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Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy

Nature Biotechnology volume 26, pages 1035–1040 (2008)Cite this article

Abstract

Fluorescent proteins that can be reversibly photoswitched between a fluorescent and a nonfluorescent state are important for innovative microscopy schemes, such as protein tracking1, fluorescence resonance energy transfer imaging2, sub-diffraction resolution microscopy3,4,5,6,7,8,9 and others. However, all available monomeric reversibly switchable fluorescent proteins (RSFPs) have similar properties and switching characteristics10,11,12, thereby limiting their use. Here, we introduce two bright green fluorescent RSFPs, bsDronpa and Padron, generated by extensive mutagenesis of the RSFP Dronpa10, with unique absorption and switching characteristics. Whereas bsDronpa features a broad absorption spectrum extending into the UV, Padron displays a switching behavior that is reversed to that of all green fluorescent RSFPs known to date. These two RSFPs enable live-cell fluorescence microscopy with multiple labels using a single detection color, because they can be distinguished by photoswitching. Furthermore, we demonstrate dual-color fluorescence microscopy with sub-diffraction resolution using bsDronpa and Dronpa whose emission maxima are separated by <20 nm.

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Figure 1: Properties of rsFastLime, Padron and bsDronpa.
Figure 2: Monochromatic triple-label imaging in living E. coli cells.
Figure 3: 3D confocal time-lapse imaging of living budding yeast cells expressing rsFastLime and Padron using reversible switching to discriminate between the fluorescence signals from the two proteins.
Figure 4: Dual-color, single-molecule-switching-based nanoscopy image of cryosections of E. coli cells expressing membrane-anchored bsDronpa or Dronpa.

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References

  1. Chudakov, D.M., Chepurnykh, T.V., Belousov, V.V., Lukyanov, S. & Lukyanov, K.A. Fast and precise protein tracking using repeated reversible photoactivation. Traffic 7, 1304–1310 (2006).

    Article  CAS  Google Scholar 

  2. Jares-Erijman, E.A. & Jovin, T.M. FRET imaging. Nat. Biotechnol. 21, 1387–1395 (2003).

    Article  CAS  Google Scholar 

  3. Hell, S.W., Jakobs, S. & Kastrup, L. Imaging and writing at the nanoscale with focused visible light through saturable optical transitions. Appl. Phys. A 77, 859–860 (2003).

    Article  CAS  Google Scholar 

  4. Hell, S.W. Toward fluorescence nanoscopy. Nat. Biotechnol. 21, 1347–1355 (2003).

    Article  CAS  Google Scholar 

  5. Hofmann, M., Eggeling, C., Jakobs, S. & Hell, S.W. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc. Natl. Acad. Sci. USA 102, 17565–17569 (2005).

    Article  CAS  Google Scholar 

  6. Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    Article  CAS  Google Scholar 

  7. Hess, S.T., Girirajan, T.P. & Mason, M.D. Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys. J. 91, 4258–4272 (2006).

    Article  CAS  Google Scholar 

  8. Dedecker, P. et al. Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: numerical analysis and application to the fluorescent protein Dronpa. J. Am. Chem. Soc. 129, 16132–16141 (2007).

    Article  CAS  Google Scholar 

  9. Egner, A. et al. Fluorescence nanoscopy in whole cells by asynchronous localization of photoswitching emitters. Biophys. J. 93, 3285–3290 (2007).

    Article  CAS  Google Scholar 

  10. Ando, R., Mizuno, H. & Miyawaki, A. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306, 1370–1373 (2004).

    Article  CAS  Google Scholar 

  11. Ando, R., Flors, C., Mizuno, H., Hofkens, J. & Miyawaki, A. Highlighted generation of fluorescence signals using simultaneous two-color irradiation on Dronpa mutants. Biophys. J. 92, L97–L99 (2007).

    Article  CAS  Google Scholar 

  12. Stiel, A.C. et al. 1.8 A bright-state structure of the reversibly switchable fluorescent protein Dronpa guides the generation of fast switching variants. Biochem. J. 402, 35–42 (2007).

    Article  CAS  Google Scholar 

  13. Henderson, J.N., Ai, H.W., Campbell, R.E. & Remington, S.J. Structural basis for reversible photobleaching of a green fluorescent protein homologue. Proc. Natl. Acad. Sci. USA 104, 6672–6677 (2007).

    Article  CAS  Google Scholar 

  14. Kogure, T. et al. A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy. Nat. Biotechnol. 24, 577–581 (2006).

    Article  CAS  Google Scholar 

  15. Ai, H.W., Shaner, N.C., Cheng, Z., Tsien, R.Y. & Campbell, R.E. Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins. Biochemistry 46, 5904–5910 (2007).

    Article  CAS  Google Scholar 

  16. Andresen, M. et al. Structural basis for reversible photoswitching in Dronpa. Proc. Natl. Acad. Sci. USA 104, 13005–13009 (2007).

    Article  CAS  Google Scholar 

  17. Habuchi, S. et al. Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa. Proc. Natl. Acad. Sci. USA 102, 9511–9516 (2005).

    Article  CAS  Google Scholar 

  18. Habuchi, S. et al. Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching. Photochem. Photobiol. Sci. 5, 567–576 (2006).

    Article  CAS  Google Scholar 

  19. Chudakov, D.M., Feofanov, A.V., Mudriku, N.N., Lukyanov, S. & Lukyanov, K.A. Chromophore environment provides clue to “kindling fluorescent protein” riddle. J. Biol. Chem. 278, 7215–7219 (2003).

    Article  CAS  Google Scholar 

  20. Andresen, M. et al. Structure and mechanism of the reversible photoswitch of a fluorescent protein. Proc. Natl. Acad. Sci. USA 102, 13070–13074 (2005).

    Article  CAS  Google Scholar 

  21. Juskaitis, R. & Wilson, T. A method for characterizing longitudinal chromatic aberration of microscope objectives using a confocal optical system. J. Microsc. 195, 17–22 (1999).

    Article  Google Scholar 

  22. Mulholland, J. et al. Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane. J. Cell Biol. 125, 381–391 (1994).

    Article  CAS  Google Scholar 

  23. Wiedenmann, J. et al. A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria). Proc. Natl. Acad. Sci. USA 99, 11646–11651 (2002).

    Article  CAS  Google Scholar 

  24. Hell, S.W. Far-field optical nanoscopy. Science 316, 1153–1158 (2007).

    Article  CAS  Google Scholar 

  25. Rust, M., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods 3, 793–795 (2006).

    Article  CAS  Google Scholar 

  26. Schonle, A. & Hell, S.W. Fluorescence nanoscopy goes multicolor. Nat. Biotechnol. 25, 1234–1235 (2007).

    Article  Google Scholar 

  27. Bossi, M. et al. Multicolor far-field fluorescence nanoscopy through isolated detection of distinct molecular species. Nano Lett. 8, 2463–2468 (2008).

    Article  CAS  Google Scholar 

  28. Drew, D.E., von Heijne, G., Nordlund, P. & de Gier, J.W. Green fluorescent protein as an indicator to monitor membrane protein overexpression in Escherichia coli . FEBS Lett. 507, 220–224 (2001).

    Article  CAS  Google Scholar 

  29. Patterson, G.H., Knobel, S.M., Sharif, W.D., Kain, S.R. & Piston, D.W. Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys. J. 73, 2782–2790 (1997).

    Article  CAS  Google Scholar 

  30. De Antoni, A. & Gallwitz, D. A novel multi-purpose cassette for repeated integrative epitope tagging of genes in Saccharomyces cerevisiae . Gene 246, 179–185 (2000).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Löbermann and J. Schilde for excellent technical assistance and J. Jethwa for carefully reading the manuscript. We acknowledge J.-W.L. de Gier, Stockholm University, Sweden, for providing us with the expression vector harboring the M13-GFP fusion.

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Authors and Affiliations

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

    Martin Andresen, Andre C Stiel, Jonas Fölling, Andreas Schönle, Alexander Egner, Christian Eggeling, Stefan W Hell & Stefan Jakobs

  2. Laboratory of Electron Microscopy, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, 37077, Germany

    Dirk Wenzel

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Contributions

M.A. performed experiments, analyzed data and wrote the manuscript. A.C.S., J.F. and D.W. performed experiments. A.S. and A.E. provided algorithms and software for sub-diffraction resolution microscopy. C.E. constructed the fluorescence microscope utilized for screening. S.W.H. designed research and wrote the manuscript. S.J. analyzed data, designed research and wrote the manuscript; all authors contributed to editing the manuscript.

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Correspondence to Stefan Jakobs.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Tables 1–3, Methods (PDF 1936 kb)

Supplementary Movie 1

3D confocal time lapse imaging of live cells expressing rsFastLime and Padron (MOV 209 kb)

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Andresen, M., Stiel, A., Fölling, J. et al. Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy. Nat Biotechnol 26, 1035–1040 (2008). https://doi.org/10.1038/nbt.1493

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