6th ESACP Congress, Heidelberg, April 7-11, 1999

A075
CONFOCAL MULTIFLUORESCENCE AND UV MULTICOLOR CHARACTERIZATION OF STAINS OF CELLULAR PREPARATIONS.
Kahn E 1, Bruzzoni-Giovanelli H 2, Souchier C 3, Frouin F 1, Clément O 1, Frija G 1, Di Paola R 1, Calvo F 2, Bernengo JC 3, Linares-Cruz G 2

1) Inserm U494, CHU Pitié Salpétrière, Paris, 2) CRI Inserm 98-1, Hôpital St-Louis, Paris, 3) Centre Commun de Quantimétrie, Université Lyon1, Lyon, France

PURPOSE: To show that cellular preparations requiring multifluorescence analysis resulting in X, Y, and Z shifted images can be realigned by factor analysis of medical images (FAMIS), and correlation methods, before being superimposed. To show that that FAMIS and correlation methods which involve multifluorescence can be avoided when selected fluorochromes can be excited by only one laser. METHODS: 1) In multifluorescence studies, we used propidium iodide fluorescent beads and tetramethyl rhodamine isothyocianate (TRITC), fluorescein isothyocianate (FITC) and 4'-6 diamidino-2-phenylindole (DAPI) stained human cancer lines. They were excited using HeNe, argon, and ultraviolet (UV) argon laser bands of a confocal laser scanning microscope. 2) Multicolor experiments using UV excitation only were performed using europium as a model for magnetic resonance paramagnetic contrast agents. Nuclei of human cancer lines were counterstained by DAPI, and cytoplasms were labelled by ELF substrates (Molecular Probes). RESULTS: 1) Estimated images corrected depth differences, and correlation methods provided X, Y correction values. 2) Each fluorochrome was clearly distinguished in the group of fluorochromes. Estimated images in both studies showed colocalizations of structures. CONCLUSIONS: It is possible to characterize differences in the focus and alignment of fluorescent probes and to correct them. It is possible to study colocalization of cellular preparations via multifluorescence or multicolor experiments.