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SummaryMapping the intricate spatial relationships between the many different molecules inside a cell is essential to understanding cellular functions in all their complexity. Super-resolution fluorescence microscopy offers the required spatial resolution but struggles to reveal more than four different targets simultaneously. Exchanging labels in subsequent imaging rounds for multiplexed imaging extends this number but is limited by its low throughput. Here we present a novel imaging method for rapid multiplexed super-resolution microscopy of a nearly unlimited number of molecular targets by leveraging fluorogenic labeling in conjunction with Transient Adapter-mediated switching for high-throughput DNA-PAINT (FLASH-PAINT). We demonstrate the cell biological versatility of FLASH-PAINT in mammalian cells in four applications: i) mapping nine proteins in a single mammalian cell, ii) elucidating the functional organization of primary cilia by nine-target imaging, iii) revealing the changes in proximity of twelve different targets in unperturbed and dissociated Golgi stacks and iv) investigating inter-organelle contacts at 3D super-resolution.

Spatial omics techniques generate spatially resolved, comprehensive data about molecules that define the identity and function of cells in tissues. Epigenetic multiplexing approaches such as Multiplexed Error-robust FISH (MERFISH), introduced by Lu et al.

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.

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.

Summary The spatial organization of the genome is essential for its functions, including gene expression, DNA replication and repair, as well as chromosome segregation1. Biomolecular condensates and loop extrusion have been proposed as the principal driving forces that underlie the formation of non-random structures such as chromatin compartments and topologically associating domains2,3. However, if the actual 3D-folding of DNA in single cells is consistent with these mechanisms has been difficult to address in situ. Here, we present LoopTrace, a workflow for high-resolution reconstruction of 3D genome architecture without DNA denaturation. Classical fluorescence in situ hybridization approaches can link chromatin architecture to DNA sequence but disrupt chromatin structure at the critical nanoscale of individual loops. Our workflow employs non-denaturing enzymatic strand resection4,5, to conserve chromatin structure and can resolve the 3D-fold of chromosomal DNA with better than 5-kb-resolution in single human cells. Our results show that the chromatin fiber behaves as a random coil that can be further structured in a manner consistent with loop formation, explaining the emergence of topologically associated domain-like features in cell population averages. Mining a large amount of single-cell data computationally, we reveal chromatin folding intermediates consistent with progressive loop extrusion and stabilized loops, highlighting the power of our method to visualize the nanoscale features of genome organization in situ.

Rotaviruses transcribe eleven distinct protein-coding RNAs that must be stoichiometrically co-packaged prior to their replication to make an infectious virion. During infection, rotavirus transcripts accumulate in cytoplasmic ribonucleoprotein (RNP) condensates, termed viroplasms. Understanding the mechanisms of viroplasm assembly and RNA enrichment within is crucial to gaining greater insight into their function and stoichiometric assortment of individual transcripts. We analysed the subcellular distribution of individual RV transcripts and viroplasm transcriptome by combining multiplexed DNA-barcoded single-molecule RNA FISH of infected cells. Using DNA-PAINT microscopy, we provide evidence of the early onset of viral transcript oligomerisation that occurs prior to the formation of viroplasms. We demonstrate that viral sequences lacking the conserved terminal regions fail to undergo enrichment in rotavirus RNP condensates. We show that individual viral transcripts exhibit variable propensities to partition into viroplasms, irrespective of their absolute numbers in cells, suggesting a selective RNA enrichment mechanism distinct from other known cellular RNP granules. We suggest that rotavirus replication factories represent unique RNP condensates enriched in eleven types of cognate transcripts that may facilitate the assembly of a multi-segmented RNA genome.

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.

DNA origami, in which a long scaffold strand is assembled with a many short staple strands into parallel arrays of double helices, has proven a powerful method for custom nanofabrication. However, currently the design and optimization of custom 3D DNA-origami shapes is a barrier to rapid application to new areas. Here we introduce a modular barrel architecture, and demonstrate hierarchical assembly of a 100 megadalton DNA-origami barrel of ~90 nm diameter and ~250 nm height, that provides a rhombic-lattice canvas of a thousand pixels each, with pitch of ~8 nm, on its inner and outer surfaces. Complex patterns rendered on these surfaces were resolved using up to twelve rounds of Exchange-PAINT super-resolution microscopy. We envision these structures as versatile nanoscale pegboards for applications requiring complex 3D arrangements of matter, which will serve to promote rapid uptake of this technology in diverse fields beyond specialist groups working in DNA nanotechnology., 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.

Innovation in genomics, transcriptomics, and proteomics research has created a plethora of state-of-the-art techniques such as nucleic acid sequencing and mass-spectrometry-based proteomics with paramount impact in the life sciences. While current approaches yield quantitative abundance analysis of biomolecules on an almost routine basis, coupling this high content to spatial information in a single cell and tissue context is challenging. Here, current implementations of spatial omics are discussed and recent developments in the field of DNA-barcoded fluorescence microscopy are reviewed. Light is shed on the potential of DNA-based imaging techniques to provide a comprehensive toolbox for spatial genomics and transcriptomics and discuss current challenges, which need to be overcome on the way to spatial proteomics using high-resolution fluorescence microscopy.

Visualizing the functional interactions of biomolecules such as proteins and nucleic acids is key to understanding cellular life on the molecular scale. Spatial proximity is often used as a proxy for the direct interaction of biomolecules. However, current techniques to visualize spatial proximity are either limited by spatial resolution, dynamic range, or lack of single‐molecule sensitivity. Here, we introduce Proximity‐PAINT (pPAINT), a variation of the super‐resolution microscopy technique DNA‐PAINT. pPAINT uses a split‐docking‐site configuration to detect spatial proximity with high sensitivity, low false‐positive rates, and tunable detection distances. We benchmark and optimize pPAINT using designer DNA nanostructures and demonstrate its cellular applicability by visualizing the spatial proximity of alpha‐ and beta‐tubulin in microtubules using super‐resolution detection., A modified implementation of DNA‐PAINT microscopy is used to detect spatial proximity of biomolecules with super‐resolution capabilities. The new technique, called Proximity‐PAINT, features a precisely tunable detection range, high sensitivity and low false‐positive rates. The implementation can be applied to visualize cellular protein‐protein interactions and other biomolecules of interest, such as nucleic acids.

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.

Superauflosende Mikroskopie ermoglicht optische Bildgebung unterhalb der klassischen Beugungsgrenze von Licht mit bis zu 20-fach verbesserter raumlicher Auflosung. Jedoch ist derzeit das Beobachten mehrerer unterschiedlicher Zielmolekule (“Multiplexing”) schwierig und zeitintensiv in der Durchfuhrung. Hier stellen wir einen einfachen Ansatz fur sequenzielles Multiplexing vor, der auf einem schnellen Austausch von DNA-Sonden basiert. Dies ermoglicht eine effiziente Detektion vieler Zielmolekule mit Superauflosungsmethoden wie (d)STORM, STED und SIM. Wir evaluieren unseren Ansatz mit DNA-Origami-Nanostrukturen, um Markierung, Bildgebung und die Wascheffizienz quantitativ zu testen. Daruber hinaus demonstrieren wir die Anwendbarkeit unserer Methode zur Bildgebung mehrerer Proteine in fixierten Zellen.

We report the development of multiplexed cellular super-resolution imaging using DNA-barcoded binders., Recent advances in super-resolution fluorescence imaging allow researchers to overcome the classical diffraction limit of light, and are already starting to make an impact in biology. However, a key challenge for traditional super-resolution methods is their limited multiplexing capability, which prevents a systematic understanding of multi-protein interactions on the nanoscale. Exchange-PAINT, a recently developed DNA-based multiplexing approach, in theory facilitates spectrally-unlimited multiplexing by sequentially imaging target molecules using orthogonal dye-labeled ‘imager’ strands. While this approach holds great promise for the bioimaging community, its widespread application has been hampered by the availability of DNA-conjugated ligands for protein labeling. Herein, we report a universal approach for the creation of DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging, using a variety of affinity reagents such as primary and secondary antibodies, nanobodies, and small molecule binders. Furthermore, we extend the availability of orthogonal imager strands for Exchange-PAINT to over 50 and assay their orthogonality in a novel DNA origami-based crosstalk assay. Using our optimized conjugation and labeling strategies, we demonstrate nine-color super-resolution imaging in situ in fixed cells.

Despite the vital role of mechanical forces in biology, it still remains a challenge to image cellular force with sub-100-nm resolution. Here, we present tension points accumulation for imaging in nanoscale topography (tPAINT), integrating molecular tension probes with the DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique to map piconewton mechanical events with ~25-nm resolution. To perform live-cell dynamic tension imaging, we engineered reversible probes with a cryptic docking site revealed only when the probe experiences forces exceeding a defined mechanical threshold (~7–21 pN). Additionally, we report a second type of irreversible tPAINT probe that exposes its cryptic docking site permanently and thus integrates force history over time, offering improved spatial resolution in exchange for temporal dynamics. We applied both types of tPAINT probes to map integrin receptor forces in live human platelets and mouse embryonic fibroblasts. Importantly, tPAINT revealed a link between platelet forces at the leading edge of cells and the dynamic actin-rich ring nucleated by the Arp2/3 complex. 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.

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.

Phasing-in quality control in the nucleus The fundamental process of protein quality control in the nucleus is not well understood. The nucleus contains several non–membrane-bound subcompartments forming liquid-like condensates. The largest of these is the nucleolus, the site of ribosome biogenesis. Frottin et al. found that metastable nuclear proteins that misfold upon heat stress enter the nucleolus. In the nucleolus, they avoid irreversible aggregation and remain competent for heat shock protein 70–dependent refolding upon recovery from stress. Prolonged stress or the uptake of proteins associated with neurodegenerative diseases prevented this reversibility. Thus, the properties of a phase-separated compartment can assist in protein quality control. Science , this issue p. 342

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.

Structural DNA nanotechnology allow researchers to use the unique molecular recognition properties of DNA strands to construct nanoscale objects with almost arbitrary complexity in two and three dimensions. Abstracted as molecular breadboards, DNA nanostructures enable nanometer-precise placement of guest molecules such as proteins, fluorophores, or nanoparticles. These assemblies can be used to study biological phenomena with unprecedented control over number, spacing, and molecular identity. Here, we give a general introduction to structural DNA nanotechnology and more specifically discuss applications of DNA nanostructures in the field of fluorescence and plasmonics.

Synthetic supramolecular host-guest complexes are inherently dynamic as they employ weak and reversible noncovalent interactions for their recognition process. This dynamic behavior allows host-guest chemistry to be employed for various state of the art applications. Herein, we demonstrate the use of the dynamic supramolecular interaction to enable nanoscopic imaging inside cells and tissues. This imaging method exploits repetitive and transient binding of fluorescently labeled hexamethylenediamine (HMD) guest molecule to complementary cucurbit[7]uril (CB[7]) host to obtain stochastic switching between fluorescence ON- and OFF-states. Through connecting CB[7] hosts to targeting ligands (e.g., antibodies and small molecules), we show that this autonomous blinking enables two-dimensional (2D) and 3D super-resolution imaging of proteins in fixed cells and tissues. Finally, we exploited the capability of host-guest molecules to maintain their interaction specificity in the complexity of the live intracellular environment to obtain super-resolution actin imaging in living HeLa cell.

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.

Super-resolution techniques have begun to transform biological and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resolution microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resolution microscopy. Using the programmability and specificity of DNA molecules as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resolution techniques, making them compatible with virtually any single-molecule-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise molecule counting and ultra-high, molecular-scale (sub-5-nm) spatial resolution, reaching ∼1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample preparation, multiplexed data acquisition, data simulation, super-resolution image reconstruction and post-processing such as drift correction, molecule counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1-2 d.

Lipid metabolism is highly compartmentalized between cellular organelles that dynamically adapt their compositions and interactions in response to metabolic challenges. Here, we investigate how diet-induced hepatic lipid accumulation, observed in non-alcoholic fatty liver disease (NAFLD), affects protein localization, organelle organization, and protein phosphorylation in vivo. We develop a mass spectrometric workflow for protein and phosphopeptide correlation profiling to monitor levels and cellular distributions of similar to 6,000 liver proteins and similar to 16,000 phosphopeptides during development of steatosis. Several organelle contact site proteins are targeted to lipid droplets (LDs) in steatotic liver, tethering organelles orchestrating lipid metabolism. Proteins of the secretory pathway dramatically redistribute, including the mis-localization of the COPI complex and sequestration of the Golgi apparatus at LDs. This correlates with reduced hepatic protein secretion. Our systematic in vivo analysis of subcellular rearrangements and organelle-specific phosphorylation reveals how nutrient overload leads to organellar reorganization and cellular dysfunction.

Background Special diphenyl-pyrazole compounds and in particular anle138b were found to reduce the progression of prion and Parkinson's disease in animal models. The therapeutic impact of these compounds was attributed to the modulation of α-synuclein and prion-protein aggregation related to these diseases. Methods Photophysical and photochemical properties of the diphenyl-pyrazole compounds anle138b, anle186b and sery313b and their interaction with monomeric and aggregated α-synuclein were studied by fluorescence techniques. Results The fluorescence emission of diphenyl-pyrazole is strongly increased upon incubation with α-synuclein fibrils, while no change in fluorescence emission is found when brought in contact with monomeric α-synuclein. This points to a distinct interaction between diphenyl-pyrazole and the fibrillar structure with a high binding affinity (Kd = 190 ± 120 nM) for anle138b. Several α-synuclein proteins form a hydrophobic binding pocket for the diphenyl-pyrazole compound. A UV-induced dehalogenation reaction was observed for anle138b which is modulated by the hydrophobic environment of the fibrils. Conclusion Fluorescence of the investigated diphenyl-pyrazole compounds strongly increases upon binding to fibrillar α-synuclein structures. Binding at high affinity occurs to hydrophobic pockets in the fibrils. General significance The observed particular fluorescence properties of the diphenyl-pyrazole molecules open new possibilities for the investigation of the mode of action of these compounds in neurodegenerative diseases. The high binding affinity to aggregates and the strong increase in fluorescence upon binding make the compounds promising fluorescence markers for the analysis of aggregation-dependent epitopes.

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.