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By combining left- and right-handed DNA-PAINT probes, Unterauer et al. achieve simple, robust, and highly multiplexed super-resolution. They show 13-plex neuronal maps, revealing nanoscale organization of cytoskeleton, organelles, and synapses.

The nanoscale organization of the antigen-antibody complexes influences the therapeutic action of monoclonal antibodies. Here, the authors present a multi-target 3D RESI imaging assay for the nanometer spatial analysis of CD20 in complex with therapeutic monoclonal antibodies within intact cells, to analyse the interdependency between the mode of antibody binding and the therapeutic function.

Tension-PAINT integrates molecular tension probes with DNA-PAINT to enable ~25-nm-resolution mapping of piconewton mechanical events. Tension-PAINT can be used to study dynamic forces, and an irreversible variant integrates force history over time.

By combining bioorthogonal metabolic labelling and resolution enhancement through sequential imaging of DNA barcodes, the molecular organization of individual sugars in the native glycocalyx has been resolved at a spatial resolution of 9 ångström.

Extracting quantitative information on biomolecular oligomerisation with high resolution remains a significant challenge. Here, the authors propose SPINNA, a framework that compares nearest-neighbour distances from experimental single-protein position data with those obtained from simulations based on a model of protein oligomerisation.

Relatively little is known about cell-matrix interactions and the intracellular transduction of an initial ligand-receptor binding event on the single-molecule level. Here authors combine ligand-decorated DNA tension sensors with DNA-PAINT super-resolution microscopy to study the mechanical engagement of single integrin receptors and the downstream influence on actin bundling.

Super-resolution imaging of reference and target structures enables precise determination of the labeling efficiency of high-affinity binding proteins in cells for improved quantitative assessment of protein organization at the single-molecule level.

Lattice light-sheet-PAINT brings localization microscopy to large volumes, and SuReSim enables ground truth modeling of super-resolution experiments.

Variations on point accumulation for imaging in nanoscale topography (PAINT) for super-resolution imaging extend the technology to allow simple imaging of cellular proteins as well as synthetic DNA nanostructures and provide a high level of multiplexing.

Single-molecule localisation microscopy is limited by low labeling and detection efficiencies of the molecular probes. Here the authors report a framework to obtain absolute molecular quantities on a true molecular scale; the data reveal a ternary adhesion complex underlying cell-matrix adhesion.

The use of TIRF microscopy for DNA-PAINT experiments is limited by inhomogeneous illumination. Here the authors show that quantitative analysis of single-molecule TIRF experiments can be improved by using a segment-wise analysis approach and overcome by using a beam-shaping device to give a flat-top illumination profile.

The design and optimisation of 3D DNA-origami can be a barrier to rapid application. Here the authors design barrel structure of stacked 2D double helical rings with complex surface patterns.

Self-assembled DNA nanostructures hold potential as nanomachines or platforms for organized chemical synthesis, but methods for assembly quality control are lacking. Here the authors use DNA-PAINT to quantify the incorporation and accessibility of individual strands in a DNA origami platform with molecular resolution.

The authors introduce a single-molecule DNA-barcoding method, resolution enhancement by sequential imaging, that improves the resolution of fluorescence microscopy down to the Ångström scale using off-the-shelf fluorescence microscopy hardware and reagents.

Single-particle tracking (SPT) has revolutionised studies of protein interactions but is often limited by photobleaching. Here, the authors evolve DNA-PAINT-SPT to enable simultaneous dual-colour detection for the quantification of protein dimerization and live cell membrane protein tracking.

Single-molecule localization microscopy relies on stochastic blinking events, treated as independent events without assignment to a particular emitter. Here, BaGoL takes low precision localizations generated from multiple emitter blinkings during DNAPAINT and dSTORM and finds the underlying emitter positions with high precision.

The length of single-particle tracking experiments are limited due to photobleaching. Here the authors achieve long-term single-particle tracking with continuous fluorophore exchange in DNA-PAINT and use this to observe DNA origami on lipid bilayers for tens of minutes.

Supercritical angle localisation microscopy (SALM) allows the z-positions of single fluorophores to be extracted from the intensity of supercritical angle fluorescence. Here the authors improve the z-resolution of SALM, and report nanometre isotropic localisation precision on DNA origami structures.

Adaptation of current algorithms to 3D SMLM data is currently problematic. Here the authors report a method that increases the signal-to-noise ratio and resolution of 3D single particle analysis in localization microscopy and enables determination of the symmetry groups of macromolecular complexes.

Existing methods for nanoscale visualization of biological targets in thick samples require complex hardware. Here, the authors combine the standard spinning disk confocal (SDC) microscopy with DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) to image proteins, DNA and RNA deep in cells.

Epitope tags are widely used in various applications, but often lack versatility. Here, the authors introduce a small, alpha helical tag, which is recognized by a high affinity nanobody and can be used in a range of different applications, from protein purification to super-resolution imaging and in vivo detection of proteins.

Dietary protein dilution, where protein is reduced and replaced by other nutrient sources without caloric restriction, promotes metabolic health via the hepatokine Fgf21. Here, the authors show that essential amino acids threonine and tryptophan are necessary and sufficient to induce these effects.

The antigen-B-cell-receptor interaction is the driving force of terminal B cell development that spans from B cell activation to antibody secreting plasma cells. Here authors determine, using DNA-PAINT super-resolution microscopy, how antigen affinity and valency define antigen binding to BCR in an in vitro system allowing precision control of these parameters.

Particle fusion can improve signal-to-noise ratio in single molecule localization microscopy, but is limited by structural heterogeneity. Here, the authors demonstrate an unsupervised classification method that differentiates structurally different DNA origami structures without prior knowledge.

The dyes chosen for DNA-PAINT microscopy are pivotal for data quality. This Analysis shows a comprehensive comparison of 18 fluorescent dyes in DNA-PAINT and offers guidance for optimum dye selection in single-color and multiplexed imaging.

Basnet et al. discover that in vitro SSNA1 forms fibrils that attach along protofilaments and guide these to grow away from microtubules, forming templates for branched microtubules. SSNA1 mutations that perturb this process lead to defective axon branching in primary neurons.

The spatial organization of the genome within the nucleus impacts many processes. Here the authors combine oligo-based DNA FISH with single-molecule super-resolution microscopy to image single-copy genomic regions and, taking advantage of SNPs, distinguish allelic regions of homologous chromosomes.

Life-science research and biomedical diagnostics call for robust fluorescence barcodes of compact size and high multiplexing capability. Here DNA-origami technology was used to construct a new kind of geometrically encoded barcode with excellent structural stiffness. They hold promise for both in situ and ex situ imaging of diverse biologically relevant entities.

Synthetic polymer wires, which contain short oligonucleotides extending from each repeat, can assemble into predesigned routings on two- and three-dimensional DNA origami templates.

Hundred-fold-faster DNA-PAINT imaging is enabled by the introduction of concatenated, periodic DNA sequence motifs in the docking strand. Six orthogonal sequences are described for speed-optimized and highly multiplexed cellular imaging.

DNA-PAINT is sped up by an order of magnitude by optimizing sequences and buffer conditions, enabling faster imaging with no compromise to image quality or resolution, improved single-molecule counting and enhanced cellular imaging.

Slow off-rate modified aptamer (SOMAmer) reagents are small and versatile probes for DNA-PAINT super-resolution microscopy that enable multiplexed, quantitative, and high-resolution imaging in fixed and live cells.

DNA-PAINT, a super-resolution fluorescence microscopy technique that exploits programmable transient oligonucleotide hybridization, can be used to image densely packed triangular lattice patterns with molecular-level resolution and ångström-level precision.

Point spread function (PSF) splitting with the ‘Circulator’, which encodes the fluorophore emission band into the PSF, improves the information content of fluorescence microscopy and enables improved super-resolution imaging and single-particle tracking.

Quantitative points accumulation in nanoscale topography (qPAINT) makes use of predictable binding kinetics between DNA-PAINT imager and docking strands to achieve accurate and precise counting of molecules in spatially unresolved complexes.

The interphase centrosome protein AKNA is necessary and sufficient for the organization of centrosomal microtubules, mediates delamination in the formation of the subventricular zone and regulates exit from this zone.

SIMFLUX combines elements of MINFLUX with structured illumination to double localization precision and improve resolution in localization microscopy. The approach was demonstrated on DNA origami and on cellular microtubules.

An all-to-all registration approach allows for improved, high-resolution, template-free single-particle reconstruction from localization microscopy data under realistic experimental conditions such as low labeling density.

This Primer explains the central concepts of single-molecule localization microscopy (SMLM) before discussing experimental considerations regarding fluorophores, optics and data acquisition, processing and analysis. The Primer further describes recent high-impact discoveries made by SMLM techniques and concludes by discussing emerging methodologies.

DNA bricks with binding domains of 13 nucleotides instead of the typical 8 make it possible to self-assemble gigadalton-scale, three-dimensional nanostructures consisting of tens of thousands of unique components.

Site-specific conjugation of oligonucleotides and native proteins remains challenging. Here, the authors select covalent DNA aptamers from a library modified with N-hydroxysuccinimide esters, and show their application in the formation of antibody–oligonucleotide conjugates for protein detection.

In DNA-PAINT, transient binding of dye-labeled oligonucleotides to their target strands creates the ‘blinking’ required for stochastic nanoscopy. This protocol describes how to apply DNA-PAINT, from sample preparation to data processing.