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Nanoscopy with more than 100,000 'doughnuts'

Nature Methods volume 10, pages 737–740 (2013)Cite this article

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Abstract

We show that nanoscopy based on the principle called RESOLFT (reversible saturable optical fluorescence transitions) or nonlinear structured illumination can be effectively parallelized using two incoherently superimposed orthogonal standing light waves. The intensity minima of the resulting pattern act as 'doughnuts', providing isotropic resolution in the focal plane and making pattern rotation redundant. We super-resolved living cells in 120 μm × 100 μm–sized fields of view in <1 s using 116,000 such doughnuts.

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Figure 1: Parallelized scanning (RESOLFT) nanoscopy using orthogonally and incoherently crossed standing waves.
Figure 2: Live-cell imaging with parallelized RESOLFT nanoscopy.
Figure 3: Continuous time-lapse imaging with parallelized RESOLFT.

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  1. Hell, S.W. Nat. Biotechnol. 21, 1347–1355 (2003).

    Article  CAS  Google Scholar 

  2. Rust, M.J., Bates, M. & Zhuang, X.W. Nat. Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

  3. Betzig, E. et al. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  4. Hess, S.T., Girirajan, T.P.K. & Mason, M.D. Biophys. J. 91, 4258–4272 (2006).

    Article  CAS  Google Scholar 

  5. Hell, S.W. Nat. Methods 6, 24–32 (2009).

    Article  CAS  Google Scholar 

  6. Hell, S.W. & Wichmann, J. Opt. Lett. 19, 780–782 (1994).

    Article  CAS  Google Scholar 

  7. Heintzmann, R., Jovin, T.M. & Cremer, C. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 19, 1599–1609 (2002).

    Article  Google Scholar 

  8. Gustafsson, M.G.L. Proc. Natl. Acad. Sci. USA 102, 13081–13086 (2005).

    Article  CAS  Google Scholar 

  9. Hofmann, M., Eggeling, C., Jakobs, S. & Hell, S.W. Proc. Natl. Acad. Sci. USA 102, 17565–17569 (2005).

    Article  CAS  Google Scholar 

  10. Westphal, V. et al. Science 320, 246–249 (2008).

    Article  CAS  Google Scholar 

  11. Bingen, P., Reuss, M., Engelhardt, J. & Hell, S.W. Opt. Express 19, 23716–23726 (2011).

    Article  CAS  Google Scholar 

  12. Grotjohann, T. et al. Nature 478, 204–208 (2011).

    Article  CAS  Google Scholar 

  13. Brakemann, T. et al. Nat. Biotechnol. 29, 942–947 (2011).

    Article  CAS  Google Scholar 

  14. Testa, I. et al. Neuron 75, 992–1000 (2012).

    Article  CAS  Google Scholar 

  15. Grotjohann, T. et al. eLife 1, e00248 (2012).

    Article  Google Scholar 

  16. Schwentker, M.A. et al. Microsc. Res. Tech. 70, 269–280 (2007).

    Article  CAS  Google Scholar 

  17. Rego, E.H. et al. Proc. Natl. Acad. Sci. USA 109, E135–E143 (2012).

    Article  CAS  Google Scholar 

  18. Gustafsson, M.G.L. J. Microsc. 198, 82–87 (2000).

    Article  CAS  Google Scholar 

  19. Frohn, J.T., Knapp, H.F. & Stemmer, A. Proc. Natl. Acad. Sci. USA 97, 7232–7236 (2000).

    Article  CAS  Google Scholar 

  20. Gustafsson, M.G.L. et al. Biophys. J. 94, 4957–4970 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

M. Leutenegger (formerly at Max Planck Institute for Biophysical Chemistry, Göttingen; now at the Ecole Polytechnique Fédérale de Lausanne) is acknowledged for early discussions about technical aspects. A.C. acknowledges a postdoctoral scholarship from the Swedish Research Council. S.J. and S.W.H. acknowledge funding from the German Research Foundation (DFG)–Center of Nanoscale Microscopy and Molecular Physiology of the Brain. S.W.H. also received support from the German Ministry of Research (BMBF) and the Körber Foundation, Hamburg, through the European Science Prize.

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Authors and Affiliations

  1. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Andriy Chmyrov, Jan Keller, Tim Grotjohann, Michael Ratz, Elisa d'Este, Stefan Jakobs, Christian Eggeling & Stefan W Hell

  2. Department of Neurology, University of Göttingen, Göttingen, Germany

    Stefan Jakobs

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  1. Andriy Chmyrov
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  7. Christian Eggeling
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Contributions

A.C. built the setup, performed the experiments and analyzed data. J.K. analyzed data and performed data modeling. T.G. developed and characterized the protein rsEGFP(N205S), advised by S.J. M.R. and E.d'E. prepared samples. C.E. advised on protein switching and setup implementations. S.W.H. defined and supervised the project, wrote the paper and is, together with A.C., responsible for its main thrust.

Corresponding author

Correspondence to Stefan W Hell.

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Competing interests

The Max Planck Society and S.W.H. hold patent rights (US 7,064,824) on the RESOLFT principle.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Notes 1–3 (PDF 7242 kb)

Supplementary Software

MATLAB scripts used for parallelized RESOLFT image reconstruction (ZIP 6 kb)

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Chmyrov, A., Keller, J., Grotjohann, T. et al. Nanoscopy with more than 100,000 'doughnuts'. Nat Methods 10, 737–740 (2013). https://doi.org/10.1038/nmeth.2556

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