DNA-PAINT Probe Modifications Support High-Resolution Imaging with Shorter Binding Domains.

Rational Design of Conformation-Specific Antibodies for Tau Oligomers

Multi-target 3D single-molecule super resolution imaging throughout mammalian cells with a single-objective tilted light sheet.

Analyzing cellular structures and the relative location of molecules is essential for addressing biological questions. Super-resolution microscopy techniques that bypass the light diffraction limit have become increasingly popular to study cellular molecule dynamics in situ. However, the application of super-resolution imaging techniques to detect small RNAs (sRNAs) is limited by the choice of proper fluorophores, autofluorescence of samples, and failure to multiplex. Here, we describe an sRNA-PAINT protocol for the detection of sRNAs at nanometer resolution. The method combines the specificity of locked nucleic acid probes and the low background, precise quantitation, and multiplexable characteristics of DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT). Using this method, we successfully located sRNA targets that are important for development in maize anthers at sub-20 nm resolution and quantitated their exact copy numbers. Graphic abstract: Multiplexed sRNA-PAINT. Multiple Vetting and Analysis of RNA for In Situ Hybridization (VARNISH) probes with different docking strands (i.e., a, b, …) will be hybridized to samples. The first probe will be imaged with the a* imager. The a* imager will be washed off with buffer C, and then the sample will be imaged with b* imager. The wash and image steps can be repeated sequentially for multiplexing.

The completion of the human genome sequencing project will greatly accelerate the development of novel and practical technologies for genome-analysis, diagnostics or therapeutics. Oligonucleotides are playing an important role in these genome technologies, because of their sequence-specific hybridization ability toward the complementary strand. Besides the sequence-specific duplex formation, oligonucleotides are able to form stable triplex structures, which is fundamental to the antigene strategy to regulate gene expression in a living cell. However, two major drawbacks are known in the triplex formation by a natural oligonucleotide: low stability of the triplex and limitations of the target DNA sequence. One promising strategy to overcome these problems is chemical modification of the oligonucleotides. We have developed various bridged nucleic acids (BNAs), and found that the oligonucleotides containing 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification form a stable parallel motif triplex with the double-stranded DNA target under physiological conditions. Some nucleobase analogues to extend the target DNA sequence were designed, synthesized and incorporated into the 2',4'-BNA structure. The obtained 2',4'-BNA derivatives containing modified nucleobases effectively recognized a pyrimidine-purine interruption. Some other examples of nucleic acid analogues to overcome the two major drawbacks in the triplex-forming oligonucleotides are also summarized.

Super-resolution fluorescence imaging based on single-molecule localization microscopy (SMLM) enables visualizing cellular structures with nanometric precision. However, its spatial and temporal resolution largely relies on the brightness of ON/OFF switchable fluorescent dyes. Moreover, in cell plasma membranes, the single-molecule localization is hampered by the fast lateral diffusion of membrane probes. Here, to address these two fundamental problems, we propose a concept of ON/OFF switchable probes for SMLM (points accumulation for imaging in nanoscale topography, PAINT) based on fluorogenic dimers of bright cyanine dyes. In these probes, the two cyanine units connected with a linker were modified at their extremities with low-affinity membrane anchors. Being self-quenched in water due to intramolecular dye H-aggregation, they displayed light up on reversible binding to lipid membranes. The charged group in the linker further decreased the probe affinity to the lipid membranes, thus accelerating its dynamic reversible ON/OFF switching. The concept was validated on cyanines 3 and 5. SMLM of live cells revealed that the new probes provided higher brightness and ∼10-fold slower diffusion at the cell surface, compared to reference probes Nile Red and DiD, which boosted axial localization precision >3-fold down to 31 nm. The new probe allowed unprecedented observation of nanoscale fibrous protrusions on plasma membranes of live cells with 40 s time resolution, revealing their fast dynamics. Thus, going beyond the brightness limit of single switchable dyes by cooperative dequenching in fluorogenic dimers and slowing down probe diffusion in biomembranes open the route to significant enhancement of super-resolution fluorescence microscopy of live cells.

Phalloidin-PAINT: Enhanced quantitative nanoscale imaging of F-actin

Fluorescence nanoscopy has become an indispensable tool for studying organelle structures, protein dynamics, and interactions in biological sciences. Single-molecule localization microscopy can now routinely achieve 10-50 nm resolution through fluorescently labeled specimens in lateral optical sections. However, visualizing structures organized along the axial direction demands scanning and imaging each of the lateral imaging planes with fine intervals throughout the whole cell. This iterative process suffers from photobleaching of tagged probes, is susceptible to alignment artifacts and also limits the imaging speed. Here, we focused on the axial plane super-resolution imaging which integrated the single-objective light-sheet illumination and axial plane optical imaging with single-molecule localization technique to resolve nanoscale cellular architectures along the axial (or depth) dimension without scanning. We demonstrated that this method is compatible with DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) and exchange-PAINT by virtue of its light-sheet illumination, allowing multiplexed super-resolution imaging throughout the depth of whole cells. We further demonstrated this proposed system by resolving the axial distributions of intracellular organelles such as microtubules, mitochondria, and nuclear pore complexes in both COS-7 cells and glioblastoma patient-derived tumor cells.

Supramolecular block copolymers are becoming attractive materials in nascent optoelectronic and catalytic technologies. However, their dynamic nature precludes the straightforward tuning and analysis of the polymer’s structure. Here we report the elucidation on the microstructure of triarylamine triamide-based supramolecular block copolymers through a comprehensive battery of spectroscopic, theoretical, and super-resolution microscopic techniques. Via spectroscopic analysis we demonstrate that the direct mixing of preassembled homopolymers and the copolymerization induced by slow cooling of monomers lead to the formation of the same copolymer’s architecture. The small but pronounced deviation of the experimental spectra from the linear combination of the homopolymers’ spectra hints at the formation of block copolymers. A mass balance model is introduced to further unravel the microstructure of the copolymers formed, and it confirms that stable multiblock supramolecular copolymers can be accessed from different routes. The multiblock structure of the supramolecular copolymers originates from the fine balance between favorable hydrogen-bonding interactions and a small mismatch penalty between two different monomers. Finally, we visualized the formation of the supramolecular block copolymers by adapting a recently developed super-resolution microscopy technique, interface point accumulation for imaging in nanoscale topography (iPAINT), for visualizing the architectures formed in organic media. Combining multiple techniques was crucial to unveil the microstructure of these complex dynamic supramolecular systems.

Folding a polymer chain into a well-defined single-chainpolymericnanoparticle (SCPN) is a fascinating approach to obtaining structuredand functional nanoparticles. Like all polymeric materials, SCPNsare heterogeneous in their nature due to the polydispersity of theirsynthesis: the stochastic synthesis of polymer backbone length andstochastic functionalization with hydrophobic and hydrophilic pendantgroups make structural diversity inevitable. Therefore, in a singlebatch of SCPNs, nanoparticles with different physicochemical propertiesare present, posing a great challenge to their characterization ata single-particle level. The development of techniques that can elucidatedifferences between SCPNs at a single-particle level is imperativeto capture their potential applications in different fields such ascatalysis and drug delivery. Here, a Nile Red based spectral pointaccumulation for imaging in nanoscale topography (NR-sPAINT) super-resolutionfluorescence technique was implemented for the study of SCPNs at asingle-particle level. This innovative method allowed us to (i) mapthe small-molecule binding rates on individual SCPNs and (ii) mapthe polarity of individual SCPNs for the first time. The SCPN designsused here have the same polymeric backbone but differ in the numberof hydrophobic groups. The experimental results show notable interparticledifferences in the binding rates within the same polymer design. Moreover,a marked polarity shift between the different designs is observed.Interestingly, interparticle polarity heterogeneity was unveiled,as well as an intraparticle diversity, information which has thusfar remained hidden by ensemble techniques. The results indicate thatthe addition of hydrophobic pendant groups is vital to determine bindingproperties and induces single-particle polarity diversity. Overall,NR-sPAINT represents a powerful approach to quantifying the single-particlepolarity of SCPNs and paves the way to relate the structural heterogeneityto functionality at the single-particle level. This provides an importantstep toward the aim of rationally designing SCPNs for the desiredapplication.

DNA-PAINT (Point Accumulation for Imaging in Nanoscale Topography) exploits the transient hybridization of a short fluorescent DNA imager strand to a complementary docking strand to achieve super-resolution imaging. However, its application is hampered by the background noise caused by unbound imager strands and the subsequent slow image acquisition. Several variants of DNA-PAINT have already been developed to successfully overcome these limitations. Here we design and validate a new alternative, based on intermolecular dark resonance energy transfer (DRET) and the use of a fluorenyl nucleobase (X) substitute incorporated into the imager strand, acting as a dark donor. Upon annealing, the fluorescence of the bright acceptor (ATTO 647N) bound to the docking strand is turned on via DRET with X. Single-molecule experiments showed that an 11 nt X-labeled imager strand provides appropriate hybridization rates with the ATTO 647N-labeled docking strand to perform DNA-PAINT. Thanks to their low background, high concentrations of imager strands can be used, facilitating the acquisition of super-resolved images of cellular microtubules within 30 s. This study provides the first proof-of-concept for this novel route to fast DNA-PAINT nanoscopy.

DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution technique with relatively easy-to-implement multi-target imaging. However, image acquisition is slow as sufficient statistical data has to be generated from spatio-temporally isolated single emitters. Here, we train the neural network (NN) DeepSTORM to predict fluorophore positions from high emitter density DNA-PAINT data. This achieves image acquisition in one minute. We demonstrate multi-colour super-resolution imaging of structure-conserved semi-thin neuronal tissue and imaging of large samples. This improvement can be integrated into any single-molecule imaging modality to enable fast single-molecule super-resolution microscopy.

DNA self-assembly of single molecules (i.e., dyes) with deterministic orientations is a powerful approach for engineering molecular excitons. However, current determination methods of the dye orientation relative to DNA do not account for the orientation of the dye plane, which is a missing degree of freedom needed to define absolute three-dimensional orientations. In this work, we combine fluorescence-detected linear dichroism, defocused dipole imaging, and DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) super-resolution microscopy to determine the absolute three-dimensional orientations of single Cy5 dyes relative to host DNA duplexes, which includes the dye plane orientation. The data revealed that the absorption and emission dipoles are perpendicular to the DNA duplex, and the mean dye plane is parallel to the DNA bases, which supports the notion that Cy5 dyes intercalate between DNA base pairs. The presented methodology will inspire investigation of the dye plane orientation for controlling dye arrangement configurations beyond spontaneous π-stacking between dyes as well as achieving novel dye-DNA arrangements.

In the last decade, point accumulation for imaging in nanoscale topography (PAINT) has emerged as a versatile tool for single-molecule localization microscopy (SMLM). Currently, DNA-PAINT is the most widely used, in which a transient stochastically binding DNA docking-imaging pair is used to reconstruct specific characteristics of biological or synthetic materials on a single-molecule level. Slowly, the need for PAINT probes that are not dependent on DNA has emerged. These probes can be based on (i) endogenous interactions, (ii) engineered binders, (iii) fusion proteins, or (iv) synthetic molecules and provide complementary applications for SMLM. Therefore, researchers have been expanding the PAINT toolbox with new probes. In this review, we provide an overview of the currently existing probes that go beyond DNA and their applications and challenges.

Self‐assembly of fluorenylmethoxycarbonyl‐protected diphenylalanine (FmocFF) in water is widely known to produce hydrogels. Typically, confocal microscopy is used to visualize such hydrogels under wet conditions, that is, without freezing or drying. However, key aspects of hydrogels like fiber diameter, network morphology and mesh size are sub‐diffraction limited features and cannot be visualized effectively using this approach. In this work, we show that it is possible to image FmocFF hydrogels by Points Accumulation for Imaging in Nanoscale Topography (PAINT) in native conditions and without direct gel labelling. We demonstrate that the fiber network can be visualized with improved resolution (≈50 nm) both in 2D and 3D. Quantitative information is extracted such as mesh size and fiber diameter. This method can complement the existing characterization tools for hydrogels and provide useful information supporting the design of new materials.

Tumor cell‐surface markers are usually overexpressed or mutated protein receptors for which spatiotemporal regulation differs between and within cancers. Single‐molecule fluorescence imaging can profile individual markers in different cellular contexts with molecular precision. However, standard single‐molecule imaging methods based on overexpressed genetically encoded tags or cumbersome probes can significantly alter the native state of receptors. We introduce a live‐cell points accumulation for imaging in nanoscale topography (PAINT) method that exploits aptamers as minimally invasive affinity probes. Localization and tracking of individual receptors are based on stochastic and transient binding between aptamers and their targets. We demonstrated single‐molecule imaging of a model tumor marker (EGFR) on a panel of living cancer cells. Affinity to EGFR was finely tuned by rational engineering of aptamer sequences to define receptor motion and/or native receptor density.