Imaging In Nanoscale Topography Research Articles

In Situ Imaging of Proteins Using DNA-PAINT Super-Resolution Microscopy.

The spatial resolution of conventional light microscopy is restricted by the diffraction limit to hundreds of nanometers. Super-resolution microscopy enables single digit nanometer resolution by circumventing the diffraction limit of conventional light microscopy. DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) belongs to the family of single-molecule localization super-resolution approaches. Unique features of DNA-PAINT are that it allows for sub-nanometer resolution, spectrally unlimited multiplexing, proximity detection, and quantitative counting of target molecules. Here, we describe prerequisites for efficient DNA-PAINT microscopy.

Fluorescence Super-Resolution Imaging Chip for Gene Silencing Exosomes.

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.

PCNA as Protein-Based Nanoruler for Sub-10nm Fluorescence Imaging.

Super-resolution microscopy has revolutionized biological imaging enabling direct insight into cellular structures and protein arrangements with so far unmatched spatial resolution. Today, refined single-molecule localization microscopy methods achieve spatial resolutions in the one-digit nanometer range. As the race for molecular resolution fluorescence imaging with visible light continues, reliable biologically compatible reference structures will become essential to validate the resolution power. Here, PicoRulers (protein-based imaging calibration optical rulers), multilabeled oligomeric proteins designed as advanced molecular nanorulers for super-resolution fluorescence imaging are introduced. Genetic code expansion (GCE) is used to site-specifically incorporate three noncanonical amino acids (ncAAs) into the homotrimeric proliferating cell nuclear antigen (PCNA) at 6nm distances. Bioorthogonal click labeling with tetrazine-dyes and tetrazine-functionalized oligonucleotides allows efficient labeling of the PicoRuler with minimal linkage error. Time-resolved photoswitching fingerprint analysis is used to demonstrate the successful synthesis and DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is used to resolve 6nm PCNA PicoRulers. Since PicoRulers maintain their structural integrity under cellular conditions they represent ideal molecular nanorulers for benchmarking the performance of super-resolution imaging techniques, particularly in complex biological environments.

Assessing crosstalk in simultaneous multicolor single-molecule localization microscopy

Single-molecule localization microscopy (SMLM) can reach sub-50nm resolution using techniques such as stochastic optical reconstruction microscopy (STORM) or DNA-point accumulation for imaging in nanoscale topography (PAINT). Here we implement two approaches for faster multicolor SMLM by splitting the emitted fluorescence toward two cameras: simultaneous two-color DNA-PAINT (S2C-DNA-PAINT) that images spectrally separated red and far-red imager strands on each camera, and spectral demixing dSTORM (SD-dSTORM) where spectrally close far-red fluorophores appear on both cameras before being identified by demixing. Using S2C-DNA-PAINT as a reference for low crosstalk, we evaluate SD-dSTORM crosstalk using three types of samples: DNA origami nanorulers of different sizes, single-target labeled cells, or cells labeled for multiple targets. We then assess if crosstalk can affect the detection of biologically relevant subdiffraction patterns. Extending these approaches to three-dimensional acquisition and SD-dSTORM to three-color imaging, we show that spectral demixing is an attractive option for robust and versatile multicolor SMLM investigations.

Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes.

NMDA receptors (NMDARs) are ionotropic glutamate receptors that play a key role in excitatory neurotransmission. The number and subtype of surface NMDARs are regulated at several levels, including their externalization, internalization, and lateral diffusion between the synaptic and extrasynaptic regions. Here, we used novel anti-GFP (green fluorescent protein) nanobodies conjugated to either the smallest commercially available quantum dot 525 (QD525) or the several nanometer larger (and thus brighter) QD605 (referred to as nanoGFP-QD525 and nanoGFP-QD605, respectively). Targeting the yellow fluorescent protein-tagged GluN1 subunit in rat hippocampal neurons, we compared these two probes to a previously established larger probe, a rabbit anti-GFP IgG together with a secondary IgG conjugated to QD605 (referred to as antiGFP-QD605). The nanoGFP-based probes allowed faster lateral diffusion of the NMDARs, with several-fold increased median values of the diffusion coefficient (D). Using thresholded tdTomato-Homer1c signals to mark synaptic regions, we found that the nanoprobe-based D values sharply increased at distances over 100 nm from the synaptic edge, while D values for antiGFP-QD605 probe remained unchanged up to a 400 nm distance. Using the nanoGFP-QD605 probe in hippocampal neurons expressing the GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits, we detected subunit-dependent differences in the synaptic localization of NMDARs, D value, synaptic residence time, and synaptic-extrasynaptic exchange rate. Finally, we confirmed the applicability of the nanoGFP-QD605 probe to study differences in the distribution of synaptic NMDARs by comparing to data obtained with nanoGFPs conjugated to organic fluorophores, using universal point accumulation imaging in nanoscale topography and direct stochastic optical reconstruction microscopy.SIGNIFICANCE STATEMENT Our study systematically compared the localization and mobility of surface NMDARs containing GFP-GluN2A, GFP-GluN2B, or GFP-GluN3A subunits expressed in rodent hippocampal neurons, using anti-green fluorescent protein (GFP) nanobodies conjugated to the quantum dot 605 (nanoGFP-QD605), as well as nanoGFP probes conjugated with small organic fluorophores. Our comprehensive analysis showed that the method used to delineate the synaptic region plays an important role in the study of synaptic and extrasynaptic pools of NMDARs. In addition, we showed that the nanoGFP-QD605 probe has optimal parameters for studying the mobility of NMDARs because of its high localization accuracy comparable to direct stochastic optical reconstruction microscopy and longer scan time compared with universal point accumulation imaging in nanoscale topography. The developed approaches are readily applicable to the study of any GFP-labeled membrane receptors expressed in mammalian neurons.

Super-Resolved FRET Imaging by Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy.

Fluorescence Resonance Energy Transfer (FRET)-basedapproaches 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.

Peptide-PAINT Using a Transfected-Docker Enables Live- and Fixed-Cell Super-Resolution Imaging.

Point accumulation for imaging in nanoscale topography (PAINT) is a single-molecule technique for super-resolution microscopy, which uses exchangeable single stranded DNA oligos or peptide-pairs to create blinking phenomenon and achieves ≈5-25 nanometer resolution. Here, it is shown that by transfecting the protein-of-interest with a docker-coil, rather than by adding the docker externally-as is the norm when using DNA tethers or antibodies as dockers-similar localization can be achieved, ≈10nm. However, using a transfected docker has several experimental advances and simplifications. Most importantly, it allows Peptide-PAINT to be applied to transfected live cells for imaging surface proteins in mammalian cells and neurons under physiological conditions. The enhanced resolution of Peptide-PAINT is also shown for organelles in fixed cells to unravel structural details including ≈40-nm and ≈60-nm axial repeats in vimentin filaments in the cytoplasm, and fiber shapes of sub-100-nm histone-rich regions in the nucleus.

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

Single-molecule based super-resolution fluorescence microscopy offers an approach to sub-diffraction-limited imaging of biological samples. While these approaches have proved significant to understanding subcellular structures, numerous super-resolution systems suffer from drawbacks such as decreased localization precision due to high background fluorescence, lack of control of the extracellular environment, decreased multi-target accuracy due to chromatic offsets in multicolor imaging, and the inability to image structures in three dimensions (3D). Light sheet fluorescence microscopy, which involves illuminating the sample with a thin sheet of light, offers a gentle illumination scheme that greatly reduces photobleaching, photodamage, and out of focus background fluorescence, thereby improving the localization precision. Here, we present a single-objective tilted light sheet super-resolution setup with five added innovations: (i) a microfluidic chip with reflective sidewalls and transparent top, (ii) sequential DNA Point Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) known as Exchange-PAINT, (iii) deep learning for high density single emitter localization, (iv) double helix point spread functions for 3D imaging, and (iv) galvanometric dithering of the illumination beam to reduce scattering artifacts and provide homogenous sample illumination. By utilizing light sheet illumination in conjunction with Exchange-PAINT and deep learning, this setup allows for the use of high concentrations of imager strands given that background fluorescence is drastically reduced and sparse emitters are no longer necessary for accurate localization. Furthermore, sequential washes of imager strands are enabled through the use of microfluidics, which allows for accurate, multi-target reconstructions that are not subject to chromatic offsets. Thus, we demonstrate a setup that offers improved localization precision, extracellular environment control, multi-target information, and 3D imaging that can easily be implemented for fast, accurate, and precise multi-target super-resolution whole cell imaging.

Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy.

The specific and covalent labeling of the protein HaloTag with fluorescent probes in living cells makes it a powerful tool for bioimaging. However, the irreversible attachment of the probe to HaloTag precludes imaging applications that require transient binding of the probe and comes with the risk of irreversible photobleaching. Here, we introduce exchangeable ligands for fluorescence labeling of HaloTag (xHTLs) that reversibly bind to HaloTag and that can be coupled to rhodamines of different colors. In stimulated emission depletion (STED) microscopy, probe exchange of xHTLs allows imaging with reduced photobleaching as compared to covalent HaloTag labeling. Transient binding of fluorogenic xHTLs to HaloTag fusion proteins enables points accumulation for imaging in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT and STED microscopy. xHTLs thus open up new possibilities in imaging across microscopy platforms for a widely used labeling approach.

Optimized Coiled-Coil Interactions for Multiplexed Peptide-PAINT.

Super-resolution microscopy has revolutionized how researchers characterize samples in the life sciences in the last decades. Amongst methods employing single-molecule localization microscopy, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a relatively easy-to-implement method that uses the programmable and repetitive binding of dye-labeled DNA imager strands to their respective docking strands. Recently developed Peptide-PAINT replaces the interaction of oligonucleotides by short coiled-coil peptide sequences leading to an improved labeling scheme by reducing linkage errors to target proteins. However, only one coiled-coil pair is currently available for Peptide-PAINT, preventing multiplexed imaging. In this study, the initial Peptide-PAINT E/K coil is improved by modifying its length for optimized binding kinetics leading to improved localization precisions. Additionally, an orthogonal P3/P4 coil pair is introduced, enabling 2-plex Peptide-PAINT imaging and benchmarking its performance and orthogonality using single-molecule and DNA origami assays. Finally, the P3/P4 peptide pair is used to image the human epidermal growth factor receptors 2 (ErbB2/Her2) in 2D and 3D at the single receptor level using genetically encoded peptide tags.

Beyond DNA: new probes for PAINT super-resolution microscopy.

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.

Ultrasensitive and multiplexed miRNA detection system with DNA-PAINT

MiRNAs hold great potential as biomarkers for the early detection and monitoring of diseases based on their differential expression profiles. Therefore, the sensitive, specific and accurate detection of miRNAs represents an emerging new tool to improve diagnosis and treatment of several diseases, cancer in particular. DNA origami-based miRNA detection is particularly advantageous as it allows to incorporate multiple attachment sites to capture different target miRNAs at the nanoscale. In this work, we present a DNA origami nanoarray system providing distance-dependent recognition of miRNAs by applying super-resolution microscopy technique; DNA-PAINT (point accumulation for imaging in nanoscale topography). The sensor can detect up to 4 miRNAs either separately or in combination based on the relative distance to the boundary markers on the structure using a single imager strand. The detection is highly sensitive, with a limit of detection down to the low femtomolar range (11 fM - 388 fM) and has a large dynamic range up to 10 nM without need for amplification. Moreover, our detection system can discriminate single base mismatches with low false positive rates. Using our strategy, we demonstrate the detection of endogenous miRNAs from cell extracts of cancer cell lines and plasma from breast cancer patients. Overall, we developed an ultrasensitive and amplification-free, DNA-PAINT imaging-based miRNA detection method using DNA origami nanoarray system for the detection of breast-cancer associated miRNAs which potentially provides a sensitive and accurate alternative to the current multiplexed diagnostic technologies.

Spectrally PAINTing a Single Chain Polymeric Nanoparticle at Super-Resolution.

Folding a polymer chain into a well-defined single-chain polymeric nanoparticle (SCPN) is a fascinating approach to obtaining structured and functional nanoparticles. Like all polymeric materials, SCPNs are heterogeneous in their nature due to the polydispersity of their synthesis: the stochastic synthesis of polymer backbone length and stochastic functionalization with hydrophobic and hydrophilic pendant groups make structural diversity inevitable. Therefore, in a single batch of SCPNs, nanoparticles with different physicochemical properties are present, posing a great challenge to their characterization at a single-particle level. The development of techniques that can elucidate differences between SCPNs at a single-particle level is imperative to capture their potential applications in different fields such as catalysis and drug delivery. Here, a Nile Red based spectral point accumulation for imaging in nanoscale topography (NR-sPAINT) super-resolution fluorescence technique was implemented for the study of SCPNs at a single-particle level. This innovative method allowed us to (i) map the small-molecule binding rates on individual SCPNs and (ii) map the polarity of individual SCPNs for the first time. The SCPN designs used here have the same polymeric backbone but differ in the number of hydrophobic groups. The experimental results show notable interparticle differences 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 thus far remained hidden by ensemble techniques. The results indicate that the addition of hydrophobic pendant groups is vital to determine binding properties and induces single-particle polarity diversity. Overall, NR-sPAINT represents a powerful approach to quantifying the single-particle polarity of SCPNs and paves the way to relate the structural heterogeneity to functionality at the single-particle level. This provides an important step toward the aim of rationally designing SCPNs for the desired application.

In Situ Super-Resolution Imaging of Telomeres with DNA-PAINT

Telomeres are located at the ends of chromosomes and play an important role in maintaining the integrity of chromosomes and controlling the cycle of cell division. Studies have shown that abnormal telomere length may lead to the occurrence of some diseases. Therefore, accurate measurement of telomere length will be helpful for the prediction and diagnosis of related diseases. DNA point accumulation for imaging in nanoscale topography (PAINT) is an optical super-resolution technology that relies on the instantaneous binding of the fluorescent DNA imaging strand to the target epitope. Here, we present the first demonstration of DNA-PAINT-based in situ super-resolution imaging of telomeres as well as centromeres. For DNA-PAINT imaging, Cy5-labeled telomere DNA (5'-Cy5-TTTTTCCCTAACCCTAA-3') and Cy3-labeled centromere DNA (5'-Cy3-TTTTTAGCTTCTGTCTAGTTT-3') are utilized as the imager strands. Through an improved permeabilization strategy that we proposed, the imager strands can bind with intracellular telomeres and centromeres with high specificity, realizing super-resolution imaging of telomeres and centromeres. To check the applicability of DNA-PAINT in evaluating telomere length, we conducted an experiment using azidothymidine (AZT)-treated tumor cells as the imaging target. The DNA-PAINT imaging results clearly revealed the telomerase inhibition effect of AZT. Compared with single-molecule localization microscopy (SMLM) with peptide nucleic acid (PNA)-based fluorescence in situ hybridization (FISH), our method has the advantages of low cost, low toxicity, and simple equipment. Such a DNA-PAINT-based imaging strategy holds great potential in measuring telomere length with high accuracy, which would play an important role in the study of telomere-related diseases such as cancer.

Fluorogenic Dimers as Bright Switchable Probes for Enhanced Super-Resolution Imaging of Cell Membranes.

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.

Fast DNA-PAINT imaging using a deep neural network

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-PAINT Super-Resolution Imaging for Characterization of Nucleic Acid Nanostructures.

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.

The Role of Nanoscale Distribution of Fibronectin in the Adhesion of Staphylococcus aureus Studied by Protein Patterning and DNA-PAINT.

Staphylococcus aureus is a widespread and highly virulent pathogen that can cause superficial and invasive infections. Interactions between S. aureus surface receptors and the extracellular matrix protein fibronectin mediate the bacterial invasion of host cells and is implicated in the colonization of medical implant surfaces. In this study, we investigate the role of distribution of both fibronectin and cellular receptors on the adhesion of S. aureus to interfaces as a model for primary adhesion at tissue interfaces or biomaterials. We present fibronectin in patches of systematically varied size (100–1000 nm) in a background of protein and bacteria rejecting chemistry based on PLL-g-PEG and studied S. aureus adhesion under flow. We developed a single molecule imaging assay for localizing fibronectin binding receptors on the surface of S. aureus via the super-resolution DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) technique. Our results indicate that S. aureus adhesion to fibronectin biointerfaces is regulated by the size of available ligand patterns, with an adhesion threshold of 300 nm and larger. DNA-PAINT was used to visualize fibronectin binding receptor organization in situ at ∼7 nm localization precision and with a surface density of 38–46 μm–2, revealing that the engagement of two or more receptors is required for strong S. aureus adhesion to fibronectin biointerfaces.

Fluorogenic DNA-PAINT for faster, low-background super-resolution imaging.

DNA-based points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution microscopy method that can acquire high-fidelity images at nanometer resolution. It suffers, however, from high background and slow imaging speed, both of which can be attributed to the presence of unbound fluorophores in solution. Here we present two-color fluorogenic DNA-PAINT, which uses improved imager probe and docking strand designs to solve these problems. These self-quenching single-stranded DNA probes are conjugated with a fluorophore and quencher at the terminals, which permits an increase in fluorescence by up to 57-fold upon binding and unquenching. In addition, the engineering of base pair mismatches between the fluorogenic imager probes and docking strands allowed us to achieve both high fluorogenicity and the fast binding kinetics required for fast imaging. We demonstrate a 26-fold increase in imaging speed over regular DNA-PAINT and show that our new implementation enables three-dimensional super-resolution DNA-PAINT imaging without optical sectioning.

Site-specific nanobody-oligonucleotide conjugation for super-resolution imaging.

Camelid single-domain antibody fragments, also called nanobodies, constitute a class of binders that are small in size (~15 kDa) and possess antigen-binding properties similar to their antibody counterparts. Facile production of recombinant nanobodies in several microorganisms has made this class of binders attractive within the field of molecular imaging. Particularly, their use in super-resolution microscopy has improved the spatial resolution of molecular targets due to a smaller linkage error. In single-molecule localization microscopy techniques, the effective spatial resolution can be further enhanced by site-specific fluorescent labeling of nanobodies owing to a more homogeneous protein-to-fluorophore stoichiometry, reduced background staining and a known distance between dye and epitope. Here, we present a protocol for site-specific bioconjugation of DNA oligonucleotides to three distinct nanobodies expressed with an N- or C-terminal unnatural amino acid, 4-azido-L-phenylalanine (pAzF). Using copper-free click chemistry, the nanobody-oligonucleotide conjugation reactions were efficient and yielded highly pure bioconjugates. Target binding was retained in the bioconjugates, as demonstrated by bio-layer interferometry binding assays and the super-resolution microscopy technique, DNA points accumulation for imaging in nanoscale topography (PAINT). This method for site-specific protein-oligonucleotide conjugation can be further extended for applications within drug delivery and molecular targeting where site-specificity and stoichiometric control are required.