Automated topographical cell proliferation analysis

Background: Cell proliferation is often studied using the incorporation of bromodeoxyuridine (BrdU). Immunohistochemical staining is then used to dete ct BrdU in the nucleus. To circumvent the observer bias and labor-intensive nature of manually counting BrdU-labeled nuclei, an automated topographica l cell proliferation analysis method is developed. Methods: Sections stained with fluorescein-labeled anti-BrdU and countersta ined with To-Pro-3 are scanned using confocal laser scanning microscopy (CL SM). For every point in the image, the nucleus density of BrdU-labeled nucl ei and the total nucleus density of the neighborhood of that point are calc ulated from the BrdU and the To-Pro-3 signal, respectively. The ratio of th ese densities gives an indication of the amount of cell proliferation at th at point. The automated measure is validated by comparing it with the ratio of BrdU-stained nuclei to the total number of nuclei obtained from a manua l count. Results: A positive correlation is found between the automated measure and the ratios calculated from the manual counting (r = 0.86, P < 0.001). Calcu lating the topographical cell proliferation using the automated method is f aster and does not suffer from interobserver variability. Conclusions: Automated topographical cell proliferation analysis is a fast method to objectively find differences in cell proliferation within a tissu e. This can be visualized by a topographical map that corresponds to the ti ssue under study. Cytometry 45:13-18, 2001. (C) 2001 Wiley-Liss, Inc.