Brief Communication | Published:

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

Nature Methods volume 5, pages 527529 (2008) | Download Citation

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    Science 316, 1153–1158 (2007).

  2. 2.

    , & Biophys. J. 91, 4258–4272 (2006).

  3. 3.

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

  4. 4.

    , & Nat. Methods 3, 793–795 (2006).

  5. 5.

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

  6. 6.

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

  7. 7.

    & Annu. Rev. Biophys. Biomol. Struct. 36, 349–369 (2007).

  8. 8.

    , & Proc. Natl. Acad. Sci. USA 103, 18137–18142 (2006).

  9. 9.

    , , & Nat. Methods 4, 915–918 (2007).

  10. 10.

    , , & Science 319, 810–813 (2008).

  11. 11.

    , , & IEEE Trans. Nanobioscience 3, 237–242 (2004).

  12. 12.

    , , & Nano Lett. 7, 2043–2045 (2007).

  13. 13.

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

  14. 14.

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

  15. 15.

    , , , & Biophys. J. 89, 1346–1352 (2005).

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

Affiliations

  1. Institute for Molecular Biophysics, The Jackson Laboratory, 600 Main Street, Bar Harbor, Maine 04609, USA.

    • Manuel F Juette
    • , Mark D Lessard
    • , Michael J Mlodzianoski
    • , Bhupendra S Nagpure
    •  & Joerg Bewersdorf
  2. Department of Biophysical Chemistry, University of Heidelberg, and Department of New Materials and Biosystems, Max Planck Institute for Metals Research, Heisenbergstr. 3, 70569 Stuttgart, Germany.

    • Manuel F Juette
  3. Department of Physics and Astronomy, University of Maine, 120 Bennett Hall, Orono, Maine 04469, USA.

    • Travis J Gould
    • , Bhupendra S Nagpure
    •  & Samuel T Hess
  4. Institute for Molecular Biophysics, University of Maine, 5737 Jenness Hall, Orono, Maine 04469, USA.

    • Travis J Gould
    •  & Samuel T Hess
  5. Active Motif, Inc., 1914 Palomar Oaks Way, Carlsbad, California 92008, USA.

    • Brian T Bennett

Authors

  1. Search for Manuel F Juette in:

  2. Search for Travis J Gould in:

  3. Search for Mark D Lessard in:

  4. Search for Michael J Mlodzianoski in:

  5. Search for Bhupendra S Nagpure in:

  6. Search for Brian T Bennett in:

  7. Search for Samuel T Hess in:

  8. Search for Joerg Bewersdorf in:

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.

Corresponding author

Correspondence to Joerg Bewersdorf.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–3, Supplementary Methods

About this article

Publication history

Received

Accepted

Published

DOI

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

Further reading