Most plasmalemmal proteins organize in submicrometer-sized clusters whose architecture and dynamics are still enigmatic. With syntaxin 1 as an example, we applied a combination of far-field optical nanoscopy, biochemistry, fluorescence recovery after photobleaching (FRAP) analysis, and simulations to show that clustering can be explained by self-organization based on simple physical principles. On average, the syntaxin clusters exhibit a diameter of 50 to 60 nanometers and contain 75 densely crowded syntaxins that dynamically exchange with freely diffusing molecules. Self-association depends on weak homophilic protein-protein interactions. Simulations suggest that clustering immobilizes and conformationally constrains the molecules. Moreover, a balance between self-association and crowding-induced steric repulsions is sufficient to explain both the size and dynamics of syntaxin clusters and likely of many oligomerizing membrane proteins that form supramolecular structures.

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 a substantial signal gain in fluorescence microscopy by ensuring that transient molecular dark states with lifetimes >1 micros, such as the triplet state relax between two molecular absorption events. For GFP and Rhodamine dye Atto532, we observed a 5-25-fold increase in total fluorescence yield before molecular bleaching when strong continuous-wave or high-repetition-rate pulsed illumination was replaced with pulses featuring temporal pulse separation >1 micros. The signal gain was observed both for one- and two-photon excitation. Obeying dark or triplet state relaxation in the illumination process signifies a major step toward imaging with low photobleaching and strong fluorescence fluxes.