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microscopie de fluorescence par excitation à deux photons : application à des études de corrélations et de déclins de fluorescence en milieu biologique

Elvire Guiot 1
1 Laboratoire Charles Fabry de l'Institut d'Optique / Elsa
LCFIO - Laboratoire Charles Fabry de l'Institut d'Optique
Abstract : This thesis presents the development of a fluorescence microscope based on the non linear process of two-photon absorption. This new type of fluorescence microscopy characterized by a intrinsic three-dimensional spatial resolution comparable to those of the confocal microscope, offers also many advantages for biological studies, in particular in terms of restriction of the photodamages. Combined with time resolved techniques, two-photon microscopy allows imaging with high space and time resolutions and opens up new opportunities for biological dynamics studies. In this context, two time resolved techniques for fluorescence analysis under two-photon excitation have been developed. The first time resolved technique, the fluorescence correlation microscopy, based on measurements in micro-volumes with weak molecular concentration, has been essentially applied to the study of translational diffusion processes. In particular, the technique has allowed the determination, at the single molecule level, of the diffusion coefficient of various fluorescent biological probes : FITC-dextran, GFP (Green Fluorescent Protein) and fluoresceine. Moreover, our two-photon fluorescence correlation technique has been originally applied to the study molecular diffusion processes inside bacterial biofilms A second time resolved technique, fluorescence lifetime imaging under two-photon excitation, have been developed. The fluorescence lifetime, or lifetime of the first singlet excited state is an important characteristic of the physico-chemical state and of the environment of a molecule. Thus, measurement of fluorescence lifetime can provide quantitative information about the system under study and can bring fundamental information on intracellular mechanisms. The first results obtained with this technique applied in the pharmacological domain to the study of molecule activity in vitro and in vivo are presented.
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Submitted on : Friday, September 2, 2005 - 11:48:52 AM
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  • HAL Id : tel-00010025, version 1

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Elvire Guiot. microscopie de fluorescence par excitation à deux photons : application à des études de corrélations et de déclins de fluorescence en milieu biologique. Biophysique [physics.bio-ph]. Université Paris Sud - Paris XI, 2001. Français. ⟨tel-00010025⟩

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