S W Hell

..pacs: 87.16.tb, 87.85.rs...

..A comparison with unrestored two-photon confocal images reveals a total reduction of the uncertainty volume up to a factor of 15...

..When imaging rotation-impaired fluorophores of unknown random orientation, the average localization accuracy in three-dimensional samples is typically limited to about ±32 nm, restricting the attainable resolution accordingly...

..Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization...

..Furthermore, we demonstrate dual-color fluorescence microscopy with sub-diffraction resolution using bsDronpa and Dronpa whose emission maxima are separated by <20 nm...

..This strong interaction is different from that responsible for forming cholesterol-dependent, liquid-ordered domains in model membranes...

..In aging bacterial cultures DsRed appeared to aggregate within the cells, accompanied by a strong reduction in its fluorescence lifetime as determined by fluorescence lifetime imaging microscopy...

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..We conclude that MMM proves to be a technically simple and very effective method for fast 3-D live cell imaging at high resolution...

..Photoswitching expands the range of single-molecule detection based experiments such as fluorescence fluctuation spectroscopy to large entity concentrations in the micromolar range...

..Future synthesis of similar compounds holds great promise for cost-effective fluorescence nanoscopy with noninvasive optical sectioning...

..We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules...

..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...

..Matrix constrictions frequently occurring in wild type cells as well as in Deltafis1 and Deltadnm1 mutants do not interfere with luminal continuity...

..Theoretical calculations did not yield a transition in the observed range, even for large aggregates (J aggregates). Therefore, other interactions may be responsible for the appearance of this band...

..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...

..Reaching almost T-Rex conditions this excitation scheme mimics fast scanning of the illumination beam and has the potential to improve a whole range of analytical tools that suffer from photobleaching and low signal levels...

..Optical images with resolutions on the nanometer scale illustrate the potential of the labels in the colocalization of biological objects and the two-photon activation technique with optical sectioning...

..g., amines and thiols) in complex mixtures. High-resolution GSDIM images and live-cell STED-FCS experiments on labeled microtubules and lipids prove the versatility of the novel probes for modern fluorescence microscopy and nanoscopy...

..Finally, FRET-MMM records performed continuously over 2 h allowed to analyze intracellular retrograde transport and sorting events and to discuss these mechanisms on a single cell level...

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..The obtained results can be further improved by mathematical restoration algorithms. The far-field optical nanoscale resolution is attained in a variety of biological samples featuring strong variations in the local density of features...

..Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization...

..The obtained resolution of 50 nm ( approximately lambda/12) is limited only by the spectroscopic properties of the proteins and the imperfections of the optical implementation, but not on principle grounds...

..Time-lapse STED recordings document morphological changes of the ER over time. Thus, nanoscale 3D imaging of organelles in the interior of living cells greatly expands the scope of light microscopy in cell biology...

..The study of ReISC not only results in a better understanding of a fluorescent label's photophysics, but the method is a possible approach to optimize fluorescence emission in experiments, where signal strength is a critical parameter...

..We find a pronounced heterogeneity in the cristae arrangements even within individual mitochondrial tubules...

..The data suggest that different molecular machineries are responsible for the separation of the matrix and the fission of the outer membrane in budding yeast...

..Hence fusion proteins that bind modified organic dyes expand widely the application range of far-field fluorescence nanoscopy of living cells...

..4-fold improvement over the diffraction barrier...

..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...

..Subdiffraction resolution images of tubulin filaments of mammalian cells stained with these dyes illustrate their applicability as labels for stimulated emission depletion microscopy and other fluorescence techniques...

..Some applications of these particles in fluorescence microscopy are also demonstrated. In particular, subdiffraction images of nanoparticles were obtained, in the focal plane of a confocal microscope...

..Our study demonstrates the emerging ability of optical microscopy to investigate intracellular physiological processes on the nanoscale in real time...

..Fully relying on focused light, this lens-based fluorescence nanoscope unravels the interior of cells noninvasively, uniquely dissecting their sub-lambda-sized organelles...

..Distinct absorption cross-sections for the switching wavelengths in the fluorescent and the nonfluorescent state are not essential for efficient photochromism in fluorescent proteins, although they may facilitate the switching process...

..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...

..Finally, we exemplify the potential of using nanodiamonds containing NV centers as luminescence tags in STED microscopy. Our results offer new experimental avenues in nanooptics, nanotechnology, and the life sciences...

..Our experimental approach demonstrates that fast molecular movements can be tracked with minimal invasion, which can reveal new important details of cellular nano-organization...

..Our results mark the advent of nanoscale biological microscopy with genetically encoded markers...

..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...

..5-3.5 microm), which possibly are related to the abolition of cytoskeletal physical barriers preventing the lateral diffusion of AChR nanoclusters...

..In conclusion, with the current aperture angles and fluorescence signal strengths, it is not advisable to trade in the suppression of the sidelobes for a larger image signal...

..Both p24a and p23 interact with ARF1, but only p24 interacts with ARFGAP. These findings suggest a model for how cargo-induced oligomerization of ERD2 regulates its sorting into COPI-coated buds...

..The findings reported in the present study support the view that a cis-trans isomerization is one of the key events common to the switching mechanism in RSFPs...

..Superresolved 3D-fluorescence imaging is exemplified with the first representation of the Golgi apparatus in a live cell at approximately 100 nm resolution...

..Sub-populations of fluorescent markers can thus be separated based on their interaction with the sample. We applied this new functional nanoscopy to imaging of living mammalian cells...

..Formed on the basis of reversible saturable optical transitions, these concepts might eventually allow us to investigate hitherto inaccessible details within live cells...

..The combination of far-field fluorescence nanoscopy with the recording of a single switchable molecular species at a time opens up a new class of functional imaging techniques...

..We propose that this mechanism is essential for myelin to perform its insulating function during nerve conduction...

..Reversible photoswitching of the protein chromophore system within intact crystals also constitutes a step toward the use of fluorescent proteins in three-dimensional data recording...

..We present the optical and chemical properties of this new medium as well as its application to a variety of differently stained cells and cellular substructures...

..Our method significantly extends the potential of far-field FFS, including for the noninvasive investigation of molecular reactions at higher concentrations...

..We suggest a comprehensive model for the light-induced switching mechanism, connecting a cascade of structural rearrangements with different protonation states of the chromophore...

..Tentatively providing a larger photon flux, CW beams should facilitate extending fast STED imaging towards imaging fainter living samples...

..One can counteract photobleaching by employing STED pulses that range from 150 ps to approximately half of the lifetime of the excited state. The results also have implications for multiphoton excitation microscopy...

..Relying on saturated optical transitions, these concepts are limited only by the attainable saturation level. As strong saturation should be feasible at low light intensities, nanoscale imaging with focused light may be closer than ever...

..Moreover, this change is associated with a 2.8-fold increase of the surface of the reticulum, resulting in an average increase in volume of the mitochondrial compartment by a factor of 3.0 +/- 0.2...

..The presence of dark states in virtually any fluorescent molecule opens up a new venue for far-field microscopy with resolution that is no longer limited by diffraction...

..The unique advantages of these new lenses in high-resolution microscopy with two coherently used opposing lenses, such as 4 Pi-microscopy, are discussed...

..In particular, we visualize the three-dimensional entanglement of the two networks with unprecedented detail...

..We conclude that both high- and low-mobility states are characteristic of synaptic vesicle movement...

..The reported performance of diffraction-unlimited fluorescence microscopy opens up a pathway for addressing fundamental problems in the life sciences...

..Here I discuss the principles of these methods together with their differences in implementation and operation. Finally, I outline potential developments...

..The mapping of a model system of spheres organized by confined convective assembly unambiguously identified face-centered cubic, hexagonal close-packed, random hexagonal close-packed, and body-centered cubic structures...

..Our study also demonstrates that questions involving cellular structures with dimensions of a few tens of nanometres can be resolved with conventional far-field optics and visible light...

..This approach allows a 3D image reconstruction with a resolution <80 nm in all directions using available state-of-the art STED microscopes...

..For relevant saturation values, the generalized square-root law is shown to predict the practical resolution with high accuracy...

..2 s...

..The imaging of dense nanoparticles and of the microtubular network of mammalian cells evidences a spatial resolution of 30-50 nm in the focal plane, i.e. by a factor of 8-9 beyond the diffraction barrier...

..Thus, syntaxin clustering represents a mechanism of membrane patterning that is based on protein-protein interactions...

..Flow-induced conversion of endothelial cells into an elongated arterial phenotype requires a coordinated regulation of cell junctions. Here we investigated the effect of acute and chronic flow on junction regulation...

..We have demonstrated not only that an antibody-tagged label is stable enough to be recorded in this microscopy mode, but also that subdiffraction resolution can be obtained using a standard immunofluorescence preparation...

..Noninvasive axial sections of 80-160 nm thickness deliver more faithful 3D-images of subcellular features, providing a new opportunity to significantly enhance our understanding of cellular structure and function...

..Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and live cells at approximately 80 nm axial resolution...

..The joint improvement of resolution and colocalization demonstrates the emerging potential of far-field fluorescence nanoscopy to study the spatial organization of macromolecules in cells...

..The closed forms of the methoxy-substituted photochromic units 2 and 3 are less resistant to UV light (313 nm) than the closed form of 1...