3-D fluorescence and reflectance imaging in cell pathology, using confocal scanning laser microscopy on multiply-labeled specimens

3-D Images were obtained by confocal laser microscopy from specimens of human breast cancers and breast cancer cell cultures and from rat livers. Fluorescence imaging was used for the analysis of nuclear DNA, Brdu and several cytoskeletal micofilament proteins. Reflectance imaging was used for the analysis of the expression of the Rho B gene by immunogold-labeling of a 0.5 Kb riboprobe. Optimized imaging, including discrete photon counting and mathematical correction for the attenuation of emission intensity in depth, allowed the study of thick 3-D images. After 3-D image segmentation, various quantitative parameters were estimated, including nuclear DNA histograms. Segmentation and quantitation of images after in situ hybridization were particularly straightforward, due to the use of reflectance imaging.