1. Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
2. LIMES Membrane Biology and Lipid Biochemistry Unit, University of Bonn, Gerhard-Domagk-Strasse 1, 53121 Bonn, Germany
3. These authors contributed equally to this work.

Correspondence to: Christian Eggeling^1,3Stefan W. Hell¹ Correspondence and requests for materials should be addressed to S.W.H. (Email: shell@gwdg.de) or C.E. (Email: ceggeli@gwdg.de).

Abstract

Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events^{1, 2, 3, 4, 5}. Although several experiments indicate their existence, lipid nanodomains ('rafts') remain controversial owing to the lack of suitable detection techniques in living cells^{4, 6, 7, 8, 9}. The controversy is reflected in their putative size of 5–200 nm, spanning the range between the extent of a protein complex and the resolution limit of optical microscopy. Here we demonstrate the ability of stimulated emission depletion (STED) far-field fluorescence nanoscopy¹⁰ to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes 70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently (10–20 ms) trapped in cholesterol-mediated molecular complexes dwelling within <20-nm diameter areas. The non-invasive optical recording of molecular time traces and fluctuation data in tunable nanoscale domains is a powerful new approach to study the dynamics of biomolecules in living cells.

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