Abstract
We show that nanoscopy based on the principle called RESOLFT (reversible saturable optical fluorescence transitions) or nonlinear structured illumination can be effectively parallelized using two incoherently superimposed orthogonal standing light waves. The intensity minima of the resulting pattern act as 'doughnuts', providing isotropic resolution in the focal plane and making pattern rotation redundant. We super-resolved living cells in 120 μm × 100 μm–sized fields of view in <1 s using 116,000 such doughnuts.
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Acknowledgements
M. Leutenegger (formerly at Max Planck Institute for Biophysical Chemistry, Göttingen; now at the Ecole Polytechnique Fédérale de Lausanne) is acknowledged for early discussions about technical aspects. A.C. acknowledges a postdoctoral scholarship from the Swedish Research Council. S.J. and S.W.H. acknowledge funding from the German Research Foundation (DFG)–Center of Nanoscale Microscopy and Molecular Physiology of the Brain. S.W.H. also received support from the German Ministry of Research (BMBF) and the Körber Foundation, Hamburg, through the European Science Prize.
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Contributions
A.C. built the setup, performed the experiments and analyzed data. J.K. analyzed data and performed data modeling. T.G. developed and characterized the protein rsEGFP(N205S), advised by S.J. M.R. and E.d'E. prepared samples. C.E. advised on protein switching and setup implementations. S.W.H. defined and supervised the project, wrote the paper and is, together with A.C., responsible for its main thrust.
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The Max Planck Society and S.W.H. hold patent rights (US 7,064,824) on the RESOLFT principle.
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Supplementary Text and Figures
Supplementary Figures 1–12 and Supplementary Notes 1–3 (PDF 7242 kb)
Supplementary Software
MATLAB scripts used for parallelized RESOLFT image reconstruction (ZIP 6 kb)
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Chmyrov, A., Keller, J., Grotjohann, T. et al. Nanoscopy with more than 100,000 'doughnuts'. Nat Methods 10, 737–740 (2013). https://doi.org/10.1038/nmeth.2556
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DOI: https://doi.org/10.1038/nmeth.2556
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