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Coordinate-targeted fluorescence nanoscopy with multiple off states

Nature Photonics volume 10, pages 122–128 (2016)Cite this article

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Abstract

Far-field super-resolution fluorescence microscopy discerns fluorophores residing closer than the diffraction barrier by briefly transferring them in different (typically ON and OFF) states before detection. In coordinate-targeted super-resolution variants, such as stimulated emission depletion (STED) microscopy, this state difference is created by the intensity minima and maxima of an optical pattern, causing all fluorophores to assume the off state, for instance, except at the minima. Although strong spatial confinement of the on state enables high resolution, it also subjects the fluorophores to excess intensities and state cycles at the maxima. Here, we address these issues by driving the fluorophores into a second off state that is inert to the excess light. By using reversibly switchable fluorescent proteins as labels, our approach reduces bleaching and enhances resolution and contrast in live-cell STED microscopy. Using two or more transitions to off states is a useful strategy for augmenting the power of coordinate-targeted super-resolution microscopy.

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Figure 1: MOST nanoscopy and protected STED.
Figure 2: Protection by deactivated state OFF2.
Figure 3: Reduced bleaching in protected STED imaging.
Figure 4: Synergistic action of two off transitions on resolution and contrast.
Figure 5: 3D-subdiffraction resolution and dual colour imaging.
Figure 6: 3D-protected STED imaging of neuron in living brain tissue.

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Acknowledgements

We thank T. Gilat and E. Rothermel (both MPI) for help with preparing samples, and J. Keller for discussion. J.G.D. acknowledges support by the European Union through a Marie Curie fellowship PIEF-GA-2011-299283. S.W.H. acknowledges support by the Körber Foundation.

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  1. Johann G. Danzl and Sven C. Sidenstein: These authors contributed equally to this work

Authors and Affiliations

  1. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, 37077, Germany

    Johann G. Danzl, Sven C. Sidenstein, Carola Gregor, Nicolai T. Urban, Peter Ilgen, Stefan Jakobs & Stefan W. Hell

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Contributions

J.G.D. and S.C.S. built the setup, planned the experiments, and evaluated the data. S.C.S. performed the measurements shown. C.G., N.T.U., and P.I. provided samples. S.J. advised on actin and protein labelling. S.W.H. laid out the concept, and initiated and supervised the project. The paper was written by J.G.D. and S.W.H. All authors commented on the data and on the final version of the manuscript.

Corresponding authors

Correspondence to Johann G. Danzl or Stefan W. Hell.

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Competing interests

S.W.H. owns shares in the company Abberior Instruments that supplies STED and RESOLFT systems and benefits through related patents owned by the Max Planck Society.

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Danzl, J., Sidenstein, S., Gregor, C. et al. Coordinate-targeted fluorescence nanoscopy with multiple off states. Nature Photon 10, 122–128 (2016). https://doi.org/10.1038/nphoton.2015.266

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