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The integration of DNA origami structures with opto-plasmonic whispering gallery mode (WGM) sensors offers a significant advancement in label-free biosensing, overcoming the limitations of traditional fluorescence-based techniques, and providing enhanced sensitivity and specificity for detecting DNA hybridization events. In this study, DNA origami acts as a scaffold for the precise assembly of plasmonic dimers, composed of gold nanorods (AuNRs), which amplify detection sensitivity by generating strong near-field enhancements in the nanogap between the nanorods. By leveraging the strong electromagnetic fields generated within the nanogap of the plasmonic dimer, this platform enables the detection of transient hybridization events between DNA docking strands and freely diffusing complementary sequences. Our findings demonstrate that the salt concentration critically influences DNA hybridization kinetics. Higher ionic strengths reduce electrostatic repulsion between negatively charged DNA strands, thereby stabilizing duplex formation and prolonging interaction times. These effects are most pronounced at salt concentrations around 300–500 mM, where optimal conditions for duplex stability and reduced dissociation rates are achieved. By thoroughly investigating the hybridization kinetics under varying environmental conditions, this study contributes to a deeper understanding of DNA interactions and offers a robust tool for single-molecule detection with real-time capabilities. [ABSTRACT FROM AUTHOR]

Abstract Single-molecule localization microscopy (SMLM) surpasses the diffraction limit by randomly switching fluorophores between fluorescent and dark states, precisely pinpointing the resulted isolated emission patterns, thereby reconstructing the super-resolution images based on the accumulated locations of thousands to millions of single molecules. This technique achieves a ten-fold improvement in resolution, unveiling the intricate details of molecular activities and structures in cells and tissues. Multicolor SMLM extends this capability by imaging distinct protein species labeled with various fluorescent probes, providing insights into structural intricacies and spatial relationships among different targets. This review explores recent advancements in multicolor SMLM, evaluates the strengths and limitations of each variant, and discusses the future prospects.

Single-molecule localization microscopy (SMLM) surpasses the diffraction limit by randomly switching fluorophores between fluorescent and dark states, precisely pinpointing the resulted isolated emission patterns, thereby reconstructing the super-resolution images based on the accumulated locations of thousands to millions of single molecules. This technique achieves a ten-fold improvement in resolution, unveiling the intricate details of molecular activities and structures in cells and tissues. Multicolor SMLM extends this capability by imaging distinct protein species labeled with various fluorescent probes, providing insights into structural intricacies and spatial relationships among different targets. This review explores recent advancements in multicolor SMLM, evaluates the strengths and limitations of each variant, and discusses the future prospects. [ABSTRACT FROM AUTHOR]

MAGUK scaffold proteins play a central role in maintaining and modulating synaptic signaling, providing a framework to retain and position receptors, signaling molecules, and other synaptic components. In particular, the MAGUKs SAP102 and PSD-95 are essential for synaptic function at distinct developmental timepoints and perform both overlapping and unique roles. While their similar structures allow for common binding partners, SAP102 is expressed earlier in synapse development and is required for synaptogenesis, whereas PSD-95 expression peaks later and is associated with synapse maturation. PSD-95 and other key synaptic proteins organize into subsynaptic nanodomains that have a significant impact on synaptic transmission, but the nanoscale organization of SAP102 is unknown. How SAP102 is organized within the synapse, and how it relates spatially to PSD-95 on a nanometer scale, could underlie its unique functions and impact how SAP102 scaffolds synaptic proteins. Here we used DNA-PAINT super-resolution microscopy to measure SAP102 nanoorganization and its spatial relationship to PSD-95 at individual synapses in mixed-sex rat cultured neurons. We found that like PSD-95, SAP102 accumulates in high-density subsynaptic nanoclusters (NCs). However, SAP102 NCs were smaller and denser than PSD-95 NCs across development. Additionally, only a subset of SAP102 NCs co-organized with PSD-95, revealing MAGUK nanodomains within individual synapses containing either one or both proteins. These MAGUK nanodomain types had distinct NC properties and were differentially enriched with the presynaptic release protein Munc13-1. This organization into both shared and distinct subsynaptic nanodomains may underlie the ability of SAP102 and PSD-95 to perform both common and unique synaptic functions. [ABSTRACT FROM AUTHOR]

The pyoverdine siderophore is produced by Pseudomonas aeruginosa to access iron. Its synthesis involves the complex coordination of four nonribosomal peptide synthetases (NRPSs), which are responsible for assembling the pyoverdine peptide backbone. The precise cellular organization of these NRPSs and their mechanisms of interaction remain unclear. Here, we used a combination of several single-molecule microscopy techniques to elucidate the spatial arrangement of NRPSs within pyoverdine-producing cells. Our findings reveal that PvdL differs from the three other NRPSs in terms of localization and mobility patterns. PvdL is predominantly located in the inner membrane, while the others also explore the cytoplasmic compartment. Leveraging the power of multicolor single-molecule localization, we further reveal co-localization between PvdL and the other NRPSs, suggesting a pivotal role for PvdL in orchestrating the intricate biosynthetic pathway. Our observations strongly indicates that PvdL serves as a central orchestrator in the assembly of NRPSs involved in pyoverdine biosynthesis, assuming a critical regulatory function. [ABSTRACT FROM AUTHOR]

Super‐resolution techniques like single‐molecule localisation microscopy (SMLM) and stimulated emission depletion (STED) microscopy have been extended by the use of non‐covalent, weak affinity‐based transient labelling systems. DNA‐based hybrid systems are a prominent example among these transient labelling systems, offering excellent opportunities for multi‐target fluorescence imaging. However, these techniques suffer from higher background relative to covalently bound fluorophores, originating from unbound fluorophore‐labelled single‐stranded oligonucleotides. Here, we introduce short‐distance self‐quenching in fluorophore dimers as an efficient mechanism to reduce background fluorescence signal, while at the same time increasing the photon budget in the bound state by almost 2‐fold. We characterise the optical and thermodynamic properties of fluorophore‐dimer single‐stranded DNA, and show super‐resolution imaging applications with STED and SMLM with increased spatial resolution and reduced background. [ABSTRACT FROM AUTHOR]

Fluorescence Resonance Energy Transfer (FRET)‐based approaches are unique tools for sensing the immediate surroundings and interactions of (bio)molecules. FRET imaging and Fluorescence Lifetime Imaging Microscopy (FLIM) enable the visualization of the spatial distribution of molecular interactions and functional states. However, conventional FLIM and FRET imaging provide average information over an ensemble of molecules within a diffraction‐limited volume, which limits the spatial information, accuracy, and dynamic range of the observed signals. Here, an approach to obtain super‐resolved FRET imaging based on single‐molecule localization microscopy using an early prototype of a commercial time‐resolved confocal microscope is demonstrated. DNA Points Accumulation for Imaging in Nanoscale Topography with fluorogenic probes provides a suitable combination of background reduction and binding kinetics compatible with the scanning speed of usual confocal microscopes. A single laser is used to excite the donor, a broad detection band is employed to retrieve both donor and acceptor emission, and FRET events are detected from lifetime information. [ABSTRACT FROM AUTHOR]

Single-molecule microscopy is an effective tool for the characterization of fluorophore–plasmonic structure interaction. However, sophisticated evaluation is required as specific information is hidden in the emission. Here, we theoretically and experimentally investigated the emission polarization of rotationally mobile fluorophores near plasmonic Au–Ag alloy nanospheres. We developed an elaborate calculation based on the Mie theory, which considers the rotational mobility of dyes near spherical plasmonic nanoparticles of any size. Furthermore, we have created a simplified model that describes the emission's degree of polarization within 10% accuracy in the case of small nanoparticles, when dipole approximation is valid. Our results indicate the close relation of the polarization degree of the emission, which can reach up to be ∼ 0. 8 in the resonant case, to fluorescence scattering. DNA-PAINT single-molecule localization microscopy experiments conducted using AF488 and Atto647N dyes revealed that the measured polarization degree distribution followed the tendency predicted by calculations. [ABSTRACT FROM AUTHOR]

Upon initial immune challenge, dendritic cells (DCs) migrate to lymph nodes and interact with fibroblastic reticular cells (FRCs) via C-type lectin-like receptor 2 (CLEC-2). CLEC-2 binds to the membrane glycoprotein podoplanin (PDPN) on FRCs, inhibiting actomyosin contractility through the FRC network and permitting lymph node expansion. The hyaluronic acid receptor CD44 is known to be required for FRCs to respond to DCs but the mechanism of action is not fully elucidated. Here, we use DNA-PAINT, a quantitative single molecule super-resolution technique, to visualize and quantify how PDPN clustering is regulated in the plasma membrane of FRCs. Our results indicate that CLEC-2 interaction leads to the formation of large PDPN clusters (i.e. more than 12 proteins per cluster) in a CD44-dependent manner. These results suggest that CD44 expression is required to stabilize large pools of PDPN at the membrane of FRCs upon CLEC-2 interaction, revealing the molecular mechanism through which CD44 facilitates cellular crosstalk between FRCs and DCs.

Rapid release of calcium from internal stores via ryanodine receptors (RyRs) is one of the fastest types of cytoplasmic second messenger signalling in excitable cells. In the heart, rapid summation of the elementary events of calcium release, 'calcium sparks', determine the contraction of the myocardium. We adapted a correlative super-resolution microscopy protocol to correlate sub-plasmalemmal spontaneous calcium sparks in rat right ventricular myocytes with the local nanoscale RyR2 positions. This revealed a steep relationship between the integral of a calcium spark and the sum of the local RyR2s. Segmentation of recurring spark sites showed evidence of repeated and triggered saltatory activation of multiple local RyR2 clusters. In myocytes taken from failing right ventricles, RyR2 clusters themselves showed a dissipated morphology and fragmented (smaller) clusters. They also featured greater heterogeneity in both the spark properties and the relationship between the integral of the calcium spark and the local ensemble of RyR2s. While fragmented (smaller) RyR2 clusters were rarely observed directly underlying the larger sparks or the recurring spark sites, local interrogation of the channel-to-channel distances confirmed a clear link between the positions of each calcium spark and the tight, non-random clustering of the local RyR2 in both healthy and failing ventricles.

DNA‐PAINT enabled super‐resolution imaging through the transient binding of fluorescently‐labelled single‐stranded DNA (ssDNA) imagers to target ssDNA. However, its performance is constrained by imager background fluorescence, resulting in relatively long image acquisition and potential artifacts. We designed a molecular beacon (MB) as the PAINT imager. Unbound MB in solution reduces the background fluorescence due to its natively quenched state. They are fluorogenic upon binding to target DNA to create individual fluorescence events. We demonstrate that MB‐PAINT provides localization precision similar to traditional linear imager DNA‐PAINT. We also show that MB‐PAINT is ideally suited for fast super‐resolution imaging of molecular tension probes in living cells, eliminating the potential of artifacts from free‐diffusing imagers in traditional DNA‐PAINT at the cell‐substrate interface. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) continues to show no improvement in survival rates. One aspect of PDAC is elevated ATP levels, pointing to the purinergic axis as a potential attractive therapeutic target. Mediated in part by highly druggable extracellular proteins, this axis plays essential roles in fibrosis, inflammation response, and immune function. Analyzing the main members of the PDAC extracellular purinome using publicly available databases discerned which members may impact patient survival. P2RY2 presents as the purinergic gene with the strongest association with hypoxia, the highest cancer cell-specific expression, and the strongest impact on overall survival. Invasion assays using a 3D spheroid model revealed P2Y2 to be critical in facilitating invasion driven by extracellular ATP. Using genetic modification and pharmacological strategies, we demonstrate mechanistically that this ATP-driven invasion requires direct protein-protein interactions between P2Y2 and αV integrins. DNA-PAINT super-resolution fluorescence microscopy reveals that P2Y2 regulates the amount and distribution of integrin αV in the plasma membrane. Moreover, receptor-integrin interactions were required for effective downstream signaling, leading to cancer cell invasion. This work elucidates a novel GPCR-integrin interaction in cancer invasion, highlighting its potential for therapeutic targeting.

Summary: DNA-point accumulation for imaging at nanoscale topography (DNA-PAINT) can image fixed biological specimens with nanometer resolution and absolute stoichiometry. In living systems, however, the usage of DNA-PAINT has been limited due to high salt concentration in the buffer required for specific binding of the imager to the docker attached to the target. Here, we used multiple binding motifs of the docker, from 2 to 16, to accelerate the binding speed of the imager under physiological buffer conditions without compromising spatial resolution and maintaining the basal level homeostasis during the measurement. We imaged endogenous α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in cultured neurons—critical proteins involved in nerve communication—by DNA-PAINT in 3-dimensions using a monovalent single-chain variable fragment (scFv) to the GluA1 subunit of AMPAR. We found a heterogeneous distribution of synaptic AMPARs: ≈60% are immobile, primarily in nanodomains, defined as AMPARs that are within 0.3 μm of the Homer1 protein in the postsynaptic density; the other ∼40% of AMPARs have restricted mobility and trajectory. Motivation: Single-molecule localization microscopy can provide nanoscale resolution of molecular structure of biological samples, yet the application of the technique in live samples is limited. Here, we developed a live-cell DNA-PAINT technique to image and quantify neuronal receptor molecules in live hippocampal neurons.

Sarcomeres are the force-producing units of all striated muscles. Their nanoarchitecture critically depends on the large titin protein, which in vertebrates spans from the sarcomeric Z-disc to the M-band and hence links actin and myosin filaments stably together. This ensures sarcomeric integrity and determines the length of vertebrate sarcomeres. However, the instructive role of titins for sarcomeric architecture outside of vertebrates is not as well understood. Here, we used a series of nanobodies, the Drosophila titin nanobody toolbox, recognising specific domains of the two Drosophila titin homologs Sallimus and Projectin to determine their precise location in intact flight muscles. By combining nanobodies with DNA-PAINT super-resolution microscopy, we found that, similar to vertebrate titin, Sallimus bridges across the flight muscle I-band, whereas Projectin is located at the beginning of the A-band. Interestingly, the ends of both proteins overlap at the I-band/A-band border, revealing a staggered organisation of the two Drosophila titin homologs. This architecture may help to stably anchor Sallimus at the myosin filament and hence ensure efficient force transduction during flight.

Tumor cell-derived extracellular vesicles and their cargo of bioactive substances have gradually been recognized as novel biomarkers for cancer diagnosis. Meanwhile, the PD-L1 (Programmed Death-Ligand 1) protein, as an immune checkpoint molecule, is highly expressed on certain tumor cells and holds significant potential in immune therapy. In comparison to PD-L1 monoclonal antibodies, the inhibitory effect of PD-L1 siRNA (small interfering RNA) is more advantageous. In this article, we introduced a microfluidic chip integrating cell cultivation and exosome detection modules, which were intended for the investigation of the gene silencing effect of PD-L1 siRNA. Basically, cells were first cultured with PD-L1 siRNA in the chip. Then, the secreted exosomes were detected via super-resolution imaging, to validate the inhibitory effect of siRNA on PD-L1 expression. To be specific, a “sandwich” immunological structure was employed to detect exosomes secreted from HeLa cells. Immunofluorescence staining and DNA-PAINT (DNA Point Accumulation for Imaging in Nanoscale Topography) techniques were utilized to quantitatively analyze the PD-L1 proteins on HeLa exosomes, which enabled precise structural and content analysis of the exosomes. Compared with other existing PD-L1 detection methods, the advantages of our work include, first, the integration of microfluidic chips greatly simplifying the cell culture, gene silencing, and PD-L1 detection procedures. Second, the utilization of DNA-PAINT can provide an ultra-high spatial resolution, which is beneficial for exosomes due to their small sizes. Third, qPAINT could allow quantitative detection of PD-L1 with better precision. Hence, the combination of the microfluidic chip with DNA-PAINT could provide a more powerful integrated platform for the study of PD-L1-related tumor immunotherapy.

Numerous nucleic acid nanostructures of unique addressability and programmability have been fabricated for emerging applications. Structural characterization with atomic force microscopy and electron microscopy can provide information on the structural morphology and precision of these nanostructures. However, either structural information of native nucleic acid nanostructures in hydrated environment or the availability of addressable sites on these nanostructures could not be determined. Alternatively, DNA points accumulation for imaging in nanoscale topography (DNA‐PAINT) enables direct optical visualization of nucleic acid nanostructures in native forms, as well as evaluation of the accessibility of addressable sites on them. In this Review, the working principle of DNA‐PAINT is introduced, followed by the summary on advances of DNA‐PAINT characterization of various nucleic acid nanostructures. Finally, the current challenges and prospects for DNA‐PAINT characterization are presented. We envision DNA‐PAINT to be a potent characterization tool for functional nanomaterials. [ABSTRACT FROM AUTHOR]

Recent work suggests that Ras small GTPases interact with the anionic lipid phosphatidylserine (PS) in an isoform-specific manner, with direct implications for their biological functions. Studies on PS-Ras associations in cells, however, have relied on immuno-EM imaging of membrane sheets. To study their spatial relationships in intact cells, we have combined the use of Lact-C2-GFP, a biosensor for PS, with multicolor super resolution imaging based on DNA-PAINT. At ~20 nm spatial resolution, the resulting super resolution images clearly show the nonuniform molecular distribution of PS on the cell membrane and its co-enrichment with caveolae, as well as with unidentified membrane structures. Two-color imaging followed by spatial analysis shows that KRas-G12D and HRas-G12V both co-enrich with PS in model U2OS cells, confirming previous observations, yet exhibit clear differences in their association patterns. Whereas HRas-G12V is almost always co-enriched with PS, KRas-G12D is strongly co-enriched with PS in about half of the cells, with the other half exhibiting a more moderate association. In addition, perturbations to the actin cytoskeleton differentially impact PS association with the two Ras isoforms. These results suggest that PS-Ras association is context-dependent and demonstrate the utility of multiplexed super resolution imaging in defining the complex interplay between Ras and the membrane. [ABSTRACT FROM AUTHOR]

Nanoarchitectural control of matter is crucial for next-generation technologies. DNA origami templates are harnessed to accurately position single molecules; however, direct single molecule evidence is lacking regarding how well DNA origami can control the orientation of such molecules in three-dimensional space, as well as the factors affecting control. Here, we present two strategies for controlling the polar (θ) and in-plane azimuthal (ϕ) angular orientations of cyanine Cy5 single molecules tethered on rationally-designed DNA origami templates that are physically adsorbed (physisorbed) on glass substrates. By using dipolar imaging to evaluate Cy5′s orientation and super-resolution microscopy, the absolute spatial orientation of Cy5 is calculated relative to the DNA template. The sequence-dependent partial intercalation of Cy5 is discovered and supported theoretically using density functional theory and molecular dynamics simulations, and it is harnessed as our first strategy to achieve θ control for a full revolution with dispersion as small as ±4.5°. In our second strategy, ϕ control is achieved by mechanically stretching the Cy5 from its two tethers, being the dispersion ±10.3° for full stretching. These results can in principle be applied to any single molecule, expanding in this way the capabilities of DNA as a functional templating material for single-molecule orientation control. The experimental and modeling insights provided herein will help engineer similar self-assembling molecular systems based on polymers, such as RNA and proteins. [ABSTRACT FROM AUTHOR]

DNA point accumulation in nanoscale topography (DNA-PAINT) is an easy-to-implement approach for localization-based super-resolution imaging. Conventional DNA-PAINT imaging typically requires tens of thousands of frames of raw data to reconstruct one super-resolution image, which prevents its potential application for live imaging. Here, we introduce a new DNA-PAINT labeling method that allows for imaging of microtubules with both DNA-PAINT and widefield illumination. We develop a U-Net-based neural network, namely, U-PAINT to accelerate DNA-PAINT imaging from a widefield fluorescent image and a sparse single-molecule localization image. Compared with the conventional method, U-PAINT only requires one-tenth of the original raw data, which permits fast imaging and reconstruction of super-resolution microtubules and can be adopted to analyze other SMLM datasets. We anticipate that this machine learning method enables faster and even live-cell DNA-PAINT imaging in the future.

Super-resolution (SR) microscopy techniques have been advancing the understanding of neuronal protein networks and interactions. Unraveling the arrangement of proteins with molecular resolution provided novel insights into neuron cytoskeleton structure and actin polymerization dynamics in synaptic spines. Recent improvements in quantitative SR imaging have been applied to synaptic protein clusters and with improved multiplexing technology, the interplay of multiple protein partners in synaptic active zones has been elucidated. While all SR techniques come with benefits and drawbacks, true molecular quantification is a major challenge with the most complex requirements for labeling reagents and careful experimental design. In this perspective, we provide an overview of quantitative SR multiplexing and discuss in greater detail the quantification and multiplexing capabilities of the SR technique DNA-PAINT. Using predictable binding kinetics of short oligonucleotides, DNA-PAINT provides two unique approaches to address multiplexed molecular quantification: qPAINT and Exchange-PAINT. With precise and accurate quantification and spectrally unlimited multiplexing, DNA-PAINT offers an attractive route to unravel complex protein interaction networks in neurons. Finally, while the SR community has been pushing technological advances from an imaging technique perspective, the development of universally available, small, efficient, and quantitative labels remains a major challenge in the field.

Single-molecule localization microscopy (SMLM) can provide nanoscale resolution in thin samples but has rarely been applied to tissues because of high background from out-of-focus emitters and optical aberrations. Here, we describe a line scanning microscope that provides optical sectioning for SMLM in tissues. Imaging endogenously-tagged nucleoporins and F-actin on this system using DNA- and peptide-point accumulation for imaging in nanoscale topography (PAINT) routinely gives 30 nm resolution or better at depths greater than 20 αm. This revealed that the nuclear pores are nonrandomly distributed in most Drosophila tissues, in contrast to what is seen in cultured cells. Lamin Dm0 shows a complementary localization to the nuclear pores, suggesting that it corrals the pores. Furthermore, ectopic expression of the tissue-specific Lamin C causes the nuclear pores to distribute more randomly, whereas lamin C mutants enhance nuclear pore clustering, particularly in muscle nuclei. Given that nucleoporins interact with specific chromatin domains, nuclear pore clustering could regulate local chromatin organization and contribute to the disease phenotypes caused by human lamin A/C laminopathies. [ABSTRACT FROM AUTHOR]

Telomerase plays an important role in maintaining the length of telomere during cell division and is recognized as a new kind of biomarkers for cancer diagnosis. In this work, we present a brand new telomerase detection strategy based on a DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) like strategy. With an extraordinary spatial resolution (∼10 nm), the DNA-PAINT based strategy offers several advantages. First, it avoids complicated polymerase chain reaction and electrophoresis procedures. Second, it enables super resolution imaging of the reaction products with a high signal-to-noise ratio and facilitates the location of telomeric elongation sites on the single particle level, which results in a high sensitivity. Third, the detection scheme of the DNA-PAINT strategy allows direct in situ visualization of the telomeric elongation process, which has never been achieved before. All these advantages make the DNA-PAINT telomerase detection strategy significant for dynamic investigation of telomerase related physiological processes as well as cancer diagnosis. [ABSTRACT FROM AUTHOR]

Single-molecule localization microscopy (SMLM) is a potent tool to examine biological systems with unprecedented resolution, enabling the investigation of increasingly smaller structures. At the forefront of these developments is DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT), which exploits the stochastic and transient binding of fluorescently labeled DNA probes. In its early stages the implementation of DNA-PAINT was burdened by low-throughput, excessive acquisition time, and difficult integration with live-cell imaging. However, recent advances are addressing these challenges and expanding the range of applications of DNA-PAINT. We review the current state of the art of DNA-PAINT in light of these advances and contemplate what further developments remain indispensable to realize live-cell imaging. In recent years the performance, utility, and ease-of-use of DNA-based point accumulation in nanoscale topography (DNA-PAINT) have greatly improved by increasing compatibility with existing technology and diversifying the usable probes. Multiplexing with DNA-PAINT has become possible, which allows users to probe many targets simultaneously and paves the way for high-throughput methods. Advances have alleviated the limiting factors that govern the binding frequency, thereby accelerating imaging acquisition and enabling DNA-PAINT imaging to be performed in several minutes. The complex environment in living cells creates numerous challenges for standard DNA-PAINT imaging. These challenges are being addressed through improved probe design such as using modified DNA nucleotides or amino acid-based backbones for the imager and docking probes. [ABSTRACT FROM AUTHOR]

[Display omitted] • DNA nanotechnology was correlated with multiplexed single-molecule super-resolution imaging. • A procedure for near-molecular visualization and analysis of membrane receptor networks was established. • This approach enabled quantitative microscopy of the FGFR1-4 receptor network at the native cellular expression level. • Information on receptor clustering and distance relationships was extracted. • The imaging and analysis pipeline can be easily adapted to other protein networks. Fibroblast growth factor receptors (FGFRs) are a subfamily of receptor tyrosine kinases and central players in health and disease. Following ligand binding and the formation of homo- and heteromeric complexes, FGFRs initiate a cellular response. Challenges in studying FGFR activation are inner-subfamily interactions and a complex heterogeneity of these in the cell membrane, which demand for observation techniques that can resolve individual protein complexes and that are compatible with endogenous protein levels. Here, we established an imaging and analysis pipeline for multiplexed single-molecule localization microscopy (SMLM) of the FGFR network at the plasma membrane. Using DNA-labeled primary antibodies, we visualize all four FGFRs in the same cell with near-molecular spatial resolution. From the super-resolution imaging data, we extract information on FGFR density, spatial distribution, and inner-subfamily colocalization. Our approach is straightforward and easily adaptable to other multiplexed SMLM data of membrane proteins. [ABSTRACT FROM AUTHOR]

• The imaging protocol visualises intracellular calcium signals and their sources. • It correlates calcium spark TIRF images with DNA-PAINT images of RyR channels. • It is a powerful method for studying nanoscale structure-function relationship. Nanometre-scale cellular information obtained through super-resolution microscopies are often unaccompanied by functional information, particularly transient and diffusible signals through which life is orchestrated in the nano-micrometre spatial scale. We describe a correlative imaging protocol which allows the ubiquitous intracellular second messenger, calcium (Ca2+), to be directly visualised against nanoscale patterns of the ryanodine receptor (RyR) Ca2+ channels which give rise to these Ca2+ signals in wildtype primary cells. This was achieved by combining total internal reflection fluorescence (TIRF) imaging of the elementary Ca2+ signals, with the subsequent DNA-PAINT imaging of the RyRs. We report a straightforward image analysis protocol of feature extraction and image alignment between correlative datasets and demonstrate how such data can be used to visually identify the ensembles of Ca2+ channels that are locally activated during the genesis of cytoplasmic Ca2+ signals. [ABSTRACT FROM AUTHOR]

The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350–400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy (SMLM). In a single labeling step, antibodies conjugated with short DNA oligonucleotides visualized multiple targets by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. This approach avoids potential effects on structural integrity when using multiple rounds of immunolabeling and eliminates chromatic aberration, because all targets are imaged using a single excitation laser wavelength. This method proved robust for multi-target imaging in semi-thin tissue sections with a lateral resolution better than 25 nm, paving the way toward structural cell biology with single-molecule SRM.

Surface functionalization with targeting ligands confers to nanomaterials the ability of selectively recognize a biological target. Therefore, a quantitative characterization of surface functional molecules is critical for the rational development of nanomaterials-based applications, especially in nanomedicine research. Single-molecule localization microscopy can provide visualization of surface molecules at the level of individual particles, preserving the integrity of the material and overcoming the limitations of analytical methods based on ensemble averaging. Here we provide single-molecule localization data obtained on streptavidin-coated polystyrene particles, which can be exploited as a model system for surface-functionalized materials. After loading of the active sites of streptavidin molecules with a biotin-conjugated probe, they were imaged with a DNA-PAINT imaging approach, which can provide single-molecule imaging at subdiffraction resolution and molecule counting. Both raw records and analysed data, consisting in a list of space-time single-molecule coordinates, are shared. Additionally, Matlab functions are provided that analyse the single-molecule coordinates in order to quantify features of individual particles. These data might constitute a valuable reference for applications of similar quantitative imaging methodologies to other types of functionalized nanomaterials.

Super‐resolution optical fluctuation imaging (SOFI) is a super‐resolution microscopy technique that overcomes the diffraction limit by analyzing intensity fluctuations of statistically independent emitters in a time series of images. The final images are background‐free and show confocality and enhanced spatial resolution (super‐resolution). Fluorophore photobleaching, however, is a key limitation for recording long time series of images that will allow for the calculation of higher order SOFI results with correspondingly increased resolution. Here, we demonstrate that photobleaching can be circumvented by using fluorophore labels that reversibly and transiently bind to a target, and which are being replenished from a buffer which serves as a reservoir. Using fluorophore‐labeled short DNA oligonucleotides, we labeled cellular structures with target‐specific antibodies that contain complementary DNA sequences and record the fluctuation events caused by transient emitter binding. We show that this concept bypasses extensive photobleaching and facilitates two‐color imaging of cellular structures with SOFI. [ABSTRACT FROM AUTHOR]

Super-resolution microscopy has revolutionised the way we observe biological systems. These methods are now a staple of fluorescence microscopy. Researchers have used super-resolution methods in myriad systems to extract nanoscale spatial information on multiple interacting parts. These methods are continually being extended and reimagined to further push their resolving power and achieve truly single protein resolution. Here, we explore the most recent advances at the frontier of the ‘super-resolution’ limit and what opportunities remain for further improvements in the near future. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Die Visualisierung funktionaler Interaktionen von Biomolekülen wie Proteinen und Nukleinsäuren ist der Schlüssel zum Verständnis zellulären Lebens auf molekularer Ebene. Räumliche Nähe wird oft stellvertretend für die direkte Interaktion von Biomolekülen angesehen. Aktuelle Techniken zur Visualisierung räumlicher Nähe sind jedoch entweder durch die erreichbare Auflösung, den dynamischen Bereich oder einen Mangel an Einzelmolekülsensitivität begrenzt. Wir haben Proximity‐PAINT (pPAINT) entwickelt, eine Variante des superauflösenden Mikroskopieverfahrens DNA‐PAINT. pPAINT verwendet eine geteilte DNA‐PAINT‐Bindestelle, um räumliche Nähe mit hoher Empfindlichkeit, wenig Fehlern und präzise einstellbaren Detektionsabständen zu erkennen. Wir testen und optimieren pPAINT mit Hilfe von DNA‐Nanostrukturen und demonstrieren die zelluläre Anwendbarkeit, indem wir die räumliche Nähe von Alpha‐ und Beta‐Tubulin in Mikrotubuli in Zellen superaufgelöst visualisieren. [ABSTRACT FROM AUTHOR]

In DNA points accumulation in nanoscale topography (DNA‐PAINT), capable of single‐molecule localization microscopy with sub‐10‐nm resolution, the high background stemming from the unbound fluorescent probes in solution limits the imaging speed and throughput. Herein, we reductively cage the fluorescent DNA probes conjugated with a cyanine dye to hydrocyanine, acting as a photoactivatable dark state. The additional dark state from caging lowered the fluorescent background while enabling optically selective activation by total internal reflection (TIR) illumination at 405 nm. These benefits from "reductive caging" helped to increase the localization density or the imaging speed while preserving the image quality. With the aid of high‐density analysis, we could further increase the imaging speed of conventional DNA‐PAINT by two orders of magnitude, making DNA‐PAINT capable of high‐throughput super‐resolution imaging. [ABSTRACT FROM AUTHOR]

Abstract Three-dimensional (3D) reconstruction of thick samples using superresolution fluorescence microscopy remains challenging due to high level of background noise and fast photobleaching of fluorescence probes. We develop superresolution fluorescence microscopy that can reconstruct 3D structures of thick samples with both high localization accuracy and no photobleaching problem. The background noise is reduced by optically sectioning the sample using line-scan confocal microscopy, and the photobleaching problem is overcome by using the DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography). As demonstrations, we take 3D superresolution images of microtubules of a whole cell, and two-color 3D images of microtubules and mitochondria. We also present superresolution images of chemical synapse of a mouse brain section at different z-positions ranging from 0 μm to 100 μm.

Abstract Super-resolution fluorescence microscopy in the current form is hard to be used to image the neural connectivity of thick tissue samples due to problems such as slow imaging speed, severe photobleaching of fluorescent probes, and high background noise. Recently developed DNA-PAINT solved the photobleaching problem, but its imaging speed is extremely low. We report accelerated super-resolution fluorescence microscopy named FRET-PAINT. Compared to conventional DNA-PAINT, the imaging speed of the microscopy increases up to ~30-fold. As demonstrations, we show that 25-50 second imaging time is long enough to provide super-resolution reconstruction of microtubules and mitochondria of COS-7 cells.

• Single Molecule Localization Microscopy (SMLM) resolves the nanoscale arrangement of cellular structures. • Optimized procedures are required to obtain good SMLM data and make the most of the technique. • We provide detailed procedures and tips for SMLM sample preparation, imaging and processing. Super-resolution microscopy has profoundly transformed how we study the architecture of cells, revealing unknown structures and refining our view of cellular assemblies. Among the various techniques, the resolution of Single Molecule Localization Microscopy (SMLM) can reach the size of macromolecular complexes and offer key insights on their nanoscale arrangement in situ. SMLM is thus a demanding technique and taking advantage of its full potential requires specifically optimized procedures. Here we describe how we perform the successive steps of an SMLM workflow, focusing on single-color Stochastic Optical Reconstruction Microscopy (STORM) as well as multicolor DNA Points Accumulation for imaging in Nanoscale Topography (DNA-PAINT) of fixed samples. We provide detailed procedures for careful sample fixation and immunostaining of typical cellular structures: cytoskeleton, clathrin-coated pits, and organelles. We then offer guidelines for optimal imaging and processing of SMLM data in order to optimize reconstruction quality and avoid the generation of artifacts. We hope that the tips and tricks we discovered over the years and detail here will be useful for researchers looking to make the best possible SMLM images, a pre-requisite for meaningful biological discovery. [ABSTRACT FROM AUTHOR]

• Routine testing and calibration are essential for high quality super-resolution imaging. • We present a number of time and cost effective DNA-PAINT based test samples that have good stability. • We demonstrate procedures for validating SMLM 3D system performance. • We describe a procedure to establish and test quantitative analysis of DNA-PAINT data. Assessment of the imaging quality in localisation-based super-resolution techniques relies on an accurate characterisation of the imaging setup and analysis procedures. Test samples can provide regular feedback on system performance and facilitate the implementation of new methods. While multiple test samples for regular, 2D imaging are available, they are not common for more specialised imaging modes. Here, we analyse robust test samples for 3D and quantitative super-resolution imaging, which are straightforward to use, are time- and cost-effective and do not require experience beyond basic laboratory and imaging skills. We present two options for assessment of 3D imaging quality, the use of microspheres functionalised for DNA-PAINT and a commercial DNA origami sample. A method to establish and assess a qPAINT workflow for quantitative imaging is demonstrated with a second, commercially available DNA origami sample. [ABSTRACT FROM AUTHOR]

Photobleaching is a major challenge in fluorescence microscopy, in particular if high excitation light intensities are used. Signal‐to‐noise and spatial resolution may be compromised, which limits the amount of information that can be extracted from an image. Photobleaching can be bypassed by using exchangeable labels, which transiently bind to and dissociate from a target, thereby replenishing the destroyed labels with intact ones from a reservoir. Here, we demonstrate confocal and STED microscopy with short, fluorophore‐labeled oligonucleotides that transiently bind to complementary oligonucleotides attached to protein‐specific antibodies. The constant exchange of fluorophore labels in DNA‐based STED imaging bypasses photobleaching that occurs with covalent labels. We show that this concept is suitable for targeted, two‐color STED imaging of whole cells. [ABSTRACT FROM AUTHOR]

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 super‐resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC proteins is thus far elusive. Herein, we combine genetically‐encoded self‐labeling enzymes such as SNAP‐tag and HaloTag with DNA‐PAINT microscopy. We resolve single copies of nucleoporins in the human Y‐complex in three dimensions with a precision of circa 3 nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy. [ABSTRACT FROM AUTHOR]

To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution. Using SUM-PAINT, we generated 30-plex single-molecule resolved datasets in neurons and adapted omics-inspired analysis for data exploration. This allowed us to reveal the complexity of synaptic heterogeneity, leading to the discovery of a distinct synapse type. We not only provide a resource for researchers, but also an integrated acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution. [Display omitted] • Development of SUM-PAINT enables high-throughput DNA-PAINT multiplexing • 30-plex, 3D neuron atlas at single-protein resolution • AI-guided analysis enables evaluation of high-dimensional SUM-PAINT datasets • Discovery of a new synapse subtype characterized by VGlut1+ and Gephyrin+ Development of SUM-PAINT, an approach that is capable of potentially unlimited super-resolution multiplexing, enables rapid visualization of distinct synapse types at single-molecule resolution. [ABSTRACT FROM AUTHOR]

Mapping 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 method for rapid multiplexed super-resolution microscopy that can, in principle, be applied to 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 versatility of FLASH-PAINT with four applications: mapping nine proteins in a single mammalian cell, elucidating the functional organization of primary cilia by nine-target imaging, revealing the changes in proximity of thirteen different targets in unperturbed and dissociated Golgi stacks, and investigating and quantifying inter-organelle contacts at 3D super-resolution. [Display omitted] • FLASH-PAINT enables highly multiplexed super-resolution imaging • Transient adapters and erasers allow for fast, efficient, and gentle label exchange • Multiplexed super-resolution imaging reveals complex cilia and Golgi organization • 3D FLASH-PAINT allows for quantification of organelle contact site numbers and areas Development of FLASH-PAINT enables rapid and efficient visualization of cellular organelles, using a potentially unlimited number of labels. [ABSTRACT FROM AUTHOR]

Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore's response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as "frequency multiplexed DNA-PAINT," or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure. [ABSTRACT FROM AUTHOR]

The optical super-resolution technique DNA-PAINT (Point Accumulation Imaging in Nanoscale Topography) provides a flexible way to achieve imaging of nanoscale structures at ∼10-nanometer resolution. In DNA-PAINT, fluorescently labeled DNA “imager” strands bind transiently and with high specificity to complementary target “docking” strands anchored to the structure of interest. The localization of single binding events enables the assembly of a super-resolution image, and this approach effectively circumvents photobleaching. The solution exchange of imager strands is the basis of Exchange-PAINT, which enables multiplexed imaging that avoids chromatic aberrations. Fluid exchange during imaging typically requires specialized chambers or washes, which can disturb the sample. Additionally, diffusional washout of imager strands is slow in thick samples such as biological tissue slices. Here, we introduce Quencher-Exchange-PAINT—a new approach to Exchange-PAINT in regular open-top imaging chambers—which overcomes the comparatively slow imager strand switching via diffusional imager washout. Quencher-Exchange-PAINT uses “quencher” strands, i.e., oligonucleotides that prevent the imager from binding to the targets, to rapidly reduce unwanted single-stranded imager concentrations to negligible levels, decoupled from the absolute imager concentration. The quencher strands contain an effective dye quencher that reduces the fluorescence of quenched imager strands to negligible levels. We characterized Quencher-Exchange-PAINT when applied to synthetic, cellular, and thick tissue samples. Quencher-Exchange-PAINT opens the way for efficient multiplexed imaging of complex nanostructures, e.g., in thick tissues, without the need for washing steps. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) expression profiles hold promise as biomarkers for diagnostics and prognosis of complex diseases. Herein, we report a super‐resolution fluorescence imaging‐based digital profiling method for specific, sensitive, and multiplexed detection of miRNAs. In particular, we applied the DNA‐PAINT (point accumulation for imaging in nanoscale topography) method to implement a super‐resolution geometric barcoding scheme for multiplexed single‐molecule miRNA capture and digital counting. Using synthetic DNA nanostructures as a programmable miRNA capture nano‐array, we demonstrated high‐specificity (single nucleotide mismatch discrimination), multiplexed (8‐plex, 2 panels), and sensitive measurements on synthetic miRNA samples, as well as applied one 8‐plex panel to measure endogenous miRNAs levels in total RNA extract from HeLa cells. PAINT by numbers: A super‐resolution fluorescence imaging‐based digital profiling method for specific, sensitive, and multiplexed detection of miRNAs is presented. Specifically, the DNA‐PAINT method was applied to implement a super‐resolution geometric barcoding scheme for multiplexed single‐molecule miRNA capture and digital counting. [ABSTRACT FROM AUTHOR]

MicroRNA (miRNA) expression profiles hold promise as biomarkers for diagnostics and prognosis of complex diseases. Herein, we report a super‐resolution fluorescence imaging‐based digital profiling method for specific, sensitive, and multiplexed detection of miRNAs. In particular, we applied the DNA‐PAINT (point accumulation for imaging in nanoscale topography) method to implement a super‐resolution geometric barcoding scheme for multiplexed single‐molecule miRNA capture and digital counting. Using synthetic DNA nanostructures as a programmable miRNA capture nano‐array, we demonstrated high‐specificity (single nucleotide mismatch discrimination), multiplexed (8‐plex, 2 panels), and sensitive measurements on synthetic miRNA samples, as well as applied one 8‐plex panel to measure endogenous miRNAs levels in total RNA extract from HeLa cells. [ABSTRACT FROM AUTHOR]

Abstract: 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. [ABSTRACT FROM AUTHOR]

Abstract: Superauflösende Mikroskopie ermöglicht Bildgebung weit unterhalb des klassischen Beugungslimits von Licht. Technisch gesehen erreichen aktuelle Methoden sogar eine räumliche Auflösung auf Einzelmolekülebene. Dieser beeindruckende Erfolg kann jedoch nicht einfach auf die Bildgebung von zellulären Komponenten übertragen werden, da Sonden, die zur Markierung von Molekülen verwendet werden, entweder durch deren Größe (Antikörper) oder wegen begrenzter Verfügbarkeit limitiert sind (Nanokörper, Aptamere, genetisch‐kodierende Marker). In dieser Arbeit kombinieren wir kürzlich entwickelte Affimer‐Reagenzien mit einer ortsspezifischen DNA‐Funktionalisierung, um eine Färbung mit anschließender Bildgebung durch DNA‐PAINT zu erreichen. Hierfür benutzen wir ein Aktin‐bindendes Affimer. Diese kleinen, mit DNA konjugierten Affinitätsmarker könnten eine zukünftige Lösung für superauflösende Bildgebung mit hoher Multiplexkapazität darstellen. [ABSTRACT FROM AUTHOR]

We present a simple and versatile single-molecule-based method for the accurate determination of binding rates to surfaces or surface bound receptors. To quantify the reversible surface attachment of fluorescently labeled molecules, we have modified previous schemes for fluorescence correlation spectroscopy with total internal reflection illumination (TIR-FCS) and camera-based detection. In contrast to most modern applications of TIR-FCS, we completely disregard spatial information in the lateral direction. Instead, we perform correlation analysis on a spatially integrated signal, effectively converting the illuminated surface area into the measurement volume. In addition to providing a high surface selectivity, our new approach resolves association and dissociation rates in equilibrium over a wide range of time scales. We chose the transient hybridization of fluorescently labeled single-stranded DNA to the complementary handles of surface-immobilized DNA origami structures as a reliable and well-characterized test system. We varied the number of base pairs in the duplex, yielding different binding times in the range of hundreds of milliseconds to tens of seconds, allowing us to quantify the respective surface affinities and binding rates. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are considered to be important biomarkers for the diagnosis of numerous diseases and basic biomedical research. Therefore, a novel rapid diagnostic technique with high sensitivity and specificity is essential for miRNAs detection in clinical trials. Herein, we proposed a sensing platform based on CRISPR/Cas13a and DNA point accumulation in nanoscale topology (DNA-PAINT) to enable an ultra-sensitive analysis of miRNA at the single-molecule level. In this platform, mismatch bases are artificially introduced into the crRNA spacer region, which reduces the mismatch tolerance of the Cas13a/crRNA complex, thus providing high specificity for miRNA detection. Furthermore, signal amplification can be achieved by the dynamic repetitive process of binding and unbinding between fluorescent probes and capture probes in DNA-PAINT imaging, with a detection limit (LOD) as low as 1.12 fM. Convincingly, the practical application capability of the platform for miRNA quantification in complex biological samples is validated. More importantly, given the flexible design of the crRNA spacer region sequence, the strategy could be easily applied to the rapid detection of other miRNAs with excellent reliability, sensitivity and specificity, which has great potential for early diagnosis of miRNA-related diseases. [Display omitted] • The combination of CRISPR and DNA-PAINT can detect miRNAs with high sensitivity and specificity. • Repeated 'binding-unbinding' event based on DNA-PAINT provide high sensitivity. • Both the high fidelity of Cas13a and the programmability of crRNA provided high specificity. • The detection platform has the ability to detect complex biological samples. [ABSTRACT FROM AUTHOR]