title: Direct observation of the nanoscale dynamics of membrane lipids in a living cell.
abstract: Cholesterol-mediated lipid interactions are thought to have a functional role in many membrane-associated processes such as signalling events. Although several experiments indicate their existence, lipid nanodomains ('rafts') remain controversial owing to the lack of suitable detection techniques in living cells. 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 nanoscopy to detect single diffusing (lipid) molecules in nanosized areas in the plasma membrane of living cells. Tuning of the probed area to spot sizes approximately 70-fold below the diffraction barrier reveals that unlike phosphoglycerolipids, sphingolipids and glycosylphosphatidylinositol-anchored proteins are transiently ( approximately 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.
authors.affiliation: Department of Nanobiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany. ceggeli@gwdg.de
authors.forename: Christian
authors.initials: C
authors.lastname: Eggeling
authors.forename: Christian
authors.initials: C
authors.lastname: Ringemann
authors.forename: Rebecca
authors.initials: R
authors.lastname: Medda
authors.forename: Günter
authors.initials: G
authors.lastname: Schwarzmann
authors.forename: Konrad
authors.initials: K
authors.lastname: Sandhoff
authors.forename: Svetlana
authors.initials: S
authors.lastname: Polyakova
authors.forename: Vladimir N
authors.initials: VN
authors.lastname: Belov
authors.forename: Birka
authors.initials: B
authors.lastname: Hein
authors.forename: Claas
authors.initials: C
authors.lastname: von Middendorff
authors.forename: Andreas
authors.initials: A
authors.lastname: Schönle
authors.forename: Stefan W
authors.initials: SW
authors.lastname: Hell
mesh_terms: D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002470:Cell Survival; D002784:Cholesterol / Q000032:analysis / Q000378:metabolism; D004058:Diffusion; D004847:Epithelial Cells / Q000166:cytology; D004983:Ethanolamines / Q000032:analysis / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D008563:Membrane Lipids / Q000032:analysis* / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D013109:Sphingomyelins / Q000032:analysis / Q000378:metabolism; D013997:Time Factors
chemical_list: D004983:Ethanolamines; D017261:Glycosylphosphatidylinositols; D008563:Membrane Lipids; D013109:Sphingomyelins; C005448:phosphorylethanolamine; D002784:Cholesterol