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The design of dynamic, reconfigurable devices is crucial for the bottom-up construction of artificial biological systems. DNA can be used as an engineering material for the de-novo design of such dynamic devices. A self-assembled DNA origami switch is presented that uses the transition from double- to single-stranded DNA and vice versa to create and annihilate an entropic force that drives a reversible conformational change inside the switch. It is distinctively demonstrated that a DNA single-strand that is extended with 0.34 nm per nucleotide - the extension this very strand has in the double-stranded configuration - exerts a contractive force on its ends leading to large-scale motion. The operation of this type of switch is demonstrated via transmission electron microscopy, DNA-PAINT super-resolution microscopy and darkfield microscopy. The work illustrates the intricate and sometimes counter-intuitive forces that act in nanoscale physical systems that operate in fluids.

DNA points accumulation in nanoscale topography (DNA-PAINT) is a relatively easy-to-implement super-resolution technique. However, image acquisition is slow compared to most other approaches. Here, we overcome this limitation by designing optimized DNA sequences and buffer conditions. We demonstrate our approach in vitro with DNA origami and in situ using cell samples, and achieve an order of magnitude faster imaging speeds without compromising image quality or spatial resolution. This improvement now makes DNA-PAINT applicable to high-throughput studies.

Synthetic host-guest complexes are inherently dynamic as they employ weak and reversible noncovalent interactions for their recognition processes. We strategically exploited dynamic supramolecular recognition between fluorescently labeled guest molecules to complementary cucurbit[7]uril hosts to obtain stochastic switching between fluorescence ON- and OFF-states, enabling PAINT-based nanoscopic imaging in cells and tissues.

Particle fusion for single molecule localization microscopy improves signal-to-noise ratio and overcomes underlabeling, but ignores structural heterogeneity or conformational variability. We present a-priori knowledge-free unsupervised classification of structurally different particles employing the Bhattacharya cost function as dissimilarity metric. We achieve 96% classification accuracy on mixtures of up to four different DNA-origami structures, detect rare classes of origami occuring at 2% rate, and capture variation in ellipticity of nuclear pore complexes., 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.

Single-molecule localization microscopy (SMLM) describes a family of powerful imaging techniques that dramatically improve spatial resolution over standard, diffraction-limited microscopy techniques and can image biological structures at the molecular scale. In SMLM, individual fluorescent molecules are computationally localized from diffraction-limited image sequences and the localizations are used to generate a super-resolution image or a time course of super-resolution images, or to define molecular trajectories. In this Primer, we introduce the basic principles of SMLM techniques before describing the main experimental considerations when performing SMLM, including fluorescent labelling, sample preparation, hardware requirements and image acquisition in fixed and live cells. We then explain how low-resolution image sequences are computationally processed to reconstruct super-resolution images and/or extract quantitative information, and highlight a selection of biological discoveries enabled by SMLM and closely related methods. We discuss some of the main limitations and potential artefacts of SMLM, as well as ways to alleviate them. Finally, we present an outlook on advanced techniques and promising new developments in the fast-evolving field of SMLM. We hope that this Primer will be a useful reference for both newcomers and practitioners of SMLM. 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.

Single molecule localization microscopy offers in principle resolution down to the molecular level, but in practice this is limited primarily by incomplete fluorescent labeling of the structure. This missing information can be completed by merging information from many structurally identical particles. In this work, we present an approach for 3D single particle analysis in localization microscopy which hugely increases signal-to-noise ratio and resolution and enables determining the symmetry groups of macromolecular complexes. Our method does not require a structural template, and handles anisotropic localization uncertainties. We demonstrate 3D reconstructions of DNA-origami tetrahedrons, Nup96 and Nup107 subcomplexes of the nuclear pore complex acquired using multiple single molecule localization microscopy techniques, with their structural symmetry deducted from the data., 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.

MINFLUX offers a breakthrough in single molecule localization precision, but is limited in field of view. Here we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micrometer-sized field of view. We show a near two-fold improvement in precision over standard localization with the same photon count on DNA-origami nanostructures and tubulin in cells, using DNA-PAINT and STORM imaging.

Optical super-resolution techniques allow fluorescence imaging below the classical diffraction limit of light. From a technology standpoint, recent methods are approaching molecular-scale spatial resolution. However, this remarkable achievement is not easily translated to imaging of cellular components, since current labeling approaches are limited by either large label sizes (antibodies) or the sparse availability of small and efficient binders (nanobodies, aptamers, genetically-encoded tags). In this work, we combined recently developed Affimer reagents with site-specific DNA modification for high-efficiency labeling and imaging using DNA-PAINT. We assayed our approach using an actin Affimer. The small DNA-conjugated affinity binders could provide a solution for efficient multitarget super-resolution imaging in the future.

We present an approach for 3D particle fusion in localization microscopy which dramatically increases signal-to-noise ratio and resolution in single particle analysis. Our method does not require a structural template, and properly handles anisotropic localization uncertainties. We demonstrate 3D particle reconstructions of the Nup107 subcomplex of the nuclear pore complex (NPC), cross-validated using multiple localization microscopy techniques, as well as two-color 3D reconstructions of the NPC, and reconstructions of DNA-origami tetrahedrons.

Super-resolution (SR) techniques have extended the optical resolution down to a few nanometers. However, quantitative treatment of SR data remains challenging due to its complex dependence on a manifold of experimental parameters. Among the different SR variants, DNA-PAINT is relatively straightforward to implement, since it achieves the necessary ‘blinking’ without the use of rather complex optical or chemical activation schemes. However, it still suffers from image and quantification artifacts caused by inhomogeneous optical excitation. Here we demonstrate that several experimental challenges can be alleviated by introducing a segment-wise analysis approach and ultimately overcome by implementing a flat-top illumination profile for TIRF microscopy using a commercially-available beam-shaping device. The improvements with regards to homogeneous spatial resolution and precise kinetic information over the whole field-of-view were quantitatively assayed using DNA origami and cell samples. Our findings open the door to high-throughput DNA-PAINT studies with thus far unprecedented accuracy for quantitative data interpretation., 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.

MINFLUX offers a breakthrough in single molecule localization precision, but suffers from a tiny field-of-view and a lack of practical parallelism. Here, we combine centroid estimation and illumination pattern induced photon count variations in a conventional widefield imaging setup to extract position information over a typical micron sized field-of-view. We show a near twofold improvement in precision over standard localization with the same photon count on DNA-origami nano-structures.

[Image: see text] Single-molecule localization microscopy (SMLM) has revolutionized optical microscopy, extending resolution down to the level of individual molecules. However, the actual counting of molecules relies on preliminary knowledge of the blinking behavior of individual targets or on a calibration to a reference. In particular for biological applications, great care has to be taken because a plethora of factors influence the quality and applicability of calibration-dependent approaches to count targets in localization clusters particularly in SMLM data obtained from heterogeneous samples. Here, we present localization-based fluorescence correlation spectroscopy (lbFCS) as the first absolute molecular counting approach for DNA-points accumulation for imaging in nanoscale topography (PAINT) microscopy and, to our knowledge, for SMLM in general. We demonstrate that lbFCS overcomes the limitation of previous DNA-PAINT counting and allows the quantification of target molecules independent of the localization cluster density. In accordance with the promising results of our systematic proof-of-principle study on DNA origami structures as idealized targets, lbFCS could potentially also provide quantitative access to more challenging biological targets featuring heterogeneous cluster sizes in the future.

Optical super-resolution microscopy is rapidly changing the way imaging studies in the biological and biomedical sciences are conducted. Due to the unique capability of achieving molecular contrast using fluorescent labels and sub-diffraction resolution down to a few tens of nanometers, super-resolution is developing as an attractive imaging modality. While the increased spatial resolution has already enabled structural studies at unprecedented molecular detail, the wide-spread use of super-resolution approaches as a standard characterization technique in biological laboratories has thus far been prevented by mainly two issues: (1) Intricate sample preparation and image acquisition and (2) costly and complex instrumentation. We here introduce a combination of the recently developed super-resolution technique DNA-PAINT (DNA points accumulation for imaging in nanoscale topography) with an easy-to-replicate, custom-built 3D single-molecule microscope (termed liteTIRF) that is an order of magnitude more economic in cost compared to most commercial systems. We assay the performance of our system using synthetic two- and three-dimensional DNA origami structures and show the applicability to single- and multiplexed cellular imaging.

Methods that fuse multiple localization microscopy images of a single structure can improve signal-to-noise ratio and resolution, but they generally suffer from template bias or sensitivity to registration errors. We present a template-free particle-fusion approach based on an all-to-all registration that provides robustness against individual misregistrations and underlabeling. We achieved 3.3-nm Fourier ring correlation (FRC) image resolution by fusing 383 DNA origami nanostructures with 80% labeling density, and 5.0-nm resolution for structures with 30% labeling density.

Single-molecule localization microscopy (SMLM) can visualize biological targets on the nanoscale, but complex hardware is required to perform SMLM in thick samples. Here, we combine 3D DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) with spinning disk confocal (SDC) hardware to overcome this limitation. We assay our achievable resolution with two- and three-dimensional DNA origami structures and demonstrate the general applicability by imaging a large variety of cellular targets including proteins, DNA and RNA deep in cells. We achieve multiplexed 3D super-resolution imaging at sample depths up to ~10 µm with up to 20 nm planar and 80 nm axial resolution, now enabling DNA-based super-resolution microscopy in whole cells using standard instrumentation., 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.

Optical super-resolution techniques reach unprecedented spatial resolution down to a few nanometers. However, efficient multiplexing strategies for the simultaneous detection of hundreds of molecular species are still elusive. Here, we introduce an entirely new approach to multiplexed super-resolution microscopy by designing the blinking behavior of targets with engineered binding frequency and duration in DNA-PAINT. We assay this kinetic barcoding approach in silico and in vitro using DNA origami structures, show the applicability for multiplexed RNA and protein detection in cells, and finally experimentally demonstrate 124-plex super-resolution imaging within minutes. We thank Martin Spitaler and the imaging facility of the MPI of Biochemistry for confocal imaging support.

The Nuclear Pore Complex (NPC) is one of the largest and most complex protein assemblies in the cell and – among other functions – serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and superresolution studies advancing our understanding of the NPC's complex architecture. However, the specific and direct visualization of single copies of NPC proteins and thus the ability to observe single-molecule heterogeneities of these complex structures is thus far elusive. Here, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We employ the high localization precision in DNA-PAINT and molecular contrast of these protein tags to optically resolve single copies of nucleoporins in the human Y-complex in three dimensions with a precision of ~3 nm. This technological advancement now enables structural studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy.

Super-resolution microscopy allows optical imaging below the classical diffraction limit of light with currently up to 20 × higher spatial resolution. However, the detection of multiple targets (multiplexing) is still hard to implement and time-consuming to conduct. Here, we report a straightforward sequential multiplexing approach based on the fast exchange of DNA probes which enables efficient and rapid multiplexed target detection with common super-resolution techniques such as (d)STORM, STED, and SIM. We assay our approach using DNA origami nanostructures to quantitatively assess labeling, imaging, and washing efficiency. We furthermore demonstrate the applicability of our approach by imaging multiple protein targets in fixed cells.