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Nanoscale view of cell membrane lipids Cholesterol-mediated lipid interactions, such as nanodomain formation, are considered vital in a cell, but because of the lack of suitable detection techniques, their spatiotemporal range remained highly controversial. Here, Eggeling et al . use subdiffraction-resolution STED (stimulated emission depletion) fluorescence microscopy to detect the diffusion of single lipids or glycosylphosphatidylinositol (GPI)-anchored proteins on the plasma membrane of a living cell. Tuning the probing spot area up to about 70-fold below that of a confocal microscope reveals that unlike phosphoglycerolipids, sphingolipids and GPI-anchored proteins are trapped for about 10 ms in cholesterol-mediated complexes within less than 20 nm space. Optical probing in nanosized areas is a powerful new approach to study biomolecular function. Here, subdiffraction-resolution STED fluorescence microscopy is used to detect the diffusion of single lipids or GPI-anchored proteins on the plasma membrane of a living cell. Tuning the probing spot area ∼70-fold below that of a confocal microscope reveals that unlike phosphoglycerolipids, sphingolipids and GPI-anchored proteins are trapped for ∼10 ms in cholesterol-mediated complexes within <20 nm space. 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 nanoscopy 10 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.

We demonstrate nanoscale resolution in far-field fluorescence microscopy using reversible photoswitching and localization of individual fluorophores at comparatively fast recording speeds and from the interior of intact cells. These advancements have become possible by asynchronously recording the photon bursts of individual molecular switching cycles. We present images from the microtubular network of an intact mammalian cell with a resolution of 40 nm.

Analyzing the first and higher-order moments of the diffraction spot of a 4Pi fluorescence detection scheme facilitates two-color, three-dimensional super-resolution microscopy with ~6 nm axial and ~8–23 nm lateral resolution in a layer ~650 nm thick. We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorophores distributed across whole cells. By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample. By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores. Offering a combination of multicolor recording, nanoscale resolution and extended axial depth, our method substantially advances the noninvasive 3D imaging of cells and of other transparent materials.