Imaging In Nanoscale Topography Research Articles

PAINT-ing Fluorenylmethoxycarbonyl (Fmoc)-Diphenylalanine Hydrogels.

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.

Super-Resolution Imaging of Self-Assembled Nanocarriers Using Quantitative Spectroscopic Analysis for Cluster Extraction.

Self-assembled nanocarriers have inspired a range of applications for bioimaging, diagnostics, and drug delivery. The noninvasive visualization and characterization of these nanocarriers are important to understand their structure to function relationship. However, the quantitative visualization of nanocarriers in the sample's native environment remains challenging with the use of existing technologies. Single-molecule localization microscopy (SMLM) has the potential to provide both high-resolution visualization and quantitative analysis of nanocarriers in their native environment. However, nonspecific binding of fluorescent probes used in SMLM can introduce artifacts, which imposes challenges in the quantitative analysis of SMLM images. We showed the feasibility of using spectroscopic point accumulation for imaging in nanoscale topography (sPAINT) to visualize self-assembled polymersomes (PS) with molecular specificity. Furthermore, we analyzed the unique spectral signatures of Nile Red (NR) molecules bound to the PS to reject artifacts from nonspecific NR bindings. We further developed quantitative spectroscopic analysis for cluster extraction (qSPACE) to increase the localization density by 4-fold compared to sPAINT; thus, reducing variations in PS size measurements to less than 5%. Finally, using qSPACE, we quantitatively imaged PS at various concentrations in aqueous solutions with ∼20 nm localization precision and 97% reduction in sample misidentification relative to conventional SMLM.

Super-resolved Measurement of Piconewton Receptor Forces via Tension-PAINT

Piconewton receptor forces exerted by cellular receptors regulate critical cellular processes, including development, migration, immune recognition, and hemostasis. Despite the vital role of mechanical forces in biology, no current technique can image cellular force organization with sub-100 nm resolution. Here, we present tension-PAINT, which leverages DNA-based molecular tension probes in combination with the stochastic super-resolution microscopy technique DNA-Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT), to enable super-resolved receptor tension measurements. DNA-PAINT relies on the transient binding of fluorophore-labeled oligonucleotide “imager” sequences to complementary “docking” sequences to produce the single-molecule fluorescence required for stochastic super-resolution imaging. We reasoned that encoding a cryptic docking sequence within DNA-based molecular tension probes would allow these probes to function like receptor force-triggered switches. These DNA based molecular force probes reveal a single-stranded DNA docking site only under the influence of pN receptor forces. The resulting technique, which we have named tension-PAINT, provides maps of receptor tension with up to 25 nm spatial resolution and pN force sensitivity. Here, we present variants of tension-PAINT capable of real-time super-resolved tension measurement in living cells, and of integrating cellular force history over time. We apply tension-PAINT to map the integrin forces of living platelets, revealing that platelets exert integrin mediated forces in a 100-200 nm wide spreading “ring” pattern at the lamellipodial edge. Additionally, we reveal the dynamics of fibroblast focal-adhesion and filopodial retraction forces. Due to its compatibility with widely available, unmodified fluorescence microscopes, we anticipate that tension-PAINT will provide a valuable, widely accessible tool to quantify the role of the nanoscale distribution of receptor forces in biological function.

Rational Design of Conformation-Specific Antibodies for Tau Oligomers

Antibodies are powerful tools for biological research and clinical applications owing to their ability to bind targets with high affinity and specificity. To exploit them against neurodegeneration, we are developing antibodies to target most toxic early stage aggregates of the protein tau, whose deposition in the brain is linked with Alzheimer's disease and other tauopathies. We generated a panel of single-domain antibodies targeting selected epitopes within tau, using the ‘cascade method’. Briefly, we designed complementary peptides targeting linear epitopes within the sequence of tau using a library of beta-sheet fragments from the protein data bank. We then grafted each peptide into the CDR3 loop of a VH human scaffold. Using this approach, we produced a library of fourteen antibodies covering systematically the length of tau. We will use these antibodies to understand which epitopes are specifically exposed in toxic tau aggregates. In particular, we will employ them in super-resolved imaging, such as DNA points accumulation for imaging in nanoscale topography (DNA PAINT), of protein aggregates. DNA PAINT enables visualization of structures with resolutions below the diffraction limit using short dye-labelled oligonucleotide probes. Transient binding of a dye-labelled ‘imager strand’ to a ‘docking strand’ (DS), which we conjugated to the C-terminus of DesAbs creates a blinking effect. Hence, a highly resolved image can be reconstructed from plotting the measured positions of the hydrolyzed DNA state. Using this technique to image protein aggregates in human cerebrospinal fluid and serum will provide novel insights into the composition, structure, size and number of aggregates present in those samples.

Axial plane single-molecule super-resolution microscopy of whole cells

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.

Toward Absolute Molecular Numbers in DNA-PAINT.

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.

Graphene-based metal-induced energy transfer for sub-nanometre optical localization

Single-molecule fluorescence imaging has become an indispensable tool for almost all fields of research, from fundamental physics to the life sciences. Among its most important applications is single-molecule localization super-resolution microscopy (SMLM) (for example, photoactivated localization microscopy (PALM)1, stochastic optical reconstruction microscopy (STORM)2, fluorescent PALM (fPALM)3, direct STORM (dSTORM)4 and point accumulation for imaging in nanoscale topography (PAINT)5), which uses the fact that the centre position of a single molecule’s image can be determined with much higher accuracy than the size of that image itself. However, a big challenge of SMLM is to achieve super-resolution along the third dimension as well. Recently, metal-induced energy transfer (MIET) was introduced to axially localize fluorescent emitters6–9. This exploits the energy transfer from an excited fluorophore to plasmons in a thin metal film. Here, we show that by using graphene as the ‘metal’ layer, one can increase the localization accuracy of MIET by nearly tenfold. We demonstrate this by axially localizing single emitters and by measuring the thickness of lipid bilayers with angstrom accuracy. Using graphene as the ‘metal’ layer can increase the localization accuracy of metal-induced energy transfer, enabling axial localization of single emitters and measurement of the thickness of lipid bilayers with angstrom accuracy.

Super-resolution imaging and estimation of protein copy numbers at single synapses with DNA-point accumulation for imaging in nanoscale topography.

.In the brain, the strength of each individual synapse is defined by the complement of proteins present or the “local proteome.” Activity-dependent changes in synaptic strength are the result of changes in this local proteome and posttranslational protein modifications. Although most synaptic proteins have been identified, we still know little about protein copy numbers in individual synapses and variations between synapses. We use DNA-point accumulation for imaging in nanoscale topography as a single-molecule super-resolution imaging technique to visualize and quantify protein copy numbers in single synapses. The imaging technique provides near-molecular spatial resolution, is unaffected by photobleaching, enables imaging of large field of views, and provides quantitative molecular information. We demonstrate these benefits by accessing copy numbers of surface AMPA-type receptors at single synapses of rat hippocampal neurons along dendritic segments.

The Accuracy of Determining Cluster Size by Analyzing Ripley’s K Function in Single Molecule Localization Microscopy

Ripley’s K function was developed to analyze the spatial distribution characteristics in point pattern analysis, including geography, economics and biomedical research. In biomedical applications, it is popularly used to analyze the clusters of proteins on the cell plasma membrane in single molecule localization microscopy (SMLM), such as photo activated localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), universal point accumulation imaging in nanoscale topography (uPAINT), etc. Here, by varying the parameters of the simulated clusters on a modeled SMLM image, the effects of cluster size, cluster separation and protein ratio inside/outside the cluster on the accuracy of cluster analysis by analyzing Ripley’s K function were studied. Although the predicted radius of clusters by analyzing Ripley’s K function did not exactly correspond to the actual radius, we suggest the cluster radius could be estimated within a factor of 1.3. Employing peak analysis methods to analyze the experimental epidermal growth factor receptor (EGFR) clusters at fibroblast-like cell lines derived from monkey kidney tissue - COS7 cell surface observed by uPAINT method, the cluster properties were characterized with errors. Our results present quantification of clusters and can be used to enhance the understanding of clusters in SMLM.

About samples, giving examples: Optimized Single Molecule Localization Microscopy.

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.

Bacterially Derived Antibody Binders as Small Adapters for DNA-PAINT Microscopy.

Current optical super-resolution implementations are capable of resolving features spaced just a few nanometers apart. However, translating this spatial resolution to cellular targets is limited by the large size of traditionally employed primary and secondary antibody reagents. Recent advancements in small and efficient protein binders for super-resolution microscopy, such as nanobodies or aptamers, provide an exciting avenue for the future; however, their widespread availability is still limited. To address this issue, here we report the combination of bacterial-derived binders commonly used in antibody purification with DNA-based point accumulation for imaging in nanoscale topography (DNA-PAINT) microscopy. The small sizes of these protein binders, relative to secondary antibodies, make them an attractive labeling alternative for emerging superresolution techniques. We present here a labeling protocol for DNA conjugation of bacterially derived proteins A and G for DNA-PAINT, having assayed their intracellular performance by targeting primary antibodies against tubulin, TOM20, and the epidermal growth factor receptor (EGFR) and quantified the increases in obtainable resolution.

Super-Resolution Geometric Barcoding for Multiplexed miRNA Profiling

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.

Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors.

DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20–25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.

Single Particle Tracking and Super-Resolution Imaging of Membrane-Assisted Stop-and-Go Diffusion and Lattice Assembly of DNA Origami.

DNA nanostructures offer the possibility to mimic functional biological membrane components due to their nanometer-precise shape configurability and versatile biochemical functionality. Here we show that the diffusional behavior of DNA nanostructures and their assembly into higher order membrane-bound lattices can be controlled in a stop-and-go manner and that the process can be monitored with super-resolution imaging. The DNA structures are transiently immobilized on glass-supported lipid bilayers by changing the mono- and divalent cation concentrations of the surrounding buffer. Using DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) super-resolution microscopy, we confirm the fixation of DNA origami structures with different shapes. On mica-supported lipid bilayers, in contrast, we observe residual movement. By increasing the concentration of NaCl and depleting MgCl2, a large fraction of DNA structures restarts to diffuse freely on both substrates. After addition of a set of oligonucleotides that enables three Y-shaped monomers to assemble into a three-legged shape (triskelion), the triskelions can be stopped and super-resolved. Exchanging buffer and adding another set of oligonucleotides triggers the triskelions to diffuse and assemble into hexagonal 2D lattices. This stop-and-go imaging technique provides a way to control and observe the diffusional behavior of DNA nanostructures on lipid membranes that could also lead to control of membrane-associated cargos.

Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy.

The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site.

Assembly and Application of DNA Origami-based Fluorescence Arrays

Organic fluorescent molecules with excellent optical properties such as small size and high brightness, are widely used in the field of bioimaging and target molecule detection. Compared to monodispersed fluorescent molecules, fluorescence arrays provide spatial controllability and quantitative programmability for patterning fluorescent molecules, which open new possibilities for expanding the applications of fluorescent molecules. The development of DNA origami techniques allows sub-nanometer addressing capabilities, which is the structural basis for organizing molecules at designed positions. DNA single strands form duplex through complementary pairing, which provides simplicity, efficiency and high orthogonality for construction and diverse programming of fluorescence arrays on DNA origami. Meanwhile, the flexible design of DNA origami provides abundant templates for one-dimensional, two-dimensional and three-dimensional fluorescence arrays of different shapes, making it widely used as a platform for building nanoscale spatial molecular arrays. The characterization of topography on DNA origami tremendously relies on atomic force microscopy (AFM). However, fluorescent molecules on DNA origami usually do not introduce significant topographical change, therefore, cannot be observed well using AFM. Recently developed DNA-PAINT (point accumulation for imaging in nanoscale topography) super-resolution imaging method is based on transient interactions between DNA oligos, which realize the characterization of nanoscale structures by fluorescent imaging. It is a new routine for the characterization of the fluorescence arrays on origami. The development of detectors such as EMCCD and CMOS also provides a higher time resolution for the detection of fluorescent signals, facilitating more accurate dynamic study of interaction between molecules In this review, we summarize recent research progress on the DNA origami-based fluorescence arrays. We introduced the design and assembly principle of DNA origami as template for patterning fluorescent dyes, and reviewed the existing strategies for the construction of fluorescence arrays on DNA origami with different shapes. Then we discribe the characterization techniques of DNA origami-based fluorescence arrays, focusing on recently developed super−resolution imaging. Furthermore, we introduced the application of DNA origami-based fluorescence arrays in bioimaging, biosensing and diagnostics. We highlighted the fundamental biochemical properties of the fluorescence arrays, such as designable topography, quantized fluorescence intensity and localized reaction and summarized the progress routes and challenges in the fundamental study and applications of DNA origami-based fluorescence arrays. Finally, we offered a perspective on the roadmap of DNA origami-based fluorescence arrays as follows: (1) the fixed relative positions of each fluorescent molecule in the array could provide information for mechanical tests. (2) Combination of fluoresecen arrays with biocomputing could also be an important application direction. (3) Due to the variety of modifications of DNA molecules, fluorescence arrays can be combined with other functional molecules to develop multifunctional fluorescence arrays for in - situ intelligent regulation while performing imaging.

Nanometer-scale Multiplexed Super-Resolution Imaging with an Economic 3D-DNA-PAINT Microscope.

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.

Super-resolution Geometric Barcoding for Multiplexed miRNA Profiling.

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.

Super‐resolution Geometric Barcoding for Multiplexed miRNA Profiling

AbstractMicroRNA (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.

Bacterial cell wall nanoimaging by autoblinking microscopy

Spurious blinking fluorescent spots are often seen in bacteria during single-molecule localization microscopy experiments. Although this ‘autoblinking’ phenomenon is widespread, its origin remains unclear. In Deinococcus strains, we observed particularly strong autoblinking at the periphery of the bacteria, facilitating its comprehensive characterization. A systematic evaluation of the contributions of different components of the sample environment to autoblinking levels and the in-depth analysis of the photophysical properties of autoblinking molecules indicate that the phenomenon results from transient binding of fluorophores originating mostly from the growth medium to the bacterial cell wall, which produces single-molecule fluorescence through a Point Accumulation for Imaging in Nanoscale Topography (PAINT) mechanism. Our data suggest that the autoblinking molecules preferentially bind to the plasma membrane of bacterial cells. Autoblinking microscopy was used to acquire nanoscale images of live, unlabeled D. radiodurans and could be combined with PALM imaging of PAmCherry-labeled bacteria in two-color experiments. Autoblinking-based super-resolved images provided insight into the formation of septa in dividing bacteria and revealed heterogeneities in the distribution and dynamics of autoblinking molecules within the cell wall.