The ultimate goal of biological superresolution fluorescence microscopy is to provide threedimensional resolution at the size scale of a fluorescent marker. Here, we show that, by localizing individual switchable fluorophores with a probing doughnut-shaped excitation beam, MINFLUX nanoscopy provides 1-3 nanometer resolution in fixed and living cells. This progress has been facilitated by approaching each fluorophore iteratively with the probing doughnut minimum, making the resolution essentially uniform and isotropic over scalable fields of view. MINFLUX imaging of nuclear pore complexes of a mammalian cell shows that this true nanometer scale resolution is obtained in three dimensions and in two color channels. Relying on fewer detected photons than popular camera-based localization, MINFLUX nanoscopy is poised to open a new chapter in the imaging of protein complexes and distributions in fixed and living cells.While STED 1, 2 and PALM/STORM 3, 4 fluorescence microscopy (nanoscopy) can theoretically achieve a resolution at the size of a single fluorophore, in practice they are typically limited to about 20 nm.Owing to a synergistic combination of the specific strengths of these key superresolution concepts, the recently introduced MINFLUX nanoscopy 5 can attain a spatial resolution of about the size of a molecule, conceptually without constraints from any wavelength or numerical aperture. In MINFLUX imaging, the fluorophores are switched individually like in PALM/STORM, whereas the localization is accomplished by using a movable excitation beam featuring an intensity minimum, such as a doughnut. The minimum ideally is a zero intensity point that is targetable like a probe 6 .
We present a real-time fitter for 3D single-molecule localization microscopy using experimental point spread functions (PSFs) that achieves optimal 3D resolution on any microscope and is compatible with any PSF engineering approach. This allowed us to image cellular structures with a 3D resolution unprecedented for astigmatic PSFs. The fitter compensates for most optical aberrations and makes accurate 3D superresolution microscopy broadly accessible, even on standard microscopes without dedicated 3D optics.
Quantitative fluorescence and superresolution microscopy are often limited by insufficient data quality or artifacts. In this context, it is essential to have biologically relevant control samples to benchmark and optimize the quality of microscopes, labels and imaging conditions. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
SummaryClathrin-mediated endocytosis is an essential cellular function in all eukaryotes that is driven by a self-assembled macromolecular machine of over 50 different proteins in tens to hundreds of copies. How these proteins are organized to produce endocytic vesicles with high precision and efficiency is not understood. Here, we developed high-throughput superresolution microscopy to reconstruct the nanoscale structural organization of 23 endocytic proteins from over 100,000 endocytic sites in yeast. We found that proteins assemble by radially ordered recruitment according to function. WASP family proteins form a circular nanoscale template on the membrane to spatially control actin nucleation during vesicle formation. Mathematical modeling of actin polymerization showed that this WASP nano-template optimizes force generation for membrane invagination and substantially increases the efficiency of endocytosis. Such nanoscale pre-patterning of actin nucleation may represent a general design principle for directional force generation in membrane remodeling processes such as during cell migration and division.
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