Skip to main content

Thank you for visiting nature.com. 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.

  • Protocol
  • Published:

Super-resolution microscopy with DNA-PAINT

Abstract

Super-resolution techniques have begun to transform biological and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) offers an easy-to-implement approach to localization-based super-resolution microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking' to enable stochastic super-resolution microscopy. Using the programmability and specificity of DNA molecules as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resolution techniques, making them compatible with virtually any single-molecule-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise molecule counting and ultra-high, molecular-scale (sub-5-nm) spatial resolution, reaching 1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA-PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample preparation, multiplexed data acquisition, data simulation, super-resolution image reconstruction and post-processing such as drift correction, molecule counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1–2 d.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: DNA-PAINT.
Figure 2: Exchange-PAINT.
Figure 3: qPAINT.
Figure 4: 'Ultra-resolution' with DNA-PAINT.
Figure 5: DNA-PAINT protocol workflow.
Figure 6: Designing DNA origami structures for DNA-PAINT with 'Picasso: Design'.
Figure 7: Simulating DNA-PAINT raw data from DNA origami-like structures.

Similar content being viewed by others

References

  1. Gustafsson, M.G.L. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl. Acad. Sci. USA 102, 13081–13086 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. 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  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  4. Hess, S.T., Girirajan, T.P.K. & Mason, M.D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. 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  PubMed  PubMed Central  Google Scholar 

  6. Heilemann, M. et al. Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes. Angew. Chem. Int. Ed. Engl. 47, 6172–6176 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Hell, S.W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    Article  CAS  PubMed  Google Scholar 

  8. Sengupta, P. et al. Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis. Nat. Methods 8, 969–975 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Xu, K., Zhong, G. & Zhuang, X. Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science 339, 452–456 (2012).

    Article  PubMed  CAS  Google Scholar 

  10. Honigmann, A. et al. Phosphatidylinositol 4,5-bisphosphate clusters act as molecular beacons for vesicle recruitment. Nat. Struct. Mol. Biol. 20, 679–686 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Li, D. et al. ADVANCED IMAGING. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349, aab3500 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Galiani, S. et al. Super-resolution microscopy reveals compartmentalization of peroxisomal membrane proteins. J. Biol. Chem. 291, 16948–16962 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hell, S.W. et al. The 2015 super-resolution microscopy roadmap. J. Phys. D Appl. Phys. 48, 443001 (2015).

    Article  CAS  Google Scholar 

  14. Huang, B., Bates, M. & Zhuang, X. Super-resolution fluorescence microscopy. Annu. Rev. Biochem. 78, 993–1016 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lippincott-Schwartz, J., Jennifer, L.-S. & Patterson, G.H. Photoactivatable fluorescent proteins for diffraction-limited and super-resolution imaging. Trends Cell Biol. 19, 555–565 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Nieuwenhuizen, R.P.J. et al. Measuring image resolution in optical nanoscopy. Nat. Methods 10, 557–562 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sharonov, A. & Hochstrasser, R.M. Wide-field subdiffraction imaging by accumulated binding of diffusing probes. Proc. Natl. Acad. Sci. USA 103, 18911–18916 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Giannone, G. et al. Dynamic superresolution imaging of endogenous proteins on living cells at ultra-high density. Biophys. J. 99, 1303–1310 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Jungmann, R. et al. Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami. Nano Lett. 10, 4756–4761 (2010).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Iinuma, R. et al. Polyhedra self-assembled from DNA tripods and characterized with 3D DNA-PAINT. Science 344, 65–69 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Schlichthaerle, T., Strauss, M.T., Schueder, F., Woehrstein, J.B. & Jungmann, R. DNA nanotechnology and fluorescence applications. Curr. Opin. Biotechnol. 39, 41–47 (2016).

    Article  CAS  PubMed  Google Scholar 

  25. Agasti, S. et al. DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging. Chem. Sci. (2017) http://dx.doi.org/10.1039/c6sc05420j.

  26. Rasnik, I., McKinney, S.A. & Ha, T. Nonblinking and long-lasting single-molecule fluorescence imaging. Nat. Methods 3, 891–893 (2006).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ha, T. & Tinnefeld, P. Photophysics of fluorescent probes for single-molecule biophysics and super-resolution imaging. Annu. Rev. Phys. Chem. 63, 595–617 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Ries, J., Kaplan, C., Platonova, E., Eghlidi, H. & Ewers, H. A simple, versatile method for GFP-based super-resolution microscopy via nanobodies. Nat. Methods 9, 582–584 (2012).

    Article  CAS  PubMed  Google Scholar 

  30. Opazo, F. et al. Aptamers as potential tools for super-resolution microscopy. Nat. Methods 9, 938–939 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Tokunaga, M., Imamoto, N. & Sakata-Sogawa, K. Highly inclined thin illumination enables clear single-molecule imaging in cells. Nat. Methods 5, 159–161 (2008).

    Article  CAS  PubMed  Google Scholar 

  32. Legant, W.R. et al. High-density three-dimensional localization microscopy across large volumes. Nat. Methods 13, 359–365 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  34. Seeman, N.C. Nucleic acid junctions and lattices. J. Theor. Biol. 99, 237–247 (1982).

    Article  CAS  PubMed  Google Scholar 

  35. Seeman, N.C. An overview of structural DNA nanotechnology. Mol. Biotechnol. 37, 246–257 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Douglas, S.M. et al. Rapid prototyping of 3D DNA-origami shapes with caDNAno. Nucleic Acids Res. 37, 5001–5006 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Benson, E. et al. DNA rendering of polyhedral meshes at the nanoscale. Nature 523, 441–444 (2015).

    Article  CAS  PubMed  Google Scholar 

  38. Kim, D.-N., Kilchherr, F., Dietz, H. & Bathe, M. Quantitative prediction of 3D solution shape and flexibility of nucleic acid nanostructures. Nucleic Acids Res. 40, 2862–2868 (2012).

    Article  CAS  PubMed  Google Scholar 

  39. Douglas, S.M. et al. Self-assembly of DNA into nanoscale three-dimensional shapes. Nature 459, 414–418 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Martin, T.G. & Dietz, H. Magnesium-free self-assembly of multi-layer DNA objects. Nat. Commun. 3, 1103 (2012).

    Article  PubMed  CAS  Google Scholar 

  41. Sobczak, J.-P.J., Martin, T.G., Gerling, T. & Dietz, H. Rapid folding of DNA into nanoscale shapes at constant temperature. Science 338, 1458–1461 (2012).

    Article  CAS  PubMed  Google Scholar 

  42. Bellot, G., Gaëtan, B., McClintock, M.A., Chenxiang, L. & Shih, W.M. Recovery of intact DNA nanostructures after agarose gel–based separation. Nat. Methods 8, 192–194 (2011).

    Article  CAS  PubMed  Google Scholar 

  43. Lin, C., Perrault, S.D., Kwak, M., Graf, F. & Shih, W.M. Purification of DNA-origami nanostructures by rate-zonal centrifugation. Nucleic Acids Res. 41, e40 (2013).

    Article  CAS  PubMed  Google Scholar 

  44. Stahl, E., Martin, T.G., Praetorius, F. & Dietz, H. Facile and scalable preparation of pure and dense DNA origami solutions. Angew. Chem. Int. Ed. Engl. 53, 12735–12740 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Steinhauer, C., Jungmann, R., Sobey, T.L., Simmel, F.C. & Tinnefeld, P. DNA origami as a nanoscopic ruler for super-resolution microscopy. Angew. Chem. Int. Ed. Engl. 48, 8870–8873 (2009).

    Article  CAS  PubMed  Google Scholar 

  46. Schmied, J.J. et al. DNA origami–based standards for quantitative fluorescence microscopy. Nat. Protoc. 9, 1367–1391 (2014).

    Article  CAS  PubMed  Google Scholar 

  47. Dean, K.M. & Palmer, A.E. Advances in fluorescence labeling strategies for dynamic cellular imaging. Nat. Chem. Biol. 10, 512–523 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Dempsey, G.T., Vaughan, J.C., Chen, K.H., Bates, M. & Zhuang, X. Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat. Methods 8, 1027–1036 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Mikhaylova, M. et al. Resolving bundled microtubules using anti-tubulin nanobodies. Nat. Commun. 6, 7933 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Los, G.V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).

    Article  CAS  PubMed  Google Scholar 

  51. Keppler, A., Pick, H., Arrivoli, C., Vogel, H. & Johnsson, K. Labeling of fusion proteins with synthetic fluorophores in live cells. Proc. Natl. Acad. Sci. USA 101, 9955–9959 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Wang, L., Xie, J. & Schultz, P.G. Expanding the genetic code. Annu. Rev. Biophys. Biomol. Struct. 35, 225–249 (2006).

    Article  PubMed  CAS  Google Scholar 

  53. Schweller, R.M. et al. Multiplexed in situ immunofluorescence using dynamic DNA complexes. Angew. Chem. Int. Ed. Engl. 51, 9292–9296 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Ullal, A.V. et al. Cancer cell profiling by barcoding allows multiplexed protein analysis in fine-needle aspirates. Sci. Transl. Med. 6, 219ra9 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  55. Gong, H. et al. Simple method to prepare oligonucleotide-conjugated antibodies and its application in multiplex protein detection in single cells. Bioconjug. Chem. 27, 217–225 (2016).

    Article  CAS  PubMed  Google Scholar 

  56. Xu, K., Babcock, H.P. & Zhuang, X. Dual-objective STORM reveals three-dimensional filament organization in the actin cytoskeleton. Nat. Methods 9, 185–188 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Whelan, D.R. & Bell, T.D.M. Image artifacts in single molecule localization microscopy: why optimization of sample preparation protocols matters. Sci. Rep. 5, 7924 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Edelstein, A.D. et al. J. Biol. Methods 1, 10 (2014).

    Article  Google Scholar 

  59. Beier, H.T. & Ibey, B.L. Experimental comparison of the high-speed imaging performance of an EM-CCD and sCMOS camera in a dynamic live-cell imaging test case. PLoS One 9, e84614 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  60. Huang, F. et al. Video-rate nanoscopy using sCMOS camera–specific single-molecule localization algorithms. Nat. Methods 10, 653–658 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Shroff, H., Galbraith, C.G., Galbraith, J.A. & Betzig, E. Live-cell photoactivated localization microscopy of nanoscale adhesion dynamics. Nat. Methods 5, 417–423 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Venkataramani, V., Herrmannsdörfer, F., Heilemann, M. & Kuner, T. SuReSim: simulating localization microscopy experiments from ground truth models. Nat. Methods 13, 319–321 (2016).

    Article  PubMed  CAS  Google Scholar 

  63. Sage, D. et al. Quantitative evaluation of software packages for single-molecule localization microscopy. Nat. Methods 12, 717–724 (2015).

    Article  CAS  PubMed  Google Scholar 

  64. Smith, C.S., Joseph, N., Rieger, B. & Lidke, K.A. Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat. Methods 7, 373–375 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Egner, A. et al. Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys. J. 93, 3285–3290 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Wang, Y. et al. Localization events-based sample drift correction for localization microscopy with redundant cross-correlation algorithm. Opt. Express 22, 15982–15991 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  67. 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  PubMed  Google Scholar 

  68. Lin, C. et al. Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA. Nat. Chem. 4, 832–839 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Bates, M., Huang, B., Dempsey, G.T. & Zhuang, X. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749–1753 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Huang, B., Jones, S.A., Brandenburg, B. & Zhuang, X. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat. Methods 5, 1047–1052 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Shin, J.Y. et al. Visualization and functional dissection of coaxial paired SpoIIIE channels across the sporulation septum. Elife 4 (2015).

  72. Puchner, E.M., Walter, J.M., Kasper, R., Huang, B. & Lim, W.A. Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory. Proc. Natl. Acad. Sci. USA 110, 16015–16020 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Lee, S.-H., Shin, J.Y., Lee, A. & Bustamante, C. Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM). Proc. Natl. Acad. Sci. USA 109, 17436–17441 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Rollins, G.C., Shin, J.Y., Bustamante, C. & Pressé, S. Stochastic approach to the molecular counting problem in superresolution microscopy. Proc. Natl. Acad. Sci. USA 112, E110–8 (2015).

    Article  CAS  PubMed  Google Scholar 

  75. Nieuwenhuizen, R.P.J. et al. Quantitative localization microscopy: effects of photophysics and labeling stoichiometry. PLoS One 10, e0127989 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  76. Sengupta, P., Jovanovic-Talisman, T. & Lippincott-Schwartz, J. Quantifying spatial organization in point-localization superresolution images using pair correlation analysis. Nat. Protoc. 8, 345–354 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Juette, M.F. et al. Three-dimensional sub-100 nm resolution fluorescence microscopy of thick samples. Nat. Methods 5, 527–529 (2008).

    Article  CAS  PubMed  Google Scholar 

  78. Broeken, J. et al. Resolution improvement by 3D particle averaging in localization microscopy. Methods Appl. Fluoresc. 3, 014003 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  79. Cheng, Y., Yifan, C., Nikolaus, G., Penczek, P.A. & Thomas, W. A primer to single-particle cryo-electron microscopy. Cell 161, 438–449 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Szymborska, A. et al. Nuclear pore scaffold structure analyzed by super-resolution microscopy and particle averaging. Science 341, 655–658 (2013).

    Article  CAS  PubMed  Google Scholar 

  81. Loschberger, A. et al. Super-resolution imaging visualizes the eightfold symmetry of gp210 proteins around the nuclear pore complex and resolves the central channel with nanometer resolution. J. Cell Sci. 125, 570–575 (2012).

    Article  PubMed  CAS  Google Scholar 

  82. Penczek, P., Radermacher, M. & Frank, J. Three-dimensional reconstruction of single particles embedded in ice. Ultramicroscopy 40, 33–53 (1992).

    Article  CAS  PubMed  Google Scholar 

  83. Perrault, S.D. & Chan, W.C.W. Synthesis and surface modification of highly monodispersed, spherical gold nanoparticles of 50-200 nm. J. Am. Chem. Soc. 131, 17042–17043 (2009).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank B. Rieger, S.S. Agasti, S. Strauss, D. Haas, J.B. Woehrstein and E. Woehrstein for helpful discussions. This work was supported by the German Research Foundation (DFG) through an Emmy Noether Fellowship (DFG JU 2957/1-1), the European Research Council (ERC) through an ERC Starting Grant (MolMap, grant agreement no. 680241), the Max Planck Society, the Max Planck Foundation and the Center for Nanoscience (CeNS). M.T.S. acknowledges support from the International Max Planck Research School for Molecular and Cellular Life Sciences (IMPRS-LS). T.S. acknowledges support from the DFG through the Graduate School of Quantitative Biosciences Munich (QBM). F.S. acknowledges support from the DFG through the SFB 1032 (Nanoagents for the spatiotemporal control of molecular and cellular reactions).

Author information

Authors and Affiliations

Authors

Contributions

J.S. and M.T.S. contributed equally to this work. J.S. designed and developed the Picasso software suite. M.T.S. developed 'Picasso: Design' and 'Simulate' and performed in vitro experiments. T.S. developed antibody labeling strategies and performed in situ experiments. F.S. performed ultra-resolution experiments. R.J. conceived the study and supervised the project. All authors contributed to the writing of the manuscript.

Corresponding author

Correspondence to Ralf Jungmann.

Ethics declarations

Competing interests

R.J. is a cofounder of Ultivue, a startup company with an interest in commercializing DNA-PAINT technology.

Integrated supplementary information

Supplementary Figure 1 Overview of “Picasso: Design”

(a) The main window showing the origami canvas with the hexagonal tiles. (b) Extensions dialog to set extensions corresponding to each selected color. (c) Plate export dialog to specify the export format of the plates. (d) Pipetting dialog to select a folder with *.csv files to generate a list of sequences that need to be pipetted and to create a visual pipetting aid. (e) Folding table to calculate volumes that are needed for pipetting.

Supplementary Figure 2 Overview of “Picasso: Simulate”

The main window has two preview windows, the left one to display the positions of structures in the full frame, the right one to display an individual structure. Structural parameters such as number and structure definition can be set in the group box “Structure”. All PAINT-related parameters, i.e. mean dark and bright times are set with the “PAINT parameters” group box. The group box “Imager parameters” is used to define properties of the simulated imaging probe.

Supplementary Figure 3 Overview of “Picasso: Localize”

(a) The main window after the analysis of a movie file. Yellow boxes indicate the identification of a spot, green crosses show the fitted subpixel coordinate. (b) The contrast setting dialog. (c) The parameters setting dialog.

Supplementary Figure 4 Overview of “Picasso: Render”

(a) The main window with two picked regions of interest (yellow circles). (b) The display settings dialog for the render scene in (a). (c) The info dialog for the picked regions in (a). (d) The tools settings dialog.

Supplementary Figure 5 Overview of “Picasso: Filter”

(a) The main window showing properties (columns) of localizations (rows). (b) Filtering in a histogram of a property column. (c) Filtering in a two-dimensional histogram of two property columns. The green areas in (b) and (c) have been selected with a pressed left mouse button. After releasing the mouse button, any localization with property values outside the green range will be removed.

Supplementary Figure 6 20 nm DNA origami grid

Supplementary Figure 7 10 nm DNA origami grid

Supplementary Figure 8 LMU Logo

Supplementary Figure 9 MPI Logo

Supplementary Figure 10 Custom-made flow chamber

(a) Two stripes of double-sided sticky tape are placed on a 76x26 mm microscopy slide with a distance of ~ 8mm. A coverglass is placed on top of the sticky tape stripes. After pressing the coverglass thoroughly against the sticky tape, overlapping tape can be removed. (b) To immobilize DNA nanostructures, fluids are pipetted from one side while simultaneously being sucked out with a lab wiper from the other side. (c) The coverglass is sealed with epoxy glue and can be used with the coverglass facing towards the objective in a microscope stage once the glue is hardened.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10, Supplementary Manual and Supplementary Tables 1–7. (PDF 2294 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schnitzbauer, J., Strauss, M., Schlichthaerle, T. et al. Super-resolution microscopy with DNA-PAINT. Nat Protoc 12, 1198–1228 (2017). https://doi.org/10.1038/nprot.2017.024

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2017.024

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

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