Search

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 futurein vivostudies 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.

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.

Type 3 secretion systems (T3SS) are essential virulence factors of numerous bacterial pathogens and inject effector proteins into host cells. The needle-like T3SS machinery consists of more than 20 components, has a length of around 100 nm and a diameter of up to 30 nm according to EM studies. Its intrabacterial components are highly dynamic and in permanent exchange with other bacterial structures. Therefore, a temporally and spatially resolved visualization of the T3SS using fluorescence microscopy techniques has been challenging. In the present study, novel labeling strategies were combined with super-resolution microscopy such as STED, STORM and MINFLUX. MINFLUX nanoscopy allowed to visualize components of the T3SS machinery such as the dynamic sorting platform component YscL and the extrabacterial pore protein YopD at unprecedented resolutions. The presented results represent the basis for an in depth investigation of T3SS structure and function and therefore gain new insights into the infection process of human pathogens in order to develop novel treatment and prevention strategies.

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, Minimal photon fluxes (MINFLUX) has enabled molecule-scale resolution in fluorescence microscopy but this had not been shown in standard, broadly applicable microscopy platforms. Here the authors report a solution to allow normal fluorescence microscopy while also providing 1-3 nm 3D resolution.

We report on an atmospheric pressure plasma (APP) treatment of fused silica and its related surface and near-surface effects. Such treatment was performed in order to improve laser micro-structuring of fused silica by a plasma-induced modification of the glass boundary layer. In this context, an APP jet applying a hydrogenous process gas was used. By the plasma treatment, the transmission of the investigated glass samples was significantly decreased. Further, a decrease in the superficial index of refraction of approx. 3.66 % at a wavelength of 636.7 nm was detected ellipsometrically. By surface energy measurements, a decrease of the surface polarity of 30.23 % was identified. These determined modifications confirm a reduction of silicon dioxide to UV-absorbing silicon suboxide as already reported in previous work. Further, a change in reflexion by maximum 0.26 % was detected which is explained by the superposition of constructive and destructive interferences due to a surface wrinkling. With the aid of atomic force microscopy, an increase of the surface root mean squared roughness by a factor of approx. 19 was determined. It was found that both the surface energy and the strength of the fused silica surface were reduced by the plasma treatment. Even though such treatment led to a clustering of carbonaceous contaminants, a surface-cleaning effect was confirmed by secondary ion mass spectroscopy and energy-dispersive X-ray spectroscopy. The increase in UV-absorption allows enhanced laser ablation results as shown in previous work.