-pyradmidal cell regions, respectively). Corrections for these limitations were made to samples from rat testes where developing spermatogonia provide nuclei of different DNA content.The contribution of photobleaching to signal degradation was determined by collecting multiple 3-D images (z-series) of the same field. Twenty-one images of 50 planes each were collected (total scans= 1050).