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Biophys. J. BioFAST: First Published November 3, 2006. doi:10.1529/biophysj.106.096586
© 2006 by the Biophysical Society.

A more recent version of this article appeared on February 1, 2007.
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MEMBRANES

Diffusion analysis within single nanometric apertures reveals the ultrafine cell membrane organization

Jerome Wenger 1, Fabien Conchonaud 2, José Dintinger 3, Laure Wawrezinieck 2, Thomas W Ebbesen 3, Hervé Rigneault 4, Didier Marguet 5 and Pierre-François Lenne 6*

1 Institut Fresnel CNRS UMR 6133
2 CIML CNRS UMR6102
3 ISIS CNRS UMR7006
4 Institut Fresnel CNRS UMR6133
5 Centre d'Immunologie de Marseille Luminy
6 CNRS

* To whom correspondence should be addressed. E-mail: lenne{at}fresnel.fr.

Submitted on September 4, 2006
Revised on October 8, 2006
Accepted on 13 October 2006


  Abstract
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 identifies the mechanism controlling the diffusion of various fluorescent lipid analogs and GFP-tagged proteins. Second, it gives an estimate of the characteristic size of the nanometric membrane heterostructures, 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.

Key Words: confined diffusion, fluorescence correlation spectroscopy, lipid rafts, membrane microdomain, nanometric aperture




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Copyright © 2006 by the Biophysical Society.