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Continuous Photobleaching in Vesicles and Living Cells: A Measure of Diffusion and Compartmentation

A. Delon ^*, Y. Usson , J. Derouard ^*, T. Biben ^* and C. Souchier 

^* Laboratoire de Spectrométrie Physique, Centre National de la Recherche Scientifique, UMR5588, Université Joseph Fourier, Saint Martin d'Hères, France; Techniques de l'Imagerie, de la Modelisation et de la Cognition, Centre National de la Recherche Scientifique, UMR5525, and Institut de la Santé et de la Recherche Médicale, U309, Université Joseph Fourier, La Tronche, France

Correspondence: Address reprint requests to Antoine Delon, Tel.: 33-4-76-63-58-01; Fax: 33-4-76-63-54-95; E-mail: adelon{at}ujf-grenoble.fr.

We present a comprehensive and analytical treatment of continuous photobleaching in a compartment, under single photon excitation. In the very short time regime (t < 0.1 ms), the diffusion does not play any role. After a transition (or short time regime), one enters in the long time regime (t > 0.1–5 s), for which the diffusion and the photobleaching balance each other. In this long time regime, the diffusion is either fast (i.e., the photobleaching probability of a molecule diffusing through the laser beam is low) so that the photobleaching rate is independent of the diffusion constant and dependent only of the laser power, or the diffusion is slow (i.e., the photobleaching probability is high) and the photobleaching rate is mainly dependent on the diffusion constant. We illustrate our theory by using giant unilamellar vesicles ranging from 10 to 100 µm in diameter, loaded with molecules of various diffusion constants (from 20 to 300 µm²/s) and various photobleaching cross sections, illuminated under laser powers between 3 and 100 µW. We also demonstrated that information about compartmentation can be obtained by this method in living cells expressing enhanced green fluorescent proteins or that were loaded with small FITC-dextrans. Our quantitative approach shows that molecules freely diffusing in a cellular compartment do experience a continuous photobleaching. We provide a generic theoretical framework that should be taken into account when studying, under confocal microscopy, molecular interactions, permeability, etc.

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