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.

  • Brief Communication
  • Published:

Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples

Abstract

Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 × 30 × 75nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.

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

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

Figure 1: BP FPALM setup and axial resolution.
Figure 2: 3D BP FPALM of 4 μm diameter beads labeled with caged fluorescein.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Toprak, E. & Selvin, P.R. Annu. Rev. Biophys. Biomol. Struct. 36, 349–369 (2007).

    Article  CAS  Google Scholar 

  8. Bewersdorf, J., Bennett, B.T. & Knight, K.L. Proc. Natl. Acad. Sci. USA 103, 18137–18142 (2006).

    Article  CAS  Google Scholar 

  9. Willig, K.I., Harke, B., Medda, R. & Hell, S.W. Nat. Methods 4, 915–918 (2007).

    Article  CAS  Google Scholar 

  10. Huang, B., Wang, W., Bates, M. & Zhuang, X. Science 319, 810–813 (2008).

    Article  CAS  Google Scholar 

  11. Prabhat, P., Ram, S., Ward, E.S. & Ober, R.J. IEEE Trans. Nanobioscience 3, 237–242 (2004).

    Article  Google Scholar 

  12. Toprak, E., Balci, H., Blehm, B.H. & Selvin, P.R. Nano Lett. 7, 2043–2045 (2007).

    Article  CAS  Google Scholar 

  13. Prabhat, P. et al. Proc. Natl. Acad. Sci. USA 104, 5889–5894 (2007).

    Article  CAS  Google Scholar 

  14. Hess, S.T. et al. Proc. Natl. Acad. Sci. USA 104, 17370–17375 (2007).

    Article  CAS  Google Scholar 

  15. Schneider, M., Barozzi, S., Testa, I., Faretta, M. & Diaspro, A. Biophys. J. 89, 1346–1352 (2005).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank L. Rocco Carpenter for purification and antibody-conjugation of the caged fluorescein, Th. Tripp for machining services, E. Burns and B. Knowles for valuable comments on the manuscript and J. Spatz for support. This work was supported by the US National Institute of Allergy and Infectious Diseases (K25-65459), the US National Cancer Institute (P30 CA034196), the US National Science Foundation (CHE-0722759 and EPS-0132384), the W.M. Keck Foundation, and start-up funds from The Jackson Laboratory and the University of Maine.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Joerg Bewersdorf.

Ethics declarations

Competing interests

M.F.J., T.J.G., S.T.H. and J.B. have filed a US patent application covering the work described in this manuscript.

B.T.B. is employed by a commercial company, Active Motif, Inc.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Methods (PDF 416 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Juette, M., Gould, T., Lessard, M. et al. Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples. Nat Methods 5, 527–529 (2008). https://doi.org/10.1038/nmeth.1211

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.1211

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