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Sharper low-power STED nanoscopy by time gating

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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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Figure 1: Principle of g-STED.
Figure 2: g-STED fluorescence nanoscopy.
Figure 3: g-STED-FCS.

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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.

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Author notes

  1. Giuseppe Vicidomini & Kyu Y Han

    Present address: 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.).,

  2. Giuseppe Vicidomini, Gael Moneron and Kyu Y Han: These authors contributed equally to this work.

Authors and Affiliations

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

    Giuseppe Vicidomini, Gael Moneron, Kyu Y Han, Volker Westphal, Haisen Ta, Christian Eggeling & Stefan W Hell

  2. Optical Nanoscopy Division, German Cancer Research Center, Heidelberg, Germany

    Matthias Reuss, Johann Engelhardt & Stefan W Hell

Authors
  1. Giuseppe Vicidomini
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  2. Gael Moneron
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  3. Kyu Y Han
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  4. Volker Westphal
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  5. Haisen Ta
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  6. Matthias Reuss
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  7. Johann Engelhardt
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  8. Christian Eggeling
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  9. 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.

Corresponding author

Correspondence to Stefan W Hell.

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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.

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Supplementary Figures 1–8 and Supplementary Note 1 (PDF 4079 kb)

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Vicidomini, G., Moneron, G., Han, K. et al. Sharper low-power STED nanoscopy by time gating. Nat Methods 8, 571–573 (2011). https://doi.org/10.1038/nmeth.1624

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