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 (Rd), the fraction of occupied membrane surface (f), in-membrane lipid diffusion inside (Din) and outside (Dout) the nanodomains as well as their self-diffusion (Dd). 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 Dd. 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.

Department of Physical and Macromolecular Chemistry Faculty of Science Charles University Hlavova 8 Prague 128 40 Czech Republic

Faculty of Mathematics and Physics Charles University Ke Karlovu 5 Prague 121 16 Czech Republic

J Heyrovský Institute of Physical Chemistry of the Czech Academy of Sciences Dolejškova 3 Prague 182 23 Czech Republic

Science for Life Laboratory Department of Applied Physics Royal Institute of Technology Solna 17165 Sweden

Science for Life Laboratory Department of Women's and Children's Health Karolinska Institutet Tomtebodavägen 23 Solna 17165 Sweden

Shemyakin Ovchinnikov Institute of Bioorganic Chemistry of the Rusian Academy of Science Moscow 117997 Russia

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