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