Revised diffusion law permits quantitative nanoscale characterization of membrane organization

Formation of functional nanoscopic domains is an inherent property of plasma membranes. Stimulated emission depletion combined with fluorescence correlation spectroscopy (STED-FCS) has been used to identify such domains, however, the information obtained by STED-FCS has been limited to presence of such domains while crucial parameters have not been accessible, such as size ( R _d ), the fraction of occupied membrane surface ( f ), in-membrane lipid diffusion inside ( D _in ) and outside ( D _out ) the nanodomains as well as their self-diffusion ( D _d ). Here, based on a revision of the ‘diffusion law’, we present an approach to retrieve these five parameters from STED-FCS data. We verify that approach on ganglioside nanodomains in giant unilamellar vesicles (GUVs), validating the Saffman-Delbrück assumption for D _d . We examined STED-FCS data in both plasma membranes of living PtK2 cells and in giant plasma membrane vesicles (GPMVs) and present a quantitative framework for molecular diffusion modes in biological membranes.