Name: Whole-cell Super-Resolution Imaging via DNA-PAINT on a Spinning Disk Confocal with Optical Photon Reassignment (Video) | JoVE Journal
Uploaded: 2026-01-06T13:00:00.000+00:00
Duration: 7 min 12 s
Description: Learn whole-cell super-resolution imaging via DNA-PAINT using a spinning disk confocal microscope with optical photon reassignment. This protocol enables deep SMLM without custom optics for neuroscience research.

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This research was funded by the Human Frontier Science Program Organization (HFSP) through a cross-disciplinary post-doctoral fellowship to CZ (LT0025/2023-C) and the Engineering and Physical Sciences Research Council to support M.T. (EP/R513143/1 and EP/W524335/1) and O.P.L.D (EP/R513143/1 and EP/T517793/1). S.S also acknowledges The Royal Society through a Dorothy Hodgkin fellowship (DHF\R1\191019 and DHF\R\251006). This work has also been supported by The Chan Zuckerberg Initiative (2023-321188) and BBSCR (BB/Y513064/1) grants to S.S.

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</ol>

The authors have nothing to declare.

Super-resolution fluorescence microscopy has broken the diffraction barrier, dramatically enhancing spatial resolution. Yet, its widespread adoption in biological research faces ongoing challenges. Notably, trade-offs between FOV, resolution, and penetration depth make it hard to optimize all three factors simultaneously. These challenges were addressed by utilizing the combined capabilities of SMLM and spinning disk confocal microscopy enhanced by optical photon reassignment (SDC-OPR). By integrating microlens arrays into a standard SDC configuration, as enabled by the commercial SDC-OPR system, photon collection was increased while the effective pinhole size was reduced, leading to significant resolution improvement. In comparison with standard SDC, the SDC-OPR system demonstrated a marked improvement in resolving power, successfully distinguishing features separated by 10 nm14 in the focal plane, whereas standard SDC could only resolve distances greater than 20 nm11. This enhanced resolving capacity underscores the benefit of photon reassignment for achieving molecular-scale separations. Specifically, sub-2 nm lateral localization precision was achieved at the focal plane (z = 0 &#181;m), with lateral precision remaining below 10 nm at depths up to 9 &#181;m, combined with a highly adaptable FOV of 53 &#215; 53 &#181;m&#178; or 76 &#215; 76 &#181;m&#178;. Even at a large FOV of 211 &#215; 211 &#181;m&#178;, the system delivered an impressive 9.5 nm average in-plane localization precision. These results highlight the system&#39;s capability for high-resolution imaging throughout whole cells.
Regarding the coupling reactions for the different antibodies used, the protocol must be carefully followed to ensure an optimal antibody-to-DNA ratio.&#160;Exceeding this ratio may reduce localization precision and could adversely affect quantitative measurements, such as those performed in qPAINT25. Therefore, it is critical to verify the ratio using a microvolume UV-Vis spectrophotometer, ensuring a final 1:1 ratio between DNA and antibody.
A critical step in obtaining high-quality super-resolution images of microtubules is proper fixation. Conventional fixation methods relying solely on high concentrations of PFA often disrupt microtubule structures. Instead, using glutaraldehyde (GA) alone or in combination with PFA significantly improves structural preservation26,27.
For optimal imaging, a high numerical aperture (NA) objective is essential to maximize photon collection per single-molecule event. In the commercial SDC-OPR system, the laser power is intentionally lower than in conventional TIRF microscopes used for SMLM to minimize sample photobleaching. While DNA-PAINT eliminates photobleaching concerns and could theoretically employ higher laser power, current commercial systems impose limitations, necessitating longer exposure times compared to TIRF illumination. Furthermore, because illumination in SDC-OPR is confocal, molecules are not continuously excited during the exposure. As a result, longer exposure times are necessary relative to continuous TIRF illumination; in this study, an exposure time of 300 ms was used. However, depending on the resolution requirements of a given experiment, shorter integration times (e.g., 150-200 ms) can be employed with minimal loss of resolution28.
Optimizing the concentration of the imager strand is a critical step to ensure that single-molecule signals remain well separated while still generating enough localizations for accurate reconstruction. This adjustment is analogous to standard SMLM acquisitions, where the balance between event density and signal overlap is carefully controlled. The optimal imager concentration should be adjusted for each different target. For example, for microtubules, an initial imager strand concentration of less than 1 nM is recommended, with adjustments made as needed.
Maintaining stable focus during prolonged acquisitions is critical, as axial (z) drift cannot be corrected post-processing. Lateral (xy) drift can be compensated via RCC or fiducial markers. In this setup, the microscope&#39;s Perfect Focus System (PFS) ensures focus stability over hours of imaging.
In summary, the integration of SMLM with SDC-OPR provides a powerful platform for high-resolution imaging deep within whole cells, overcoming traditional trade-offs between resolution, depth, and FOV. However, to fully realize the system&#39;s capabilities, meticulous attention to experimental conditions is essential, from antibody conjugation and fixation to acquisition settings and data processing. Each step, from sample preparation to drift correction, plays a critical role in achieving reliable, high-precision data. With careful protocol optimization, this approach offers a robust and versatile solution for quantitative, nanoscale imaging in complex biological samples. This protocol is expected to assist the community in adopting single-molecule localization microscopy and in obtaining super-resolved images from sample planes that are challenging to access using conventional TIRF or HILO illumination. Both objectives are facilitated using commercially available microscopes that are typically found in microscopy facilities worldwide.
In terms of other and future applications, the method is applicable to whole cell samples as well as to more complex tissue samples at greater imaging depths, as demonstrated in previous studies14. Unlike approaches that require physical sectioning, this technique enables volumetric imaging of intact tissues, preserving native architecture and minimizing artifacts introduced by physical sectioning. The use of smaller affinity probes, such as nanobodies, further enhances tissue penetration compared with full-length antibodies, allowing more uniform labeling throughout thick samples. This approach opens the door to studying cellular organization and molecular distributions in situ with higher fidelity. Beyond these applications, the strategy also lends itself to high-throughput screening of nanostructures and biomolecular assemblies, enabling systematic investigations of structural heterogeneity and function. In addition, recent developments in DNA-PAINT have yielded self-quenching imager probes, either based on dye-quencher or dye-dye interactions29,30,31, which effectively lower background signals, increase fluorescence intensity, and enhance spatial resolution, enabling faster acquisition rates. Integrating these fluorogenic probes into the SDC-OPR framework has the potential to further boost both imaging speed and resolution. Combining these advances with the strengths of the SDC-OPR system could expand its applicability to more complex biological specimens, establishing the platform as a flexible tool for detailed mapping of cellular and molecular structures in a wide variety of tissues.

Single-molecule localization microscopy (SMLM)1 is a family of super-resolution techniques that achieve nanoscale resolution by temporally separating the emission of individual fluorophores and localizing them with high precision. Successful SMLM requires a high signal-to-noise ratio (SNR) to detect single-molecule events, which has traditionally limited its use to optical setups providing intense, localized excitation. The most common configurations are total internal reflection fluorescence (TIRF)2 microscopy and highly inclined laminated optical sheet (HILO) illumination3. TIRF reduces background fluorescence by creating an evanescent field that selectively excites fluorophores within a few nanometers of the coverslip, thereby suppressing out-of-focus illumination. However, this restriction makes TIRF unsuitable for imaging deeper biological structures, such as organelles extending into the extracellular domain. HILO microscopy provides greater penetration depths, allowing imaging several hundred nanometers above the glass substrate. This comes at the cost of a reduced field-of-view (FOV)4 and uneven illumination with increasingly higher planes, limiting its applicability for larger cellular structures. More recently, light-sheet microscopy (LSM) techniques such as lattice light-sheet5,6,7 and single objective single plane illumination microscopy (soSPIM)8 have extended SMLM into volumetric and tissue-scale imaging. By shaping the excitation beam into thin, structured light sheets, LSM achieves optical sectioning with reduced background and phototoxicity. SoSPIM captures focal planes orthogonal to the detection axis in a single exposure, enabling fast volumetric imaging with near-isotropic resolution. The soSPIM application has demonstrated 3D SMLM in whole cells and small organelles (&#62;20 &#181;m depth) with down to 40 nm isotropic resolution, and with minimal out-of-focus background9. Despite their strengths, these methods typically require transparent samples, specialized optics, and complex alignment, and remain sensitive to optical aberrations and scattering in thicker specimens.
Confocal microscopy offers an alternative by rejecting out-of-focus light through the use of a pinhole in front of the detector, combined with beam-scanning mirrors10. This optical sectioning capability enables deep volumetric imaging with high SNR. In particular, spinning disk confocal (SDC) microscopy combines the speed and wide-field detection of conventional imaging with the sectioning capacity of point-scanning systems. This is achieved in conventional SDC through the incorporation of a rotating disk patterned with parallel pinholes, often combined with an aligned microlens array. The microlenses collect a substantial portion of the emitted fluorescence and focus it through the pinholes, which reduces the background signal. This configuration provides rapid optical sectioning without the need for specialized optics or additional sample mounting preparation, making it a robust choice that can be readily implemented and optimized for deep volumetric SMLM image acquisition11.
In this study, the CSU-W1 spinning disk confocal scanner unit equipped with optical photon reassignment (SDC-OPR) is employed. Unlike standard SDC, SDC-OPR introduces a second microlens array that projects the emitted photons onto a complementary pinhole pattern12, to mitigate scattering and recover photons that would otherwise be discarded. This photon reassignment effectively sharpens the point spread function (PSF), enhancing lateral resolution beyond that of conventional SDC while still rejecting out-of-focus light13. Specifically, the SDC-OPR microlens array redirects off-axis photons to their closest origin points, effectively increasing detected fluorescence brightness compared to standard SDC. Furthermore, the resolution benefits of SDC-OPR can be amplified via image deconvolution. Iterative deconvolution algorithms computationally refine the PSF, reduce background, and improve signal-to-background ratio without requiring changes to the optical configuration. This combined approach provides a significant advantage in terms of localization precision over standard SDC for single-molecule localization experiments14.
Here, a robust and reproducible protocol for single-molecule localization microscopy using a spinning disk confocal microscope equipped with optical photon reassignment (SDC-OPR) is presented. This approach combines the nanoscale precision of DNA-PAINT with the speed, FOV, and depth penetration of spinning disk imaging. DNA-PAINT, an adaptation of point accumulation for imaging nanoscale topography (PAINT)15, relies on the transient hybridization of fluorescently labelled &#34;imager&#34; strands with complementary &#34;docking&#34; strands anchored to molecular targets. These reversible binding events generate stochastic fluorescence signals that can be precisely localized, enabling high-resolution reconstruction of cellular ultrastructure. DNA-PAINT enables the use of highly photostable fluorophores, provides tunable binding kinetics for precise control of imaging conditions, and mitigates photobleaching through the continuous replenishment of fluorescent imager strands16.
To ensure reproducibility and specificity, the approach relies on custom DNA-antibody conjugates that link docking strands to secondary antibodies. Detailed sample preparation steps, including antibody-DNA conjugation, are presented to support robust experimental outcomes. The protocol is validated on well-characterized cellular structures such as nuclear pore complexes (NPCs)17 and microtubules in fixed samples, providing a reference point in estimating reconstruction accuracy. Variations in resolution, quantified via localization precision as a proxy, are further assessed as a function of FOV and imaging depth.
In addition, the efficiency of this workflow for multiplexed imaging is demonstrated using Exchange-PAINT18, a strategy that enables sequential visualization of multiple targets with the same dye and a single laser source based on DNA-PAINT. The Exchange-PAINT workflow is applied to multicolor super-resolution imaging of mitochondria, microtubules, and NPCs within the same sample.
Together, this protocol provides comprehensive guidance and key adaptability notes on antibody-DNA conjugation, sample preparation, imaging conditions, and data analysis, enabling researchers to perform reproducible DNA-PAINT imaging on SDC-OPR. By extending high-resolution imaging beyond the depth and FOV limitations of TIRF-based approaches, this method broadens the applicability of SMLM to a wide range of biological contexts.

<table><tbody><tr><td>(&amp;plusmn;)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid</td><td>Merck&amp;nbsp;</td><td>238813-5G</td><td>Trolox</td></tr><tr><td>3,4-Dihydroxybenzoic acid&amp;nbsp;</td><td>Merck&amp;nbsp;</td><td>37580-25G-F</td><td>PCA</td></tr><tr><td>60&amp;times; Apo NA 1.49 objective lens</td><td>Nikon</td><td></td><td></td></tr><tr><td>7.0 KDA spin desalting column containing size-exclusion resin&amp;nbsp;</td><td>Fisher Scientific Ltd</td><td>89882</td><td>Zeba Spin Desalting Columns for step 2.4</td></tr><tr><td>Accutase - Enzyme Cell Detachment Medium</td><td>Promo Cell</td><td>C-41310</td><td></td></tr><tr><td>Alpaca sdAb (1H1) anti-GFP coupled with custom DNA (sdAB-5&amp;rsquo;-TCCTCCTCCTCCT-3&amp;rsquo;)</td><td>Massive Photonics</td><td>Custom product</td><td>Anti-GFP nanobody for Drosophila</td></tr><tr><td>Alpaca sdAb (1H1) anti-GFP coupled with DNA</td><td>Massive Photonics</td><td>MASSIVE-TAG-Q-FAST anti-GFP - F3</td><td>Anti-GFP nanobody for NUP</td></tr><tr><td>Anti-alpha tubulin (YL1/2)</td><td>Thermo Fisher Scientific</td><td>MA1-80017</td><td></td></tr><tr><td>Anti-rabbit IgG</td><td>Massive Photonics</td><td>Massive-sdAB-FAST 2-Plex, Secondary sdAB F2</td><td></td></tr><tr><td>Anti-TCR&amp;zeta; (6B10.2)</td><td>BioLegend</td><td>644102</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Merck&amp;nbsp;</td><td>A4503-10G</td><td>BSA</td></tr><tr><td>Conical centrifugal tube 15 mL</td><td>Fisher Scientific Ltd</td><td>17705004</td><td></td></tr><tr><td>Conical centrifugal tube 50 mL</td><td>VWR International</td><td>734-0448</td><td></td></tr><tr><td>CSU-W1 SoRa</td><td>Nikon</td><td></td><td>SDC-OPR microscope</td></tr><tr><td>Desalting spin columns with Sephadex G-25 (size-exclusion resin for DNA purification)&amp;nbsp;</td><td>Fisher Scientific Ltd</td><td>11743309</td><td>Brand: Cytiva. G-25 columns for step 2.5</td></tr><tr><td>D-glucose</td><td>Merck&amp;nbsp;</td><td>G8270-1KG</td><td></td></tr><tr><td>DMEM media</td><td>Thermo Fisher Scientific</td><td>11965092</td><td></td></tr><tr><td>DMSO, Anhydrous</td><td>Thermo Fisher Scientific</td><td>D12345</td><td></td></tr><tr><td>DTT&amp;nbsp;(Dithiothreitol)</td><td>Thermo Fisher Scientific</td><td>15976082</td><td></td></tr><tr><td>EDTA</td><td>Thermo Fisher Scientific</td><td>15575020</td><td></td></tr><tr><td>EGTA</td><td>Marck</td><td>324626</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>A5256701</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Serva</td><td>2311402</td><td></td></tr><tr><td>Glycerol</td><td>Merck&amp;nbsp;</td><td>G5516-500ML&amp;nbsp;</td><td></td></tr><tr><td>Glycine</td><td>MP Biomedicals</td><td>219482580</td><td></td></tr><tr><td>Maleimide-PEG2-succinimidyl&amp;nbsp;</td><td>Merck&amp;nbsp;</td><td>746223-50MG</td><td></td></tr><tr><td>McCoy&#039;s 5A media</td><td>Fisher Scientific Ltd</td><td>16600082</td><td></td></tr><tr><td>MES (2-(N-morpholino)ethanesulfonic acid)</td><td>Merck</td><td>M3671-50G</td><td></td></tr><tr><td>Methanol</td><td>Fisher Scientific Ltd</td><td>32213-25L</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Fisher Scientific Ltd</td><td>10418464</td><td></td></tr><tr><td>Microcentrifuge tubes 0.5 mL</td><td>Fisher Scientific Ltd</td><td>11508232</td><td></td></tr><tr><td>Microcentrifuge tubes 1.5 mL</td><td>Eppendorf</td><td>10509691&amp;nbsp;</td><td></td></tr><tr><td>Microvolume UV-Vis Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>ND-ONE-W</td><td>Nanodrop</td></tr><tr><td>NaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S6546-1L&amp;nbsp;</td><td></td></tr><tr><td>Nanoparticles 90 nm -Gold</td><td>Cytodiagnostics</td><td>G-90-100</td><td></td></tr><tr><td>Orca Fusion-BT cmos camera</td><td>Hamamatsu</td><td></td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15070063</td><td>P/S</td></tr><tr><td>Phosphate-buffered saline (1x solution)</td><td>Fisher Scientific Ltd</td><td>11530546</td><td>PBS</td></tr><tr><td>Pierce 16% formaldehyde (w/v), methanol free</td><td>Fisher Scientific Ltd</td><td>11586711</td><td>PFA</td></tr><tr><td>Potassium Chloride&amp;nbsp;</td><td>Merck&amp;nbsp;</td><td>&amp;nbsp;P9541-500G</td><td></td></tr><tr><td>Protocatechuate 3,4-Dioxygenase &amp;nbsp;</td><td>Merck&amp;nbsp;</td><td>P8279-25UN&amp;nbsp;</td><td>PCD</td></tr><tr><td>Sodium&amp;nbsp;hydroxide</td><td>Fisher Scientific Ltd</td><td>12963614</td><td></td></tr><tr><td>TOM20 antibody</td><td>Abcam</td><td>Ab186735</td><td></td></tr><tr><td>Tris buffer</td><td>VWR International</td><td>A4577.1000</td><td></td></tr><tr><td>Triton&amp;nbsp;X-100&amp;nbsp;&amp;nbsp;</td><td>VWR International</td><td>1086031000</td><td></td></tr><tr><td>Tween-20</td><td>Merck&amp;nbsp;</td><td>P9416-50ML</td><td></td></tr><tr><td>Ultrafiltration centrifugal filter unit, 100 kDa MWCO</td><td>Merck&amp;nbsp;</td><td>UFC510024</td><td>Manufacturer: Amicon Ultra</td></tr><tr><td>&amp;mu;-Slide 8 Well High Glass Bottom</td><td>Ibidi</td><td>IB-80807</td><td></td></tr><tr><td>&amp;mu;-Slide VI 0.5 Glass Bottom</td><td>Ibidi</td><td>IB-80607</td><td></td></tr></tbody></table>

whole-cell super-resolution imaging via dna-paint on a spinning disk confocal with optical photon reassignment

Whole-cell Super-Resolution Imaging via DNA-PAINT on a Spinning Disk Confocal with Optical Photon Reassignment

Single-molecule localization microscopy (SMLM) enables nanoscale imaging of cellular structures but is typically restricted to imaging near the coverslip due to the limitations of total internal reflection fluorescence (TIRF) and highly inclined and optical sheet (HILO) illumination. Here, a protocol that leverages a spinning disk confocal microscope equipped with optical photon reassignment (SDC-OPR) to perform SMLM in whole cells is presented. This method enables high-precision single-molecule imaging throughout the full cell volume using DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) without the need for custom optics or complex illumination schemes. The protocol provides a stepwise guide on configuring a commercially available spinning disk microscope (e.g., CSU-W1 SoRA) for SMLM imaging, including acquisition parameters optimized for deep imaging. Sample preparation steps are outlined for labeling intracellular targets with DNA-conjugated probes to perform DNA-PAINT imaging, including strategies for multicolor imaging of several targets. Image acquisition is followed by single-molecule localization and reconstruction using standard software packages. Critical considerations for minimizing background, optimizing lateral resolution, and ensuring imaging quality across the full cell depth are also discussed. This accessible protocol allows researchers to perform deep, whole-cell SMLM using standard confocal equipment, expanding the range of biological questions addressable by single-molecule imaging beyond the near-membrane regions.

To demonstrate the super-resolution imaging capabilities of the SDC-OPR system (Figure 1A), its in-plane resolution was assessed by performing DNA-PAINT imaging experiments (Figure 1B) on nuclear pore complexes (NPCs), which are a biological reference structure17. Figure 1C shows a representative DNA-PAINT SDC-OPR image of nucleoporin 96 (Nup96), tagged with monomeric enhanced green fluorescent protein (mEGFPs) and labeled with DNA-conjugated anti-GFP nanobodies, in U2OS cells. The zoomed-in panels reveal NUP substructure, and Figure 1D shows paired Nup96 proteins (indicated by arrows) consistent with the expected 8-fold symmetry of nuclear pores. In Figure 1E, the Euclidean distance between Nup96 pairs was measured by aligning them and plotting the cross-sectional histogram of the summed image (n = 16 pairs). The analysis revealed a peak-to-peak separation of (13 ± 2) nm (Figure 1E), in agreement with results from EM models as well as HILO and TIR imaging23. Each peak fit displayed a standard deviation of 4 nm, confirming the high localization precision (σSMLM = 3.3 nm, NeNA = 4.4 nm) achieved using DNA-PAINT on SDC-OPR. Notably, this precision was maintained across the entire 53 × 53 µm² FOV, which is exceptional for confocal-based systems.
To demonstrate the capability for multicolor imaging, Nup96, mitochondria, and microtubules in U2OS cells were DNA-labeled for acquisition using Exchange-PAINT18. This technique employs orthogonal DNA imager strands conjugated to a single fluorophore, enabling all targets to be excited with the same laser source (Figure 2A). Rather than acquiring signals simultaneously, it separates them across sequential imaging rounds, facilitating straightforward integration into any commercial SDC-OPR system. Figure 2B shows Nup96 in blue labeled with an anti-GFP nanobody, mitochondria in red with a secondary anti-TOM20 antibody, and microtubules in green with a primary anti-α-tubulin antibody. Notably, the outstanding single-molecule localization precision obtained for single-color Nup96 imaging was preserved across all targets in the multi-target experiment, yielding localization precision values of 3.9 nm for the NPC, 4.0 nm for α-tubulin, and 3.3 nm for mitochondria (Figure 2B panels 1, 2, and 3).
For large FOV imaging, microtubules in HeLa cells were imaged using various magnifications available on the SDC-OPR system, combined with different camera binning settings to maintain pixel sizes between 78 nm and 108 nm (Figure 3). Figure 3A shows the microtubule cytoskeleton of 10 HeLa cells, with colors representing localization precision. At 1× magnification, the average localization precision is noticeably worse compared to higher magnifications (Figure 3B-D), with greater variability observed. This is due to the reduced laser power density over the larger FOV, where the Gaussian illumination profile becomes apparent, leading to decreased precision toward the edges of the FOV. Nonetheless, achieving an average localization precision of 9.5 nm across the expansive 211 × 211 µm² FOV is impressive for confocal-based systems and enables high-resolution investigation of biological heterogeneity.
Finally, the possibility of imaging whole cells is demonstrated by acquiring DNA-PAINT images of the microtubule network in fixed HeLa cells of a confocal volume of ~500 nm thickness and 1 µm z-step using the system's highest magnification (4×, 53 × 53 µm² FOV). Figure 4A presents the single-molecule reconstruction images for each z-plane, along with the corresponding localization precision and NeNA values. In addition, a 3D color-rendered visualization of the microtubule network in the HeLa cell is provided in Figure 4C. As depth increases, the number of detected photons diminishes due to scattering and optical aberrations, resulting in reduced localization precision for both the localization precisions derived from Cramér-Rao Lower Bound (CRLB) (Figure 4A, right) and those calculated using the NeNA metric. Still, the high level of photon collection possible by the SDC-OPR system enables σSMLM ≤ 10 nm for up to 9 µm imaging depth with narrow distributions. Indeed, double-walled filamentous microtubule structures were clearly resolved at various depths: near the coverslip-cell interface, at intermediate axial positions, and at the top of the cell, with peak-to-peak distance between 30 nm and 40 nm (Figure 4C), consistent with reported values24. These results highlight the SDC-OPR's ability to deliver high-resolution imaging across large FOVs and throughout the entire height of cells.
<img alt="SOD-CPR microscopy and DNA-PAINT diagram for protein localization using nanometer precision." class="xfigimg" src="/files/ftp_upload/69531/69531fig1.jpg" data-altupdatedat="1784001604000" data-alt="Figure 1"> 
Figure 1: In-plane single-molecule localization microscopy of different cell samples and multicolor super-resolution imaging. (A) Schematic of the Spinning Disk Confocal with Optical Photon Reassignment (SDC-OPR) system, featuring a microlens array designed to maximize photon collection. (B) Schematic of DNA-PAINT: Fluorescently labeled DNA strands, known as imagers strands, transiently hybridize with complementary DNA sequences (docking strands) that are anchored to the antibody/nanobody targeting the protein of interest. These short-lived binding events generate fluorescence signals that appear as stochastic blinking. (C) Representative DNA-PAINT image on the SDC-OPR system using 4× magnification of the NPC of U2OS cells. The high-resolution capability is demonstrated in the inset, where individual NPCs are clearly resolved. (D) SDC-OPR-enhanced DNA-PAINT enables visualization of single NPCs, where white arrows highlight characteristic Nup96 protein pairs within the pore architecture. (E) Distribution of distance measurements between Nup96 dimers (n=16 pairs) within single NPC symmetry units. This figure has been modified from Zaza et al.14. <a href="https://www.jove.com/files/ftp_upload/69531/69531fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microscopy diagram; cellular structures and processes; protein interactions; fluorescence visualization." class="xfigimg" src="/files/ftp_upload/69531/69531fig2.jpg" data-altupdatedat="1784001604000" data-alt="Figure 2"> 
Figure 2: Exchange-PAINT for multicolor imaging using an SDC-OPR microscope. (A) Schematic of Exchange-PAINT of three target imaged sequentially. Three protein targets were labelled with either DNA-functionalized antibodies, Fab fragments, or nanobodies. The Cy3B imager strands were imaged sequentially by strand type with wash steps between imaging rounds. (B) Three-color Exchange-PAINT imaging of U2OS cells using SDC-OPR microscopy using 4× magnification, showing microtubules (red; labeled with DNA-conjugated anti-α-tubulin), mitochondria (green; TOM20-targeting DNA-nanobodies), and NPC (blue; mEGFP-tagged Nup96 with anti-GFP DNA-nanobodies). Scale bar: 5 µm. Zoomed-in region emphasizes the enhanced resolution obtained on the SDC-OPR system, focusing on (1) mitochondria, (2) microtubules and (3) NUP. Scale bars = 1 µm. This figure has been modified from Zaza et al.14. <a href="https://www.jove.com/files/ftp_upload/69531/69531fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Cell structure microscopy; diagram showing varying magnifications; 1x, 2.8x, 4x; spectral analysis." class="xfigimg" src="/files/ftp_upload/69531/69531fig3.jpg" data-altupdatedat="1784001604000" data-alt="Figure 3"> 
Figure 3: DNA-PAINT imaging within large FOVs on an SDC-OPR microscope. Microtubule DNA-PAINT images (anti-α-tubulin labeling) acquired at multiple magnifications: (A) 1× magnification and 1 binning (FOV of 211 × 211 µm²), (B) 2.8× and 2 binning (FOV of 76 × 76 µm²), (C) 4× magnification and 4 binning (FOV of 53 × 53 µm²). Color represents the localization precision from 0-16 nm in the case of 1x magnification and 0-4 nm in the others. (D) Localization precision measured at different distances (in µm) from the center of the FOV for 1× (red), 2.8× (yellow), and 4× (green) magnifications. This figure has been modified from Zaza et al.14. <a href="https://www.jove.com/files/ftp_upload/69531/69531fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Super-resolution microscopy images and graph showing optical sectioning for nanostructure analysis." class="xfigimg" src="/files/ftp_upload/69531/69531fig4.jpg" data-altupdatedat="1784001604000" data-alt="Figure 4"> 
Figure 4: Whole cell DNA-PAINT imaging. (A) Left. Volumetric microtubule imaging in HeLa cells via DNA-PAINT acquired using SDC-OPR at 4× magnification, showing axial sections from z = 0 to 9 µm (1 µm steps). Each plane displays z position, localization precision, and NeNA metric. Scale bars: 5 µm (xy). Right. Localization precision versus penetration depth for all planes. (B) 3D render of the microtubule network on the same HeLa cell as in (A). Color represents penetration depth from 0 to 9 µm. (C) Zoomed-in images of highlighted small regions from each penetration depth indicated in (A). This figure has been modified from Zaza et al.14. <a href="https://www.jove.com/files/ftp_upload/69531/69531fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol outlines the procedure for performing deep, whole-cell single-molecule localization microscopy (SMLM) using a spinning disk confocal microscope with optical photon reassignment, thereby enabling DNA-PAINT imaging without the need for custom optics or complex illumination.

Single-molecule localization microscopy (SMLM) enables nanoscale imaging of cellular structures but is typically restricted to imaging near the coverslip ...

whole-cell-super-resolution-imaging-via-dna-paint-on-spinning-disk

Research

JoVE Journal

Biology

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Learn whole-cell super-resolution imaging via DNA-PAINT using a spinning disk confocal microscope with optical photon reassignment. This protocol enables deep SMLM without custom optics for neuroscience research.

Whole-cell Super-Resolution Imaging via DNA-PAINT on a Spinning Disk Confocal with Optical Photon Reassignment

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