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

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.