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A nanoscale 3D DNA tracing workflow visualizes Cohesin-dependent loops in single, structurally well-preserved cells. Computer simulations based on the tracing data give further insight into how Cohesin folds compact domains in the genome.

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.

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.

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.

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 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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.