Fluorescence microscopy of single autofluorescent proteins for cellular biology

Laurent Cognet; Françoise Coussen; Daniel Choquet; Brahim Lounis

doi:10.1016/S1631-0705(02)01341-5

Biophysique à l'échelle de la molécule unique/Single molecule biophysics

Fluorescence microscopy of single autofluorescent proteins for cellular biology
[Microscopie de fluorescence de protéines autofluorescentes uniques pour la biologie cellulaire]

Laurent Cognet ¹ ; Françoise Coussen ² ; Daniel Choquet ² ; Brahim Lounis ¹

¹ Centre de physique moléculaire optique et hertzienne – CNRS UMR 5798 et Université Bordeaux 1, 351, Cours de la Libération, 33405 Talence, France
² Laboratoire de physiologie cellulaire de la synapse – CNRS UMR 5091 et Université Bordeaux 2, Institut François Magendie, 1, rue Camille Saint-Saëns, 33077 Bordeaux, France

Comptes Rendus. Physique, Volume 3 (2002) no. 5, pp. 645-656.

Résumé
Abstract

Nous présentons une revue sur l'utilisation des protéines autofluorescentes en microscopie de molécules uniques en biologie. Les caractéristiques photophysiques de plusieurs mutants de la protéine fluorescente verte (la « GFP ») ainsi que celles de la DsRed y sont comparées et discutées de manière critique en vue de leur utilisation dans des cellules vivantes. Des méthodes alternatives d'excitation, telle l'excitation biphotonique, ou d'analyses, telle la méthode de spectroscopie par corrélation de fluorescence sont envisagées. Nous rendons compte d'expériences utilisant la eGFP et préférentiellement la eYFP au niveau de la molécule unique dans des cellules vivantes. Nous reportons enfin, la première utilisation au niveau de la molécule individuelle de la citrine, un mutant de la eYFP plus résistant au photoblanchiment et son application à l'étude de la dynamique de récepteurs de neurotransmetteurs individuels dans la membrane de cellules vivantes.

In this paper we review the applicability of autofluorescent proteins for single-molecule imaging in biology. The photophysical characteristics of several mutants of the Green Fluorescent Protein (GFP) and those of DsRed are compared and critically discussed for their use in cellular biology. The alternative use of two-photon excitation at the single-molecule level or Fluorescence Correlation Spectroscopy is envisaged for the study of individual autofluorescent proteins. Single-molecule experiments performed in live cells using eGFP and preferably eYFP fusion proteins are reviewed. Finally, the first use at the single-molecule level of citrine, a more photostable variant of the eYFP is reported when fused to a receptor for neurotransmitter in live cells.

Reçu le : 2002-03-18
Accepté le : 2002-04-25
Publié le : 2002-01-01

DOI : 10.1016/S1631-0705(02)01341-5

Keywords: single-molecule, GFP, DsRed, fluorescence microscopy, two-photon excitation, fluorescence correlation spectroscopy
Mot clés : détection de molécules uniques, GFP, DsRed, microscopie de fluorescence, excitation à deux photons, spectroscopie par corrélation de fluorescence

Affiliations des auteurs :

Laurent Cognet ¹ ; Françoise Coussen ² ; Daniel Choquet ² ; Brahim Lounis ¹

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Laurent Cognet; Françoise Coussen; Daniel Choquet; Brahim Lounis. Fluorescence microscopy of single autofluorescent proteins for cellular biology. Comptes Rendus. Physique, Volume 3 (2002) no. 5, pp. 645-656. doi : 10.1016/S1631-0705(02)01341-5. https://comptes-rendus.academie-sciences.fr/physique/articles/10.1016/S1631-0705(02)01341-5/

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