Brief Communication | Published:

Sharper low-power STED nanoscopy by time gating

Nature Methods volume 8, pages 571573 (2011) | Download Citation

Abstract

Applying pulsed excitation together with time-gated detection improves the fluorescence on-off contrast in continuous-wave stimulated emission depletion (CW-STED) microscopy, thus revealing finer details in fixed and living cells using moderate light intensities. This method also enables super-resolution fluorescence correlation spectroscopy with CW-STED beams, as demonstrated by quantifying the dynamics of labeled lipid molecules in the plasma membrane of living cells.

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Acknowledgements

We thank A. Schönle and M. Leutenegger for fruitful discussions, A. Schönle for support with the software Imspector, V. Müller and A. Honigmann for support with the FCS measurements, and U. Gemm for support with the electronics. C. Wurm, T. Gilat and E. Rothermel helped prepare samples.

Author information

Author notes

    • Giuseppe Vicidomini
    •  & Kyu Y Han

    Present addresses: Italian Institute of Technology, Department of Nanophysics, Genoa, Italy (G.V.) and Department of Physics, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA (K.Y.H.).

    • Giuseppe Vicidomini
    • , Gael Moneron
    •  & Kyu Y Han

    These authors contributed equally to this work.

Affiliations

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

    • Giuseppe Vicidomini
    • , Gael Moneron
    • , Kyu Y Han
    • , Volker Westphal
    • , Haisen Ta
    • , Christian Eggeling
    •  & Stefan W Hell
  2. German Cancer Research Center, Optical Nanoscopy Division, Heidelberg, Germany.

    • Matthias Reuss
    • , Johann Engelhardt
    •  & Stefan W Hell

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Contributions

G.V., G.M., J.E., C.E. and S.W.H. conceived and designed the study. G.V. performed theoretical studies. V.W. designed electronic components. G.V., G.M., K.Y.H., H.T. and M.R. performed experiments. G.V., G.M., K.Y.H. and C.E. analyzed data. G.V., G.M., C.E. and S.W.H. wrote the manuscript. All authors discussed the conceptual and practical implications of the method at all stages.

Competing interests

G.V., G.M., K.Y.H, V.W., M.R., J.E., C.E. and S.W.H. have filed a patent application on the method presented.

Corresponding author

Correspondence to Stefan W Hell.

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    Supplementary Text and Figures

    Supplementary Figures 1–8 and Supplementary Note 1

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DOI

https://doi.org/10.1038/nmeth.1624

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