Affiliations: Laboratoire de Mécanique et Ingénierie Cellulaire et Tissulaire, UMR CNRS 7563 LEMTA et IFR 111 CNRS, UHP‐INPL‐CHU, 54505 Vandoeuvre‐lès‐Nancy, France | Physiopathologie et Pharmacologie Articulaires, UMR 7561 CNRS‐UHP et IFR 111 CNRS, UHP‐INPL‐CHU Nancy 1, 54505 Vandoeuvre les Nancy, France | Grupo de Optica Aplicada, Instituto de Fisica de Rosario (CONICET‐UNR), Argentina
Note: [] Address for correspondence: Dumas Dominique, Service d'Imagerie et Biophysique Cellulaire et tissulaire de Lorraine, Laboratoire de Mécanique et Ingénierie Cellulaire et Tissulaire, UMR CNRS 7563 et IFR 111, Faculté de Médecine, B.P. 184, 54505 Vandoeuvre‐les‐Nancy Cedex, France. Tel.: +33 03 83 68 34 65; Fax: +33 03 83 59 26 43; E‐mail: [email protected]‐nancy.fr.
Abstract: Spectral and multiphoton imaging is the preferred approach for non‐invasive study allowing deeper penetration to image molecular processes in living cells. But currently available fluorescence microscopic techniques based on fluorescence intensity, such as confocal or multiphoton excitation, cannot provide detailed quantitative information about the dynamic of complex cellular structure (molecular interaction). Due to the variation of the probe concentration, photostability, cross‐talking, its effects cannot be distinguished in simple intensity images. Therefore, Time Resolved fluorescence image is required to investigate molecular interactions in biological systems. Fluorescence lifetimes are generally absolute, sensitive to environment, independent of the concentration of the probe and allow the use of probes with overlapping spectra but that not have the same fluorescence lifetime. In this work, we present the possibilities that are opened up by Fluorescence Lifetime Imaging Microscopy, firstly to collect images based on fluorescence lifetime contrast of GFP variants used as a reporter of gene expression in chondrocytes and secondly, to measure molecular proximity in erythrocyte (glycophorin/membrane) by Fluorescence Resonance Energy Transfer (FLIM‐FRET).
