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.

  • Article
  • Published:

Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores

Nature Methods volume 8, pages 353–359 (2011)Cite this article

Subjects

Abstract

We demonstrate three-dimensional (3D) super-resolution imaging of stochastically switched fluorophores distributed across whole cells. By evaluating the higher moments of the diffraction spot provided by a 4Pi detection scheme, single markers can be simultaneously localized with <10 nm precision in three dimensions in a layer of 650 nm thickness at an arbitrarily selected depth in the sample. By splitting the fluorescence light into orthogonal polarization states, our 4Pi setup also facilitates the 3D nanoscopy of multiple fluorophores. Offering a combination of multicolor recording, nanoscale resolution and extended axial depth, our method substantially advances the noninvasive 3D imaging of cells and of other transparent materials.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: The 4Pi-SMS microscope.
Figure 2: 4Pi-SMS imaging of human platelets.
Figure 3: 4Pi-SMS imaging with extended axial depth.
Figure 4: Two-color imaging.

Similar content being viewed by others

References

  1. Hell, S. & Stelzer, E.H.K. Properties of a 4pi confocal fluorescence microscope. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 9, 2159–2166 (1992).

    Article  Google Scholar 

  2. Gustafsson, M.G.L., Agard, D.A. & Sedat, J.W. I5M: 3d widefield light microscopy with better than 100 nm axial resolution. J. Microsc. 195, 10–16 (1999).

    Article  CAS  Google Scholar 

  3. Egner, A., Jakobs, S. & Hell, S.W. Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast. Proc. Natl. Acad. Sci. USA 99, 3370–3375 (2002).

    Article  CAS  Google Scholar 

  4. Egner, A. & Hell, S.W. Fluorescence microscopy with super-resolved optical sections. Trends Cell Biol. 15, 207–215 (2005).

    Article  CAS  Google Scholar 

  5. Schmidt, M., Nagorni, M. & Hell, S.W. Subresolution axial distance measurements in far-field fluorescence microscopy with precision of 1 nanometer. Rev. Sci. Instrum. 71, 2742–2745 (2000).

    Article  CAS  Google Scholar 

  6. Albrecht, B., Failla, A.V., Schweitzer, A. & Cremer, C. Spatially modulated illumination microscopy allows axial distance resolution in the nanometer range. Appl. Opt. 41, 80–87 (2002).

    Article  Google Scholar 

  7. Hell, S.W. Improvement of lateral resolution in far-field light microscopy using two-photon excitation with offset beams. Opt. Commun. 106, 19–24 (1994).

    Article  Google Scholar 

  8. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Bock, H. et al. Two-color far-field fluorescence nanoscopy based on photoswitchable emitters. Appl. Phys. B 88, 161–165 (2007).

    Article  CAS  Google Scholar 

  16. Fölling, J. et al. Fluorescence nanoscopy by ground-state depletion and single-molecule return. Nat. Methods 5, 943–945 (2008).

    Article  Google Scholar 

  17. Heilemann, M., van de Linde, S., Mukherjee, A. & Sauer, M. Super-resolution imaging with small organic fluorophores. Angew. Chem. Int. Ed. 48, 6903–6908 (2009).

    Article  CAS  Google Scholar 

  18. Von Middendorff, C., Egner, A., Geisler, C., Hell, S. & Schonle, A. Isotropic 3D nanoscopy based on single emitter switching. Opt. Express 16, 20774–20788 (2008).

    Article  Google Scholar 

  19. Hell, S.W., Schmidt, R. & Egner, A. Diffraction-unlimited three-dimensional optical nanoscopy with opposing lenses. Nat. Photonics 3, 381–387 (2009).

    Article  CAS  Google Scholar 

  20. Schmidt, R. et al. Spherical nanosized focal spot unravels the interior of cells. Nat. Methods 5, 539–544 (2008).

    Article  CAS  Google Scholar 

  21. Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl. Acad. Sci. USA 106, 3125–3130 (2009).

    Article  CAS  Google Scholar 

  22. Engelhardt, J. et al. Molecular orientation affects localization accuracy in superresolution far-field fluorescence microscopy. Nano Lett. 11, 209–213 (2011).

    Article  CAS  Google Scholar 

  23. Okamura, Y. et al. Few immobilized thrombins are sufficient for platelet spreading. Biophys. J. (in the press).

  24. Isenberg, W.M., McEver, R.P., Phillips, D.R., Shuman, M.A. & Bainton, D.F. The platelet fibrinogen receptor: an immunogold-surface replica study of agonist-induced ligand binding and receptor clustering. J. Cell Biol. 104, 1655–1663 (1987).

    Article  CAS  Google Scholar 

  25. Smith, C.M. II, Burris, S., Rao, G. & White, J. Detergent-resistant cytoskeleton of the surface-activated platelet differs from the suspension-activated platelet cytoskeleton. Blood 80, 2774–2780 (1992).

    CAS  PubMed  Google Scholar 

  26. Bossi, M. et al. Multi-color far-field fluorescence nanoscopy through isolated detection of distinct molecular species. Nano Lett. 8, 2463–2468 (2008).

    Article  CAS  Google Scholar 

  27. Egner, A., Verrier, S., Goroshkov, A., Soling, H.D. & Hell, S.W. 4Pi-microscopy of the Golgi apparatus in live mammalian cells. J. Struct. Biol. 147, 70–76 (2004).

    Article  CAS  Google Scholar 

  28. Egner, A., Schrader, M. & Hell, S.W. Refractive index mismatch induced intensity and phase variations in fluorescence confocal, multiphoton and 4pi-microscopy. Opt. Commun. 153, 211–217 (1998).

    Article  CAS  Google Scholar 

  29. Dowski, J., Edward, R. & Cathey, W.T. Extended depth of field through wave-front coding. Appl. Opt. 34, 1859–1866 (1995).

    Article  Google Scholar 

  30. Testa, I. et al. Multicolor fluorescence nanoscopy in fixed and living cells by exciting conventional fluorophores with a single wavelength. Biophys. J. 99, 2686–2694 (2010).

    Article  CAS  Google Scholar 

  31. Schoenle, A. & Hell, S.W. Fluorescence nanoscopy goes multicolor. Nat. Biotechnol. 25, 1234–1235 (2007).

    Article  CAS  Google Scholar 

  32. Testa, I. et al. Nanoscale separation of molecular species based on their rotational mobility. Opt. Express 16, 21093–21104 (2008).

    Article  CAS  Google Scholar 

  33. Okamura, Y., Kabata, K., Kinoshita, M., Saitoh, D. & Takeoka, S. Free-standing biodegradable poly(lactic acid) nanosheet for sealing operations in surgery. Adv. Mater. 21, 4388–4392 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Pick for aid in the design of optics and mechanics, T. Gilat and E. Rothermel for technical assistance, and J. Keller for helpful discussions. We acknowledge J. Jethwa for critical reading of the manuscript. V. Belov (Max Planck Institute for Biophysical Chemistry, Göttingen) provided us with Rhodamine S and S. Takeoka (Waseda University, Tokyo) provided support on the nanosheets. This work was supported by the Gottfried Wilhelm Leibniz Program of the Deutsche Forschungsgemeinschaft (to S.W.H.) and a grant of the Deutsche Forschungsgemeinschaft to A.E. and S.W.H. (SFB 755).

Author information

Author notes

  1. Claudia Geisler & Alexander Egner

    Present address: Present address: Laser Laboratory Göttingen e.V., Department of Optical Nanoscopy, Göttingen, Germany.,

Authors and Affiliations

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

    Daniel Aquino, Andreas Schönle, Claudia Geisler, Claas v Middendorff, Christian A Wurm, Stefan W Hell & Alexander Egner

  2. Membrane Biochemistry Laboratory, Life and Medical Sciences Institute, University of Bonn, Bonn, Germany

    Yosuke Okamura & Thorsten Lang

Authors
  1. Daniel Aquino
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andreas Schönle
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Claudia Geisler
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Claas v Middendorff
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christian A Wurm
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yosuke Okamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Thorsten Lang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stefan W Hell
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Alexander Egner
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.E., A.S., T.L. and S.W.H. conceived and designed the study. D.A., C.G., C.A.W. and Y.O. performed experiments. D.A., A.E., A.S., C.v.M. and C.G. analyzed data. A.E., A.S., T.L. and S.W.H. wrote the manuscript.

Corresponding authors

Correspondence to Stefan W Hell or Alexander Egner.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Notes 1–2 (PDF 3157 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Aquino, D., Schönle, A., Geisler, C. et al. Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores. Nat Methods 8, 353–359 (2011). https://doi.org/10.1038/nmeth.1583

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1583

This article is cited by

Search

Advanced 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