A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching


Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of 365 nm and 405 nm, respectively, whereas fluorescence is elicited at 515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.

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Figure 1: Properties of Dreiklang.
Figure 2: Molecular basis of Dreiklang photoswitching.
Figure 3: Applications of Dreiklang.
Figure 4: Super-resolution microscopy of living PtK2 cells using Dreiklang.

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We acknowledge access to beamline BL14.2 of the BESSY II storage ring (Berlin) through the Joint Berlin MX-Laboratory sponsored by the Helmholtz Zentrum Berlin für Materialien und Energie, the Freie Universität Berlin, the Humboldt-Universität zu Berlin, the Max-Delbrück Centrum and the Leibniz-Institut für Molekulare Pharmakologie. We thank V. Belov for insightful discussions and F. Lavoie-Cardinal for help with the set-up for targeted switching. We acknowledge A. Schönle for providing the software ImSpector. We also thank T. Gilat, S. Löbermann, R. Pick and E. Rothermel for excellent technical assistance, H.-H. Hsiao for help in ESI-MS and H. Schill and J. Jethwa for carefully reading the manuscript. We acknowledge R.Y. Tsien for sharing the plasmid pRSET-Citrine. This work was supported by the Deutsche Forschungsgemeinschaft through the Gottfried Wilhelm Leibniz Prize (to S.W.H.) and through the DFG-Research Center for Molecular Physiology of the Brain (to S.J.).

Author information




G.W., M.A. and I.T. contributed equally to this work. C.E., S.W.H. and S.J. conceived the project. T.B., A.C.S., G.W., M.A., I.T., T.G., M.L. and U.P. performed all experiments. I.T. recorded the super-resolution images. Data analysis was done by T.B., A.C.S., G.W., M.A., I.T., T.G., M.L., H.U., C.E., M.C.W., S.W.H. and S.J. The manuscript was written by S.W.H. and S.J. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Stefan W Hell or Stefan Jakobs.

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Competing interests

A patent application concerning the protein Dreiklang has been filed.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1,2 and Supplementary and Figures 1–17 (PDF 1847 kb)

Supplementary Movie 1

Animated sequence of 33 individual images as shown in Figure 3a. (MPG 232 kb)

Supplementary Movie 2

Animated sequence of 100 consecutive switching cycles of vimentin-Dreiklang in PtK2 cells, as shown in Supplementary Figure 13. (AVI 1396 kb)

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Brakemann, T., Stiel, A., Weber, G. et al. A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nat Biotechnol 29, 942–947 (2011). https://doi.org/10.1038/nbt.1952

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