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.

Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but these measurements have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in two and three dimensions using the recently introduced super-resolution technique MINFLUX. Using this approach, we resolved the precise stepping motion of the motor protein kinesin-1 as it walked on microtubules in living cells. Nanoscopic tracking of motors walking on the microtubules of fixed cells also enabled us to resolve the architecture of the microtubule cytoskeleton with protofilament resolution.

Dynamic measurements of molecular machines can provide invaluable insights into their mechanism, but have been challenging in living cells. Here, we developed live-cell tracking of single fluorophores with nanometer spatial and millisecond temporal resolution in 2D and 3D using the recently introduced super-resolution technique MINFLUX. This allowed us to resolve the precise stepping motion of the motor protein kinesin-1 as it walks on microtubules in living cells. In addition, nanoscopic tracking of motors on microtubule of fixed cells enabled us to resolve their spatial organization with protofilament resolution. Our approach will enable future in vivo studies of motor protein kinetics in complex environments and super-resolution mapping of dense microtubule arrays, and pave the way towards monitoring functional conformational changes of protein machines at high spatiotemporal resolution in living systems.

The resolution achievable with the established super-resolution fluorescence nanoscopy methods, such as STORM or STED, is in general not sufficient to resolve protein complexes or even individual proteins. Recently, minimal photon flux (MINFLUX) nanoscopy has been introduced that combines the strengths of STED and STORM nanoscopy and can achieve a localization precision of less than 5 nm. We established a generally applicable workflow for MINFLUX imaging and applied it for the first time to a bacterial molecular machine in situ, i.e., the injectisome of the enteropathogen Y. enterocolitica. We demonstrate with a pore protein of the injectisome that MINFLUX can achieve a resolution down to the single molecule level in situ. By imaging a sorting platform protein using 3D-MINFLUX, insights into the precise localization and distribution of an injectisome component in a bacterial cell could be accomplished. MINFLUX nanoscopy has the potential to revolutionize super-resolution imaging of dynamic molecular processes in bacteria and eukaryotes.

Cells assemble macromolecular complexes into scaffoldings that serve as substrates for catalytic processes. Years of molecular neurobiology research indicate that neurotransmission depends on such optimization strategies. However, the molecular topography of the presynaptic active zone (AZ), where transmitter is released upon synaptic vesicle (SV) fusion, remains to be visualized. Therefore, we implemented MINFLUX optical nanoscopy to resolve the AZ of rod photoreceptors. This was facilitated by a novel sample immobilization technique that we name heat-assisted rapid dehydration (HARD), wherein a thin layer of rod synaptic terminals (spherules) was transferred onto glass coverslips from fresh retinal slices. Rod ribbon AZs were readily immunolabeled and imaged in 3D with a precision of a few nanometers. Our 3D-MINFLUX results indicate that the SV release site in rods is a molecular complex of bassoon–RIM2–ubMunc13-2–Ca v 1.4, which repeats longitudinally on both sides of the ribbon.