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.

We introduce far-field fluorescence nanoscopy with ordinary fluorophores based on switching the majority of them to a metastable dark state, such as the triplet, and calculating the position of those left or those that spontaneously returned to the ground state. Continuous widefield illumination by a single laser and a continuously operating camera yielded dual-color images of rhodamine- and fluorescent protein-labeled (living) samples, proving a simple yet powerful super-resolution approach.

We demonstrate far-field fluorescence microscopy with a focalplane resolution of 15-20 nm in biological samples. The 10-to 12-fold multilateral increase in resolution below the diffraction barrier has been enabled by the elimination of molecular triplet state excitation as a major source of photobleaching of a number of dyes in stimulated emission depletion microscopy. Allowing for relaxation of the triplet state between subsequent excitationdepletion cycles yields an up to 30-fold increase in total fluorescence signal as compared with reported stimulated emission depletion illumination schemes. Moreover, it enables the reduction of the effective focal spot area by up to Ϸ140-fold below that given by diffraction. Triplet-state relaxation can be realized either by reducing the repetition rate of pulsed lasers or by increasing the scanning speed such that the build-up of the triplet state is effectively prevented. This resolution in immunofluorescence imaging is evidenced by revealing nanoscale protein patterns on endosomes, the punctuated structures of intermediate filaments in neurons, and nuclear protein speckles in mammalian cells with conventional optics. The reported performance of diffractionunlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences.imaging ͉ stimulated emission depletion illumination ͉ subdiffraction ͉ triplet state F or more than a century, the resolution of a lens-based (far-field) optical microscope has been limited by diffraction (1). However, in the 1990s it became evident that the limiting role of diffraction can be broken in lens-based fluorescence microscopy if certain fluorophore properties are judiciously integrated into the image formation (2). The first viable concept of this kind is stimulated emission depletion (STED) microscopy (3), which, since its experimental validation (4, 5), has been key to solving a number of problems in biophysics (6) and cell biology (7,8).STED microscopy typically uses a scanning excitation spot that is overlapped with a doughnut-shaped counterpart for deexcitation of fluorophores by light, a phenomenon referred to as stimulated emission (9, 10). Oversaturating the deexcitation squeezes the fluorescence spot to subdiffraction dimensions (Fig. 1a) so that superresolved images emerge by scanning this spot through the object (5).The rate for deexcitation by stimulated emission is given by k STED ϭ I STED , with denoting the fluorophore cross-section and I STED denoting the intensity of the stimulating beam. Oversaturating the deexcitation requires k STED be much larger than the fluorescence decay given by the inverse of the lifetime, Fl Ϸ 1-5 ns, of the fluorescent state S 1 . With Ϸ 10 Ϫ17 cm 2 , it follows that I STED Ͼ Ͼ 1͞( Fl ) ϭ 10 26 photons per second per squared centimeter, which, at a wavelength of ϭ 600 nm, amounts to Î STED Ͼ Ͼ 33 MW͞cm 2 . This intensity value is at least 10 3 -fold lower than what is required for multiphoton excitation (11), but still 10 2 -fold larger than what is used ...

We report stimulated emission depletion (STED) fluorescence microscopy with continuous wave (CW) laser beams. Lateral fluorescence confinement from the scanning focal spot delivered a resolution of 29-60 nm in the focal plane, corresponding to a 5-8-fold improvement over the diffraction barrier. Axial spot confinement increased the axial resolution by 3.5-fold. We observed three-dimensional (3D) subdiffraction resolution in 3D image stacks. Viable for fluorophores with low triplet yield, the use of CW light sources greatly simplifies the implementation of this concept of far-field fluorescence nanoscopy.