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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A075
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.
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