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Diffusion Analysis within Single Nanometric Apertures Reveals the Ultrafine Cell Membrane Organization

Jérôme Wenger ^* , Fabien Conchonaud   ^¶, José Dintinger ^|| **, Laure Wawrezinieck ^*    ¶, Thomas W. Ebbesen ^|| **, Hervé Rigneault ^* , Didier Marguet   ^¶ and Pierre-François Lenne ^* 

^* Institut Fresnel, Mosaic Group, Université Paul Cézanne Aix-Marseille III, Domaine Universitaire de Saint Jérôme, Marseille Cedex, France; CNRS UMR 6133, Marseille, France; Centre d'Immunologie de Marseille Luminy, Université de la Méditerranée, Parc Scientifique de Luminy, Case 906, Marseille Cedex, France; CNRS UMR 6102, Marseille, France; ^¶ INSERM UMR 631, Marseille, France; ^|| ISIS, Université Louis Pasteur, Strasbourg, France; and ^** CNRS UMR 7006, Strasbourg, France

Correspondence: Address reprint requests to Pierre-François Lenne, Institut Fresnel, Domaine Universitaire de Saint Jérôme, 13397 Marseille Cedex 20, France. Tel.: 33-491-288-494; Fax: 33-491-288-067; E-mail: lenne{at}fresnel.fr, jerome.wenger{at}fresnel.fr.

We describe the development of a new methodology to probe the plasma membrane organization of living cells at the nanometric scale. Single nanometric apertures in a metallic film limit the observed membrane area below the optical diffraction barrier. The new approach performs fluorescence correlation spectroscopy with increasing aperture sizes and extracts information on the diffusion process from the whole set of data. In particular, transient diffusion regimes are clearly observed when the probed area comes close to the size of the confining structures. First, this strategy allows identification of the mechanism controlling the diffusion of various fluorescent lipid analogs and green fluorescent protein-tagged proteins. Second, it gives an estimate of the characteristic size of the nanometric membrane heterogeneities, allowing a quantitative study of membrane domains such as lipid rafts. Compared to other optical techniques, this method combines the advantages of high spatio-temporal resolution and direct statistical analysis.

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