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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. 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.

Background The rapid spread of COVID-19 required swift action to provide people with rheumatic and musculoskeletal diseases (RMDs) with reliable information. People with limited health literacy constitute a vulnerable group that might have difficulty accessing, understanding and applying health information, particularly in times of crisis. Objectives This study explored (a) key aspects of crisis communication and (b) explicit consideration of people’s health literacy needs in communication to people with RMDs during the first wave of COVID-19 in the Netherlands. Methods We conducted a convergent, qualitatively driven mixed-methods study comprising seven qualitative interviews with professional representatives of organisations responsible for information provision to people with RMDs, and quantitative analysis of 15 patient information materials distributed by these organisations. The study was guided by principles of crisis communication and health literacy. We assessed understandability and actionability of information materials using the Dutch version of the Patient Education Materials Assessment Tool (PEMAT, resulting in a percentage of quality criteria met), and language difficulty level using an online application (assessing difficult words, jargon, passive, complex and long sentences, long paragraphs, and difficulty levels according to the Common European Framework of Reference for Languages (CEFR, from A1 (basic) to C2 (proficient))). Results Respondents reported lack of preparedness, challenges related to scientific uncertainty and reaching the target group, difficulty simplifying information, and uncertainty regarding adequacy of the communication approach. Patient information materials (written and video) showed variation in actionability (range 60–100%) and understandability (range 58–100%), and 69% of written materials were too difficult, mostly due to the use of long sentences and difficult words. The quantitative findings were in coherence with the limitations in communication reported by respondents. Several potential improvements were formulated in ‘lessons learned’. Conclusions Although rheumatology organisations mostly adhered to principles of crisis communication and made efforts to adapt information to their audience’s needs, we propose recommendations to improve preparedness, strategy, content, reach and consideration of health literacy needs in future crisis communication. Key messages Rheumatology organisations reported efforts to align with principles of crisis communication and health literacy. Patients’ health literacy needs were insufficiently considered and patient information materials were too difficult. Improving preparedness, collaboration between organisations and disciplines, and adaptation to patients’ needs is necessary.

Background Upper gastrointestinal pathologies are common in bariatric patients. Preoperative esophagogastroduodenal endoscopy (EGD) should detect and treat pathologies that might alter the type of bariatric surgery. However, clinical consequences of these findings are often insignificant. The aim of this study was to assess the influence of preoperative endoscopy in our cohort and its clinical consequences. Methods We conducted a retrospective analysis of endoscopic findings in patients under evaluation for bariatric surgery. Endoscopic findings were compared to preoperative risk factors as well as postoperative complications, and its clinical consequences were analyzed. Results Data was available for 801 patients. Abnormal endoscopic findings were found in 65.7% of all patients. The most common conditions were gastritis (32.1%) and gastroesophageal reflux (24.8%). Malignancies were observed in 0.5% of all patients. We observed early-stage adenocarcinoma of the esophagus in two patients through our routine preoperative evaluation. Helicobacter pylori infections were detected in preoperative biopsies in only 3.7% of all patients. Patients who reported reflux symptoms had a higher rate of pathological EGDs (74.2 vs. 64.9%, p .019). We did not find any other risk factors for a pathological endoscopy. The postoperative complication rate was 11.2%. Leakage rate was 1.1%. Mortality rate was 0.4%. We did not find any correlation between the incidence of postoperative complications and preoperative endoscopic findings. Conclusions Relevant findings in routine preoperative endoscopy are rare but have significant influence on decision-making in bariatric patients and should be assessed as a necessary diagnostic tool.

The cardiac ryanodine receptor (RyR2) constitutes the molecular basis of the process of calcium-induced calcium release where activation of RyR2s can be locally regenerative. Here, we present purely optical data of RyR2 distribution with sub-molecular resolution by applying 3D MINFLUX microscopy. Using single-domain antibodies and DNA-PAINT we determine the location of individual RyR2 subunits with high precision (~3 nm) and resolve the 3D orientations of RyR2s in-situ. We measured labeling efficiencies of ~50%, implying RyR2 tetramer detection probability approaching 95%. In HEK293 cells, RyR2 expression was dense, with some clusters containing several hundred RyR2s. Ventricular myocytes from mice contained large clusters containing many tens of close-packed RyR2s, resolving apparent discrepancies between electron microscopy and previous super-resolution microscopy data. The methodology developed here reveals the full 3D morphological complexity of RyR2 channels and is applicable to other multi-subunit complexes in a variety of cell types. Cardiac ryanodine receptors (RyR2) are critical for heart contraction. Here, the authors use 3D MINFLUX microscopy to image receptor subunits and RyR2 orientation with nanometre resolution, thereby providing a molecular view of the organisation and clustering of these cardiac muscle receptors.

The kinetochore is an essential protein complex for accurate chromosome segregation. The constitutive centromere-associated network (CCAN), a subcomplex of the kinetochore, associates with centromeric chromatin providing a platform for the kinetochore assembly. A CCAN protein, CENP-C, is thought to be a central hub for the centromere/kinetochore organization. However, the crucial role of CENP-C in centromeres remains to be elucidated. Here, we demonstrated that both the CCAN-binding domain and C-terminal Cupin domain of CENP-C are necessary and sufficient for chicken CENP-C function. Our structural and biochemical analyses revealed that the Cupin domain of chicken and human CENP-C is self-oligomerization domain, which is crucial for centromeric chromatin organization. CENP-C mutants lacking the oligomerization interface cause mislocalization of CCAN and cell death. Based on these results, we conclude that the CENP-C oligomerization plays a crucial role in centromere function via providing the robust centromeric chromatin in vertebrate cells. Competing Interest Statement I.J. is an employee of Abberior Instruments that develops and manufactures super-resolution fluorescence microscopes, including the MINFLUX system used here. All other authors declare no competing interests.

Neurotransmitter release and membrane retrieval at active zones require precise spatial and temporal coordination relying on an intricate molecular machinery. However, the exact nano-structural organization of this machinery is not yet fully elucidated. Here, we used 3D MINFLUX combined with both spectral demixing and DNA-PAINT to analyze the positioning of the scaffolding protein Bassoon at presynaptic active zones of glutamatergic spine synapses of hippocampal neurons, achieving a localization precision of 5 nm in 3D. This approach allowed us to visualize directly the distribution of N-terminal and the C-terminal regions of Bassoon, and demonstrates that Bassoon exhibits an orientation at the active zone, where the C-terminal region is directed toward the synaptic cleft and the N-terminal region towards synaptic vesicles. Having demonstrated this spatial configuration for endogenous and recombinant Bassoon molecules, our study paves the way towards molecular-scale resolution analysis of other prominent proteins of the presynaptic release machinery.

The membrane proteins Ninjurin1 (NINJ1) and Ninjurin2 (NINJ2) are upregulated by nerve injury to increase cell adhesion and promote axonal growth in neurons. NINJ1, but not NINJ2, has also been shown to play an essential role in pyroptosis by promoting plasma membrane rupture downstream of gasdermin D (GSDMD) pore formation, as well as in lytic cell death mediated by other pathways. Recombinant NINJ1 and NINJ2 purified in detergent show irregular rings of various diameters as well as curved filaments. While NINJ1 and NINJ2 both formed ring-like structures when mixed with liposomes, strikingly, only NINJ1, but not NINJ2, ruptures liposome membranes, leading to their dissolution. Because of the better feasibility, we determined the cryo-EM structure of NINJ1 ring segments from detergent by segmenting the irregular rings into shorter fragments. Each NINJ1 subunit contains a transmembrane (TM) helical hairpin (α3 and α4) that likely mediates NINJ1 membrane localization, as well as the side-by-side interaction between adjacent subunits. There are two extracellular domain amphipathic helices (α1 and α2), among which α1 crosses over to the neighboring subunit at the outside facing surface of the ring, to link NINJ1 subunits together into chains. As such, the inner face of the rings is hydrophobic whereas the outer face of the rings is hydrophilic and should repel membranes. Live cell imaging of NINJ1-deficient THP-1 cells reconstituted with NINJ1-eGFP uncovers the pinching off of NINJ1 rings from the cell surface and the loss of NINJ1 to the culture supernatant in oligomerized forms upon inflammasome activation. Formation of rings is also confirmed by super-resolution imaging of endogenous NINJ1 using anti-NINJ1 antibody. These data suggest that membrane insertion of amphipathic helices and formation of rings with a hydrophilic outer surface underlie the mechanism for NINJ1 to pinch off membranes as if it were a nanodisc-forming amphipathic polymer, leading to membrane rupture and lysis during cell death.

The cardiac type 2 ryanodine receptor (RyR2) is a large homotetramer of a ~560 kD subunit and is the molecular pathway through which the majority of Ca2+ enters the cytosol during cardiac activation. It constitutes the molecular basis of the process of calcium-induced calcium release where activation of RyR2s can be locally regenerative giving rise to local release events termed Ca2+ sparks. Accordingly, the molecular distribution of RyR2 in cardiac myocytes has been of great interest. Here we present the first purely optical data of RyR2 distribution with sub-molecular resolution by applying 3D MINFLUX fluorescence super-resolution microscopy. We demonstrate that by using single-domain antibodies (sdABs) against fluorescent protein domains in engineered RyR2 fluorescent protein fusions we can determine the location of individual RyR2 subunits with high precision (~3 nm) in all directions. Combining MINFLUX with DNA-PAINT, to maximize detection efficiency, we measured in situ labeling efficiencies using NPC structures as reference and regularly achieved efficiencies around 50%, which would translate to RyR2 detection efficiencies close to 95%, i.e. the probability that at least one subunit is detected, if target accessibility is similar. Using this approach, we detect dense and extended RyR2 expression in HEK cells with some clusters spanning several micrometres in extent and containing several hundred RyR2s. Similarly, myocytes from PA-RFP RyR2 mice contained large clusters containing many tens of RyR2s. The new data also suggests a resolution to apparent discrepancies between previous data from electron microscopy and super-resolution data. The methodology developed here will be critical to reveal the full complexity of RyR2 and related Ca2+ handling proteins in 3D as well as their relationship to contractile function. Our new approaches should be applicable to other multi-subunit complexes in cardiac muscle and other cell types.

Abstract Reversibly switchable fluorescent proteins (RSFPs) can be repeatedly transferred between a fluorescent on- and a non-fluorescent off-state in response to irradiation with light of different wavelengths. Negative switching RSFPs are switched from the on- to the off-state with the same wavelength which also excites fluorescence. Positive switching RSFPs have a reversed light response where the fluorescence excitation wavelength induces the transition from the off- to the on-state. Reversible saturable optical linear (fluorescence) transitions (RESOLFT) nanoscopy utilizes these switching states to achieve diffraction-unlimited resolution, but so far has primarily relied on negative switching RSFPs by using time sequential switching schemes. Based on the green fluorescent RSFP Padron, we engineered the positive switching RSFP Padron2. Compared to its predecessor, it can undergo 50-fold more switching cycles while displaying a contrast ratio between the on- and the off-state of more than 100:1. Because of its robust switching behavior, Padron2 supports a RESOLFT imaging scheme that entirely refrains from sequential switching as it only requires beam scanning of two spatially overlaid light distributions. Using Padron2, we demonstrate live-cell RESOLFT nanoscopy without sequential irradiation steps. Competing Interest Statement The authors have declared no competing interest. * Abbreviations RESOLFT reversible saturable optical linear (fluorescence) transitions RSFP reversibly switchable fluorescent proteins STED stimulated emission depletion

Abstract Cells assemble macromolecular complexes into scaffoldings that serve as substrates for catalytic processes. Years of molecular neurobiology indicate that neurotransmission depends on such optimization strategies, yet 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. To facilitate MINFLUX nanoscopy of the AZ, we developed and verified an immobilization technique, we name Heat Assisted Rapid Dehydration (HARD). Here fresh retinal slices are directly stamped onto glass coverslips yielding a single layer of rod AZs. These AZs exhibited excellent labeling efficiency and minimal signal redundancy in the Z-direction. Our data indicate that the SV release site is a molecular complex of bassoon-Rab3-binding molecule 2 (RIM2)-ubMunc13-2-CAST. The complexes are serially duplicated longitudinally, and reflected in register along the axis of symmetry of the synaptic ribbon. One sentence summary Structural motifs formed by active zone proteins at the photoreceptor synapse. Competing Interest Statement Abberior Instruments develops and manufactures super-resolution fluorescence microscopes, including Confocal, STED and MINFLUX systems.