Revised Diffusion Law Permits Quantitative Nanoscale Characterization of Membrane Organization

The formation of functional nanoscopic domains is an inherent property of plasma membranes. Stimulated emission depletion combined with fluorescence correlation spectroscopy (STED-FCS) has been previously used to identify such domains; however, the information obtained by STED-FCS has been limited to the presence of such domains while crucial parameters have not been accessible, such as size (<i>R</i>_d), the fraction of occupied membrane surface (<i>f</i>), in-membrane lipid diffusion inside (<i>D</i>_in) and outside (<i>D</i>_out) the nanodomains as well as their self-diffusion (<i>D</i>_d). Here, we introduce a quantitative approach based on a revised interpretation of the diffusion law. By analyzing experimentally recorded STED-FCS diffusion law plots using a comprehensive library of simulated diffusion law plots, we extract these five parameters from STED-FCS data. That approach is verified on ganglioside nanodomains in giant unilamellar vesicles, validating the Saffman-Delbrück assumption for <i>D</i>_d. STED-FCS data in both plasma membranes of living PtK2 cells and giant plasma membrane vesicles are examined, and a quantitative framework for molecular diffusion modes in biological membranes is presented.