Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Up to 100-fold speed-up and multiplexing in optimized DNA-PAINT


DNA-PAINT’s imaging speed has recently been significantly enhanced by optimized sequence design and buffer conditions. However, this implementation has not reached an ultimate speed limit and is only applicable to imaging of single targets. To further improve acquisition speed, we introduce concatenated, periodic DNA sequence motifs, yielding up to 100-fold-faster sampling in comparison to traditional DNA-PAINT. We extend this approach to six orthogonal sequence motifs, now enabling speed-optimized multiplexed imaging.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type



Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Faster DNA-PAINT through overlapping sequence motifs.
Fig. 2: Multiplexing with concatenated speed-optimized motifs.

Data availability

All raw data are available upon reasonable request from the authors.


  1. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994).

    Article  CAS  Google Scholar 

  2. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  3. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

  4. Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Super-resolution microscopy with DNA-PAINT. Nat. Protoc. 12, 1198–1228 (2017).

    Article  CAS  Google Scholar 

  5. Strauss, S. et al. Modified aptamers enable quantitative sub-10-nm cellular DNA-PAINT imaging. Nat. Methods 15, 685–688 (2018).

    Article  CAS  Google Scholar 

  6. Schlichthaerle, T. et al. Site-specific labeling of affimers for DNA-PAINT microscopy. Angew. Chem. Int. Ed. Engl. 57, 11060–11063 (2018).

    Article  CAS  Google Scholar 

  7. Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313–318 (2014).

    Article  CAS  Google Scholar 

  8. Dai, M., Jungmann, R. & Yin, P. Optical imaging of individual biomolecules in densely packed clusters. Nat. Nanotechnol. 11, 798–807 (2016).

    Article  CAS  Google Scholar 

  9. Schickinger, M., Zacharias, M. & Dietz, H. Tethered multifluorophore motion reveals equilibrium transition kinetics of single DNA double helices. Proc. Natl Acad. Sci. USA 115, E7512–E7521 (2018).

    Article  CAS  Google Scholar 

  10. Schueder, F. et al. An order of magnitude faster DNA-PAINT imaging by optimized sequence design and buffer conditions. Nat. Methods 16, 1101–1104 (2019).

    Article  CAS  Google Scholar 

  11. Filius, M. et al. High-speed super-resolution imaging using protein-assisted DNA-PAINT. Nano Lett. 20, 2264–2270 (2020).

  12. Jungmann, R. et al. Quantitative super-resolution imaging with qPAINT. Nat. Methods 13, 439–442 (2016).

    Article  CAS  Google Scholar 

  13. Rothemund, P. W. Folding DNA to create nanoscale shapes and patterns. Nature 440, 297–302 (2006).

    Article  CAS  Google Scholar 

  14. Blumhardt, P. et al. Photo-induced depletion of binding sites in DNA-PAINT microscopy. Molecules 23, 3165 (2018).

  15. Aitken, C. E., Marshall, R. A. & Puglisi, J. D. An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophysical J. 94, 1826–1835 (2008).

    Article  CAS  Google Scholar 

  16. Schlichthaerle, T. et al. Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT. Angew. Chem. Int. Ed. Engl. 58, 13004–13008 (2019).

    Article  CAS  Google Scholar 

  17. Pleiner, T. et al. Nanobodies: site-specific labeling for super-resolution imaging, rapid epitope-mapping and native protein complex isolation. eLife 4, e11349 (2015).

    Article  Google Scholar 

  18. Thevathasan, J. V. et al. Nuclear pores as versatile reference standards for quantitative superresolution microscopy. Nat. Methods 16, 1045–1053 (2019).

    Article  CAS  Google Scholar 

  19. Gwosch, K. C. et al. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat. Methods 17, 217–224 (2020).

    CAS  PubMed  Google Scholar 

  20. Sograte-Idrissi, S. et al. Nanobody detection of standard fluorescent proteins enables multi-target DNA-PAINT with high resolution and minimal displacement errors. Cells 8, 48 (2019).

  21. Endesfelder, U., Malkusch, S., Fricke, F. & Heilemann, M. A simple method to estimate the average localization precision of a single-molecule localization microscopy experiment. Histochem. Cell Biol. 141, 629–638 (2014).

    Article  CAS  Google Scholar 

  22. Wade, O. K. et al. 124-color super-resolution imaging by engineering DNA-PAINT blinking kinetics. Nano Lett. 19, 2641–2646 (2019).

    Article  CAS  Google Scholar 

  23. Chung, K. K. et al. Fluorogenic probe for fast 3D whole-cell DNA-PAINT. Preprint at bioRxiv (2020).

  24. Wagenbauer, K. F. et al. How we make DNA origami. ChemBioChem 18, 1873–1885 (2017).

    Article  CAS  Google Scholar 

  25. McInnes, L., Healy, J. & Astels, S. hdbscan: hierarchical density based clustering. J. Open Source Softw. 2, 205 (2017).

    Article  Google Scholar 

Download references


This work has been supported in part by the German Research Foundation through the Emmy Noether Program (DFG JU 2957/1-1), the SFB1032 (project A11), the European Research Council through an ERC Starting Grant (MolMap; grant agreement number 680241), the Allen Distinguished Investigator Program through the Paul G. Allen Frontiers Group, the Danish National Research Foundation (Centre for Cellular Signal Patterns, DNRF135), the Max Planck Foundation and the Max Planck Society. We thank the Ries and Ellenberg groups at EMBL for the kind gift of the cell line expressing Nup96–mEGFP. We thank T. Schlichthaerle, M. Ganji and F. Schueder for fruitful discussions. S.S. acknowledges support by the QBM Graduate School.

Author information

Authors and Affiliations



S.S. conceived and performed experiments, analyzed data and contributed to the writing of the manuscript. R.J. conceived and supervised the study, interpreted data and wrote the manuscript. Both authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Ralf Jungmann.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Imaging of 20-nm grids with 1xR1 and 5xR1 docking sites.

20-nm grids with 1×R1 and 5×R1 docking sites were measured in the same sample. The data represents linked localizations, hence the brightness scales linearly with the number of binding events. a, Overview image. (b and c) 25 selected structures of 1× and 5×R1 DNA origami, respectively. 1×R1 structures show comparable signal intensity to 5×R1 after 5-fold increase of brightness level. Scale bars, 500 nm (a), 100 nm (b, c). (Integration time: 100 ms, 40 000 frames, 500 pM R1 Cy3B, Excitation intensity: 125 W/cm2). The experiment was repeated two times independently with similar results.

Extended Data Fig. 2 Linear increase of kon with number of repeats for 6 different sequence motifs.

Apparent \(k_{on}\) values were calculated by fitting the average dark times obtained from single binding sites on DNA origami with a Gaussian function and subsequently calculating \(k_{on} = 1/\left( {\tau _d \cdot c} \right)\), where c denotes the imager strand concentration. Error bars indicate standard deviation of Gaussian fits. All overlapping binding site motifs show a linear dependency until 5x repeats. Some 10x binding motifs exhibit a lower-than-expected \(k_{on}\), which could be explained by decreased accessibility due to the increased sequence length (and thus flexibility). Mean \(k_{on}\) values are additionally denoted in Supplementary Table 1. We note that each graph consists of data gathered from three separate experiments per sequence, containing repeat pairs of 1x and 3×, 1x and 5×, and 3x and 10×, respectively (see Supplementary Fig. 2). Furthermore, higher motif repeats were performed with reduced imager strand concentration to avoid multiple binding events. Plotting apparent kon values allows us to faithfully compare sequence repeats (see Supplementary Tables 8 and 9 for details about image acquisition parameters and number of analyzed origami structures).

Extended Data Fig. 3 Mean bright time values depending on the number of repeats.

Analysis was performed with the same data sets presented in Extended Data Fig. 2 and Supplementary Fig. 2. Each data point shows the average of the calculated mean bright time (\(\tau _B\)) values of each origami structure. Error bars indicate standard deviation. Bright times increase with increasing number of repeats (at various degrees depending on the sequence). Increasing times can particularly be observed from 1 to 3 repeats, whereas it is similar/equal from 5 to 10 repeats, indicating a potential stabilization of DNA hybridization due to extended overhangs of the docking site sequences.

Extended Data Fig. 4 Quantification of sequence orthogonality.

To evaluate the orthogonality of the designed docking sites, the number of binding events of the respective imager sequences (labeled in each graph) in the DNA origami Exchange-PAINT experiment from Supplementary Fig. 4 is quantified for all six structures in the same sample. The violin plots show the distribution of the calculated mean number of binding events detected per structure. The white circle in the violin plots depicts the median number of binding events. A significant number of binding events was exclusively detected for imagers binding to their corresponding docking sites, highlighting exquisite sequence orthogonality, making the sequences applicable for multiplexing experiments. (Number of analyzed origami structures: 5×R1: n = 3515; 5×R2: n = 2436; 7×R3: n = 2619; 7×R4: n = 2502; 5×R5: n = 2962; 5×R6: n = 3381).

Extended Data Fig. 5 6-color Exchange-PAINT in 30 minutes image acquisition time.

6 different 20-nm-grid origami structures carrying 6 orthogonal concatenated docking sites were measured sequentially with an imaging time of no longer than 5 minutes per round. Each channel shows clearly resolved and sampled 20-nm-grids and NeNa values below 4 nm (see Supplementary Table 10) in two exemplary field of views. Scale bar: 500 nm. (Integration time: 50 ms, 5 500 frames, Imager concentrations: R1_6nt (5 nM), R2_6nt (2 nM), R3_2 (5 nM), R4_6nt (5 nM), R5 (10 nM), R6_6nt (10 nM), Excitation intensity: 650 W/cm2). The experiment was repeated three times independently with similar results.

Extended Data Fig. 6 Sub-5-nm resolution imaging with 5xR1 sequence.

Array of 30 selected DNA origami structures showing an MPI logo with neighboring docking sites designed to be spaced in 5 nm distances. Extended length of the 5×R1 docking site does not negatively affect the resolution capability as 5-nm-distances can be clearly resolved. Scale bar: 200 nm. (integration time: 100 ms, 40 000 frames, 200 pM R1-Cy3B, Excitation intensity: 650 W/cm2). The experiment was repeated three times independently with similar results.

Extended Data Fig. 7 Nuclear pore complex imaging using 5xR1 sequence with a GFP nanobody.

a, Overview image showing specific staining of NPCs at the nuclear envelope. b, Zoom-in area shows distinct NPC structures. c, Zoom-in of 40 picked NPCs with clearly resolved single Nup96 proteins. Scale bars, 5 µm (a), 200 nm (b), 100 nm (c). (Integration time: 100 ms, 40 000 frames, 250 pM R1 Cy3B, Excitation intensity: 180 W/cm2). The experiment was repeated three times independently with similar results.

Extended Data Fig. 8 Two-round Exchange-PAINT alpha-tubulin and vimentin imaging.

Alpha-tubulin and vimentin were labeled with primary and secondary antibodies. a, Overview image shows specific labeling of vimentin and microtubules. b, Zoom-in to the region highlighted in a. Scale bars, 2 µm (a), 500 nm (b). (Integration time: 100 ms, 20 000 frames, R3/4 50 pM, Excitation intensity: 200 W/cm2). The experiment was repeated two times independently with similar results.

Extended Data Fig. 9 4-color overview image of receptor tyrosine kinases.

Large FOV display from the data shown in Fig. 2f and g. Receptors in SKOV3 GFP-Her2 tagRFP-EGFR cells were labeled using DNA-conjugated nanobodies as shown in Supplementary Table 6 and 7. Data is represented as linked localizations. Each channel reveals distinct single molecules on the cell surface. Scale bar: 1 µm. Image acquisition parameters are shown in Supplementary Table 8. The experiment was repeated three times independently with similar results.

Extended Data Fig. 10 Improved kinetic properties for barcoding.

Concatenation of binding sequences leads to an increased bright time and a decreased dark time which can be used for barcoding based on these kinetic parameters. Potential barcoding capability was demonstrated on single 1×, 3× and 10× R3 binding sites, because the R3 sequence shows distinct separation of bright and dark time values (Extended Data Figs. 2 and 3, Supplementary Fig. 2). The barcoding capability was tested by using a HDBSCAN clustering algorithm. 84.2% of the 3492 analyzed binding sites were classified as either 1×R3 (blue), 3×R3 (green) or 10×R3 (orange). (Integration time: 100 ms, 20 000 frames, 3 nM R3 Cy3B, Excitation intensity: 25 W/cm2, all structures measured in the same sample).

Supplementary information

Supplementary Information

Supplementary Figs. 1–4 and Supplementary Tables 1–10

Reporting Summary

Supplementary Data 1

Scaffold strand sequence.

Supplementary Data 2

Staple strand sequences for single-binding-site DNA origami.

Supplementary Data 3

Staple strand sequences for 20-nm-grid DNA origami.

Supplementary Data 4

Staple strand sequences for MPI logo DNA origami.

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Strauss, S., Jungmann, R. Up to 100-fold speed-up and multiplexing in optimized DNA-PAINT. Nat Methods 17, 789–791 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing