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.

Fluorescence nanoscopy by ground-state depletion and single-molecule return

Abstract

We introduce far-field fluorescence nanoscopy with ordinary fluorophores based on switching the majority of them to a metastable dark state, such as the triplet, and calculating the position of those left or those that spontaneously returned to the ground state. Continuous widefield illumination by a single laser and a continuously operating camera yielded dual-color images of rhodamine- and fluorescent protein–labeled (living) samples, proving a simple yet powerful super-resolution approach.

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: Switching a regular fluorophore using its dark states.
Figure 2: Subdiffraction resolution images recorded by GSDIM.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Hell, S.W. & Kroug, M. Appl. Phys. B 60, 495–497 (1995).

    Article  Google Scholar 

  3. Bretschneider, S., Eggeling, C. & Hell, S.W. Phys. Rev. Lett. 98, 218103 (2007).

    Article  Google Scholar 

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

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Hell, S.W., Jakobs, S. & Kastrup, L. Appl. Phys. A 77, 859–860 (2003).

    Article  CAS  Google Scholar 

  7. Hell, S.W. Science 316, 1153–1158 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Rust, M.J., Bates, M. & Zhuang, X. Nat. Methods 3, 793–796 (2006).

    Article  CAS  Google Scholar 

  11. Heisenberg, W. The Physical Principles of the Quantum Theory. (University of Chicago Press, Chicago, 1930).

    Google Scholar 

  12. Fölling, J. et al. Angew. Chem. Int. Ed. 46, 6266–6270 (2007).

    Article  Google Scholar 

  13. Bock, H. et al. Appl. Phys. B 88, 161–165 (2007).

    Article  CAS  Google Scholar 

  14. Heilemann, M. et al. Angew. Chem. 47, 6172–6176 (2008).

    Article  CAS  Google Scholar 

  15. Zondervan, R., Kulzer, F., Orlinskii, S.B. & Orrit, M. J. Phys. Chem. A 107, 6770–6776 (2003).

    Article  CAS  Google Scholar 

  16. Bossi, M. et al. Nano Lett. 8, 2463–2468 (2008).

    Article  CAS  Google Scholar 

  17. Dickson, R.M., Cubitt, A.B., Tsien, R.Y. & Moerner, W.E. Nature 388, 355–358 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank A. Egner and A. Schönle for support with the instrumentation and data evaluation software and for valuable discussions, R.Y. Tsien (University of California, San Diego) for providing the plasmid coding for fluorescent proteins, V.N. Belov (Max Planck Institute for Biophysical Chemistry, Göttingen) for supplying the rhodamine dyes Rh-sart3b and Rh-sart3f, R. Schmitz-Salue and S. Löbermann for excellent technical assistance, S. Sahl for help with the experiments, and B. Rankin and J. Jethwa for carefully reading the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Christian Eggeling or Stefan W Hell.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1, Supplementary Table 1, Supplementary Methods (PDF 227 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fölling, J., Bossi, M., Bock, H. et al. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat Methods 5, 943–945 (2008). https://doi.org/10.1038/nmeth.1257

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1257

This article is cited by

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