Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT


Super-resolution fluorescence microscopy is a powerful tool for biological research, but obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, a variation of point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed super-resolution imaging that achieves sub-10-nm spatial resolution in vitro on synthetic DNA structures. We also report a multiplexing approach (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-color super-resolution imaging in vitro on synthetic DNA structures as well as four-color two-dimensional (2D) imaging and three-color 3D imaging of proteins in fixed cells.

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Figure 1: DNA-PAINT.
Figure 2: Spectrally multiplexed DNA-PAINT super-resolution imaging of microtubules and mitochondria inside fixed cells.
Figure 3: Exchange-PAINT.
Figure 4: Multiplexed 2D and 3D Exchange-PAINT super-resolution imaging in fixed cells.


  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

    Hell, S.W. Microscopy and its focal switch. Nat. Methods 6, 24–32 (2009).

    CAS  Article  Google Scholar 

  3. 3

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

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

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

    CAS  Article  Google Scholar 

  6. 6

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

    CAS  Article  Google Scholar 

  7. 7

    Lew, M.D. et al. Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus. Proc. Natl. Acad. Sci. USA 108, E1102–E1110 (2011).

    CAS  Article  Google Scholar 

  8. 8

    Flors, C., Ravarani, C.N. & Dryden, D.T. Super-resolution imaging of DNA labelled with intercalating dyes. ChemPhysChem 10, 2201–2204 (2009).

    CAS  Article  Google Scholar 

  9. 9

    Schoen, I., Ries, J., Klotzsch, E., Ewers, H. & Vogel, V. Binding-activated localization microscopy of DNA structures. Nano Lett. 11, 4008–4011 (2011).

    CAS  Article  Google Scholar 

  10. 10

    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).

    CAS  Article  Google Scholar 

  11. 11

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

    CAS  Article  Google Scholar 

  12. 12

    Jungmann, R., Scheible, M. & Simmel, F.C. Nanoscale imaging in DNA nanotechnology. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 4, 66–81 (2012).

    CAS  Article  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 14

    Derr, N.D. et al. Tug-of-war in motor protein ensembles revealed with a programmable DNA origami scaffold. Science 338, 662–665 (2012).

    CAS  Article  Google Scholar 

  15. 15

    Johnson-Buck, A. et al. Super-resolution fingerprinting detects chemical reactions and idiosyncrasies of single DNA pegboards. Nano Lett. 13, 728–733 (2013).

    CAS  Article  Google Scholar 

  16. 16

    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).

    CAS  Article  Google Scholar 

  17. 17

    Vaughan, J.C., Jia, S. & Zhuang, X. Ultrabright photoactivatable fluorophores created by reductive caging. Nat. Methods 9, 1181–1184 (2012).

    CAS  Article  Google Scholar 

  18. 18

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

    CAS  Article  Google Scholar 

  19. 19

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

    CAS  Article  Google Scholar 

  20. 20

    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).

    CAS  Article  Google Scholar 

  21. 21

    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).

    CAS  Article  Google Scholar 

  22. 22

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

    CAS  Article  Google Scholar 

  23. 23

    Vogelsang, J. et al. A reducing and oxidizing system minimizes photobleaching and blinking of fluorescent dyes. Angew. Chem. Int. Ed. Engl. 47, 5465–5469 (2008).

    CAS  Article  Google Scholar 

  24. 24

    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).

    CAS  Article  Google Scholar 

  25. 25

    Wu, N. et al. Molecular threading and tunable molecular recognition on DNA origami nanostructures. J. Am. Chem. Soc. 135, 12172–12175 (2013).

    CAS  Article  Google Scholar 

  26. 26

    Kao, H.P. & Verkman, A.S. Tracking of single fluorescent particles in three dimensions: use of cylindrical optics to encode particle position. Biophys. J. 67, 1291–1300 (1994).

    CAS  Article  Google Scholar 

  27. 27

    Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    CAS  Article  Google Scholar 

  28. 28

    Lubeck, E. & Cai, L. Single-cell systems biology by super-resolution imaging and combinatorial labeling. Nat. Methods 9, 743–748 (2012).

    CAS  Article  Google Scholar 

  29. 29

    Bates, M., Dempsey, G.T., Chen, K.H. & Zhuang, X. Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection. ChemPhysChem 13, 99–107 (2012).

    CAS  Article  Google Scholar 

  30. 30

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

    CAS  Article  Google Scholar 

  31. 31

    Ke, R. et al. In situ sequencing for RNA analysis in preserved tissue and cells. Nat. Methods 10, 857–860 (2013).

    CAS  Article  Google Scholar 

  32. 32

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

    CAS  Article  Google Scholar 

  33. 33

    Kazane, S.A. et al. Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR. Proc. Natl. Acad. Sci. USA 109, 3731–3736 (2012).

    CAS  Article  Google Scholar 

  34. 34

    Beliveau, B.J. et al. Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc. Natl. Acad. Sci. USA 109, 21301–21306 (2012).

    CAS  Article  Google Scholar 

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We thank J. Nicoludis and M.T. Strauss for help with DNA origami design, T. Schlichthaerle for transmission electron microscopy imaging support and M. Zhang for help with DLD1 cells. We thank C. Steinhauer for help with DNA-PAINT software development and fruitful discussions. We thank R.D. Barish for critical reading and commenting on the manuscript. This work is supported by a US National Institutes of Health (NIH) Director's New Innovator Award (1DP2OD007292), an NIH Transformative Research Award (1R01EB018659), an NIH grant (5R21HD072481), an Office of Naval Research (ONR) Young Investigator Program Award (N000141110914), ONR grants (N000141010827 and N000141310593), a US National Science Foundation (NSF) Faculty Early Career Development Award (CCF1054898), an NSF grant (CCF1162459) and a Wyss Institute for Biologically Engineering Faculty Startup Fund to P.Y., and an NIH Director's New Innovator Award (1DP2OD004641) and a Wyss Institute for Biologically Inspired Engineering Faculty Award to W.M.S. R.J. acknowledges support from the Alexander von Humboldt-Foundation through a Feodor-Lynen Fellowship. M.S.A. and M.D. acknowledge support from Howard Hughes Medical Institute International Student Research Fellowships.

Author information




R.J., M.S.A. and J.B.W. contributed equally to this work. R.J. and M.S.A. conceived of the study, designed and performed the experiments, analyzed the data and wrote the manuscript. J.B.W. designed and performed the experiments, analyzed the data and wrote the manuscript. M.D. performed the experiments, analyzed the data and developed the drift correction software. W.M.S. supervised the project, discussed the results and critiqued the paper. P.Y. conceived of, designed and supervised the study, interpreted the data and wrote the manuscript. All authors reviewed and approved the manuscript.

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Correspondence to Peng Yin.

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R.J., M.D., M.S.A., J.B.W. and P.Y. have filed a provisional US patent application regarding the current work.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14, Supplementary Tables 1–8 and Supplementary Protocol (PDF 29243 kb)

Supplementary Software

Parallelized spot finding and 2D Gaussian fitting software implemented in LabVIEW (ZIP 1769 kb)

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

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