The resolution of any linear imaging system is given by its point spread function (PSF) that quantifies the blur of an object point in the image. The sharper the PSF, the better the resolution is. In standard fluorescence microscopy, however, diffraction dictates a PSF with a cigar-shaped main maximum, called the focal spot, which extends over at least half the wavelength of light (lambda = 400-700 nm) in the focal plane and >lambda along the optical axis (z). Although concepts have been developed to sharpen the focal spot both laterally and axially, none of them has reached their ultimate goal: a spherical spot that can be arbitrarily downscaled in size. Here we introduce a fluorescence microscope that creates nearly spherical focal spots of 40-45 nm (lambda/16) in diameter. Fully relying on focused light, this lens-based fluorescence nanoscope unravels the interior of cells noninvasively, uniquely dissecting their sub-lambda-sized organelles.

The recently introduced minimal photon fluxes (MINFLUX) concept pushed the resolution of fluorescence microscopy to molecular dimensions. Initial demonstrations relied on custom made, specialized microscopes, raising the question of the method’s general availability. Here, we show that MINFLUX implemented with a standard microscope stand can attain 1–3 nm resolution in three dimensions, rendering fluorescence microscopy with molecule-scale resolution widely applicable. Advances, such as synchronized electro-optical and galvanometric beam steering and a stabilization that locks the sample position to sub-nanometer precision with respect to the stand, ensure nanometer-precise and accurate real-time localization of individually activated fluorophores. In our MINFLUX imaging of cell- and neurobiological samples, ~800 detected photons suffice to attain a localization precision of 2.2 nm, whereas ~2500 photons yield precisions <1 nm (standard deviation). We further demonstrate 3D imaging with localization precision of ~2.4 nm in the focal plane and ~1.9 nm along the optic axis. Localizing with a precision of <20 nm within ~100 µs, we establish this spatio-temporal resolution in single fluorophore tracking and apply it to the diffusion of single labeled lipids in lipid-bilayer model membranes.

Embryonic and neonatal neurons require specific trophic supplements for their survival and the induction of transmitter-synthesizing enzymes in vivo and in vitro. Acidic and basic fibroblast growth factor (aFGF, bFGF) and the closely related astroglial growth factors AGF-1 and AGF-2 were studied for putative neurotrophic functions using dissociated, highly neuron-enriched cultures from chick and rat peripheral ganglia and central nervous system tissues. Embryonic chick ciliary ganglion neurons were the only peripheral neurons that responded to bFGF and AGF-2 by enhanced survival equivalent to that obtained with ciliary neurotrophic factor. Half-maximal effects were achieved with bFGF at 360 pg/ml or AGF-2 at 3 ng/ml. Small effects seen with aFGF could be potentiated by adding heparin at 1 ,ug/ml. bFGF, but not ciliary neurotropic factor, also promoted neuron survival after the factor was bound to polyornithine and laminin. Both AGF-2 and ciliary neurotropic factor induced choline acetyltransferase activity during 48 hr. AGFs and FGFs also enhanced the long-term survival of embryonic chick spinal cord neurons, including motoneurons that had been retrogradely labeled with rhodamine isothiocyanate. These results demonstrate the potency of a class of mitogenic growth factors as neurotrophic agents for embryonic ciliary ganglion and spinal cord neurons-adding to the emerging evidence that mitogenic and neuronal growth factors are not strictly separate entities.Mitogenic, growth-promoting factors for cells derived from the mesoderm (e.g., fibroblast growth factors, FGFs) and neurotrophic factors (the prototype of which is nerve growth factor) that promote survival, differentiation, and functional maintenance of neurons have long been considered as separate entities. Emerging evidence suggests that this view can no longer strictly be maintained. Thus, nerve growth factor has been shown to act as a mitogen on rat pheochromocytoma (1) and adrenal medullary chromaffin cells (2). On the other hand, FGF and astroglial growth factors (AGF-1 and -2) that are closely related or even identical to acidic and basic FGF (aFGF, bFGF) (3) induce proliferation and differentiation of neuroectodermal astroglial cells (4), oligodendrocytes (5, 6), and neuron-like PC-12 pheochromocytoma cells (7).Large amounts of FGF are found in brain (8-10), and immunocytochemical studies using antibodies to AGF/FGF have revealed their localization in neurons of the central nervous system (11). We therefore questioned whether AGF and FGF had functions in the nervous system in addition to their established role for glial cells.We provide evidence that AGF and FGF mimic two effects typical of neurotrophic factors-promotion of in vitro neuron survival and enhancement of the activity of a transmittersynthesizing enzyme, choline acetyltransferase (ChoAcTase, acetyl-CoA:choline O-acetyltransferase, EC 2.3

3 3 a r t I C l e SA main rate-limiting step in synaptic transmission is the retrieval of synaptic vesicles from the presynaptic membrane for further rounds of use. Several modes of synaptic vesicle retrieval have been proposed, but the main pathway is considered to be clathrin-mediated endocytosis 1,2 . This process is relatively slow because after fusion the recycling machinery has to resort different vesicle membrane proteins in the right stoichiometry to generate fusion-competent synaptic vesicles 3 . As a result, this process occurs with a time constant of tens of seconds to minutes 4 . However, to sustain transmission during continuous activity, it was suggested that synaptic vesicles might 'kiss and run' with a time constant of 1-2 s, whereby the vesicles transiently fuse with the membrane without full collapse and hence retain their molecular identity [5][6][7][8][9] .The fast 'kiss and run' recycling mechanism not only would provide a kinetic advantage but would spatially and temporally couple exoand endocytosis. Using a green fluorescent protein (GFP) fused with the coat-forming clathrin light chain, we previously found evidence that during the first 10 s of prolonged stimulation, clathrin is not being recruited from the cytosol to form coated pits, although the rate of endocytosis measured with styryl (FM) dyes is high 3 , suggesting that vesicles during this first phase are either retrieved by a clathrin-independent mechanism (kiss and run) or by preassembled coat structures at the periphery of the active zone.Support for such a 'readily retrievable pool' (RRetP) of preassembled structures came from experiments using fusion constructs of the synaptic vesicle proteins synaptobrevin 2 (Syb2) and synaptotagmin 1 (Syt1) with a pH-sensitive GFP, pHluorin 10 . These studies showed that synaptic vesicles lose their protein complement after fusion, and the molecular identity of synaptic vesicles exocytosed and subsequently endocytosed is not conserved [11][12][13] . On the basis of these observations, we suggested that exocytosis and subsequent endocytosis are uncoupled and that there is a pool of preassembled vesicle proteins on the presynaptic surface that is preferentially retrieved on exocytosis 3,13 . Previous studies using activity-dependent markers in snake neuromuscular terminals have proposed that the accumulation of such probes at the bouton margins upon stimulation might represent endocytic active zones 14,15 . This is in agreement with other ultrastructural and high-resolution microscopy analyses that describe the presence of several synaptic vesicle proteins on the presynaptic membrane of resting synapses 16,17 . Likewise, the first reconstruction of the endocytic time course from electron micrographs of frog neuromuscular junctions quick-frozen at different times after stimulation revealed a first wave of clathrin-mediated endocytosis lasting ~10 s (ref. 18), in line with the notion of a preclustered pool being immediately available for this first wave of endocytosis upon stimulation 13 . In hi...