We introduce MINFLUX, a concept for localizing photon emitters in space. By probing the emitter with a local intensity minimum of excitation light, MINFLUX minimizes the fluorescence photons needed for high localization precision. In our experiments, 22 times fewer fluorescence photons are required as compared to popular centroid localization. In superresolution microscopy, MINFLUX attained ~1-nm precision, resolving molecules only 6 nanometers apart. MINFLUX tracking of single fluorescent proteins increased the temporal resolution and the number of localizations per trace by a factor of 100, as demonstrated with diffusing 30 ribosomal subunits in living As conceptual limits have not been reached, we expect this localization modality to break new ground for observing the dynamics, distribution, and structure of macromolecules in living cells and beyond.

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 .

Mitochondrial function is critically dependent on the folding of the mitochondrial inner membrane into cristae; indeed, numerous human diseases are associated with aberrant crista morphologies. With the MICOS complex, OPA1 and the F 1 F o ‐ATP synthase, key players of cristae biogenesis have been identified, yet their interplay is poorly understood. Harnessing super‐resolution light and 3D electron microscopy, we dissect the roles of these proteins in the formation of cristae in human mitochondria. We individually disrupted the genes of all seven MICOS subunits in human cells and re‐expressed Mic10 or Mic60 in the respective knockout cell line. We demonstrate that assembly of the MICOS complex triggers remodeling of pre‐existing unstructured cristae and de novo formation of crista junctions (CJs) on existing cristae. We show that the Mic60‐subcomplex is sufficient for CJ formation, whereas the Mic10‐subcomplex controls lamellar cristae biogenesis. OPA1 stabilizes tubular CJs and, along with the F 1 F o ‐ATP synthase, fine‐tunes the positioning of the MICOS complex and CJs. We propose a new model of cristae formation, involving the coordinated remodeling of an unstructured crista precursor into multiple lamellar cristae.

We used stimulated emission depletion (STED) superresolution microscopy to analyze the nanoscale organization of 12 glial and axonal proteins at the nodes of Ranvier of teased sciatic nerve fibers. Cytoskeletal proteins of the axon (betaIV spectrin, ankyrin G) exhibit a high degree of one-dimensional longitudinal order at nodal gaps. In contrast, axonal and glial nodal adhesion molecules [neurofascin-186, neuron glial-related cell adhesion molecule (NrCAM)] can arrange in a more complex, 2D hexagonal-like lattice but still feature a ∼190-nm periodicity. Such a lattice-like organization is also found for glial actin. Sodium and potassium channels exhibit a one-dimensional periodicity, with the Na v channels appearing to have a lower degree of organization. At paranodes, both axonal proteins (betaII spectrin, Caspr) and glial proteins (neurofascin-155, ankyrin B) form periodic quasi-one-dimensional arrangements, with a high degree of interdependence between the position of the axonal and the glial proteins. The results indicate the presence of mechanisms that finely align the cytoskeleton of the axon with the one of the Schwann cells, both at paranodal junctions (with myelin loops) and at nodal gaps (with microvilli). Taken together, our observations reveal the importance of the lateral organization of proteins at the nodes of Ranvier and pave the way for deeper investigations of the molecular ultrastructural mechanisms involved in action potential propagation, the formation of the nodes, axon-glia interactions, and demyelination diseases.nodes of Ranvier | STED nanoscopy | cytoskeleton | axon-glia interaction | sciatic nerve I n recent years, knowledge of the ultrastructural organization of the axon initial segment (AIS) has been greatly extended by using optical nanoscopy to visualize its molecular composition. Stochastic optical reconstruction microscopy (STORM) and stimulated emission depletion (STED) microscopy were indeed crucial for the identification of a ∼190-nm periodic organization of the key components of the AIS. In particular, a periodic spatial arrangement was discovered for cytoskeletal proteins (actin, ankyrin G, betaIV spectrin), adhesion molecules (neurofascin), and channels (voltage-gated sodium Na v channels) (1-4). In the distal part of the axon, this periodicity has been found for actin, adducin, ankyrin B, and betaII spectrin in virtually every neuron type from the central and peripheral nervous systems (CNS and PNS) (1, 2, 5, 6). Although myelination of axons does not alter the patterning of the subcortical cytoskeleton (5, 6), it changes its molecular composition, promoting the clustering of specific markers at the nodes of Ranvier, where the action potential is regenerated (reviewed in refs. 7, 8). At nodes of Ranvier, the myelin sheath formed by oligodendrocytes (in the CNS) or Schwann cells (in the PNS) is interrupted. Still, the nodal gap is surrounded by perinodal astrocytes and microvilli stemming from the Schwann cells in the CNS and PNS, respectively (9). Interestingly, the AIS an...

SignificancePopular localization of single molecules through calculating the centroid of the diffraction pattern produced by molecular fluorescence on a camera is typically limited to spatiotemporal resolutions of >10 nm per >10 milliseconds. By requiring at least 10–100 times fewer detected photons and being free of bias due to molecular orientation, the localization concept called MINFLUX propels molecular tracking to the hitherto-unachievable regime of single-digit nanometer precision within substantially less than a millisecond. Our experiments herald the feasibility to detect molecular interactions and conformational changes at microsecond timescales.

The mitochondrial contact site and cristae organizing system (MICOS) is a multisubunit protein complex that is essential for the proper architecture of the mitochondrial inner membrane. MICOS plays a key role in establishing and maintaining crista junctions, tubular or slit-like structures that connect the cristae membrane with the inner boundary membrane, thereby ensuring a contiguous inner membrane. MICOS is enriched at crista junctions, but the detailed distribution of its subunits around crista junctions is unclear because such small length scales are inaccessible with established fluorescence microscopy. By targeting individually activated fluorophores with an excitation beam featuring a central zero-intensity point, the nanoscopy method called MINFLUX delivers single-digit nanometer-scale three-dimensional (3D) resolution and localization precision. We employed MINFLUX nanoscopy to investigate the submitochondrial localization of the core MICOS subunit Mic60 in relation to two other MICOS proteins, Mic10 and Mic19. We demonstrate that dual-color 3D MINFLUX nanoscopy is applicable to the imaging of organellar substructures, yielding a 3D localization precision of ∼5 nm in human mitochondria. This isotropic precision facilitated the development of an analysis framework that assigns localization clouds to individual molecules, thus eliminating a source of bias when drawing quantitative conclusions from single-molecule localization microscopy data. MINFLUX recordings of Mic60 indicate ringlike arrangements of multiple molecules with a diameter of 40 to 50 nm, suggesting that Mic60 surrounds individual crista junctions. Statistical analysis of dual-color MINFLUX images demonstrates that Mic19 is generally in close proximity to Mic60, whereas the spatial coordination of Mic10 with Mic60 is less regular, suggesting structural heterogeneity of MICOS.

We show that RESOLFT fluorescence nanoscopy, a low light level scanning superresolution technique employing reversibly switchable fluorescent proteins (rsFPs), is capable of dual-channel live-cell imaging that is virtually free of chromatic errors and temporal offsets. This is accomplished using rsEGFP and Dronpa, two rsFPs having similar spectra but different kinetics of switching and fluorescence emission. Our approach is demonstrated by imaging protein distributions and dynamics in living neurons and neuronal tissues.

Li et al. (Research Articles, 28 August 2015, aab3500) purport to present solutions to longstanding challenges in live-cell microscopy, reporting relatively fast acquisition times in conjunction with improved image resolution. We question the methods' reliability to visualize specimen features at sub-100-nanometer scales, because the mandatory mathematical processing of the recorded data leads to artifacts that are either difficult or impossible to disentangle from real features. We are also concerned about the chosen approach of subjectively comparing images from different super-resolution methods, as opposed to using quantitative measures.(1) extend structured illumination microscopy (SIM) (2, 3) by exploiting a higher numerical aperture (NA) lens and using standing light waves to switch reversibly switchable fluorescent proteins (RSFPs). The patterned on-switching of RSFPs yields a spatially controlled distribution of fluorophores in the fluorescent "on" state, adding spatial frequencies of higher order to the fluorescence emission. These higher-order frequencies improve SIM resolution, provided that they can be disentangled from the low-frequency signals. According to Li et al., even moderate illumination intensities lead to higher frequencies of enhanced amplitudes, making their method more applicable to living cells than other super-resolution approaches. Employing Skylan-NS, an apparently quite photostable-but undisclosed-RSFP, recordings of a few tens of images were made. The resolution was reportedly improved to nominally 84 nm by SIM with an NA 1.7 lens and improved to nominally 62 nm when performing the RSFP switching with sinusoidal on-switching and readout patterns. By saturating the on-switching of the fluorescent proteins, Li et al. extended the resolution to nominally 45 nm.