METHODS FOR CELL-PROLIFERATION ANALYSIS BY FLUORESCENT IMAGE CYTOMETRY

Methods were developed for multimodal microscopic image analysis in or der to identify and analyze one cell type under various microscopic co nditions. Our purpose was to quantify both total DNA content using pro pidium iodide (PI) stain and S-phase fraction using the bromodeoxyurid ine (BrdUrd) incorporation technique in cell population subsets. The m odel chosen was plasma cells in bone marrow triply labelled with fluor escein isothiocyanate (FITC) for intracytoplasmic immunoglobulins, wit h amino-methylcoumarin-acetate (AMCA) for BrdUrd, and with PI for DNA. Image analysis included three phases. First, plasma cells were recogn ized on FITC images, and the centroid positions were stored. Second, p lasma cell nuclei were geodesically reconstructed from these stored po sitions using PI images in which DNA content was measured, and the nuc lear mask outlines were stored. Third, BrdUrd incorporation level of p lasma cells was measured on AMCA images inside PI nuclei masks and sto red. Image DNA vs. BrdUrd scatterplots were obtained for cells selecte d according to the expression of intracytoplasmic immunoglobulin. Thus , both ploidy and proliferation could be independently evaluated on a subset of the cellular population. (C) 1995 Wiley-Liss, Inc.