Skip to main content

Advertisement

SpringerLink
Log in

Higher resolution in localizationmicroscopy by slower switching of a photochromic protein

  • Paper
  • Published:
Photochemical & Photobiological Sciences Aims and scope Submit manuscript

Abstract

Photoswitchable fluorophores play an essential role in super-resolution fluorescence microscopy, including techniques such as photoactivated localizationmicroscopy (PALM). A determining factor in the precision of the images generated by PALM measurements is the photon numbers that can be detected from the fluorophores. Dronpa is a reversibly photoswitchable fluorescent protein that has been successfully used in PALM experiments. The number of photons per switching cycle that can be acquired for Dronpa depends on its off-switching rate, limiting the number of photons that can be recorded. In this study we report our discovery that the tetrameric ancestor of Dronpa, 22G, shows slower switching, and develop a mutant that displays switching kinetics between those of Dronpa and 22G. We show that the kinetics of the photoswitching are strongly related to self-association of the protein, supporting our view of dynamic flexibility as determining in the photoswitching. Similarly we find that higher-resolution PALM images can be acquired with slower-switching proteins due to their higher number of emitted photons per switching cycle.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Notes and references

  1. R. Y. Tsien, The green fluorescent protein, Annu. Rev. Biochem., 1998, 67, 509–544.

    CAS  PubMed  Google Scholar 

  2. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz and H. F. Hess, Imaging intracellular fluorescent proteins at nanometer resolution, Science, 2006, 313, 1642–1645.

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. C. Flors, J. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki and J. Hofkens, A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants, J. Am. Chem. Soc., 2007, 129, 13970–13977.

    Article  CAS  PubMed  Google Scholar 

  5. K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn and S. W. Hell, STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis, Nature, 2006, 440, 935–939

    Article  CAS  PubMed  Google Scholar 

  6. K. I. Willig, R. R. Kellner, R. Medda, B. Hein, S. Jakobs and S. W. Hell, Nanoscale resolution in GFP-based microscopy, Nat. Methods, 2006, 3, 721–723

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. P. Dedecker, J. Hotta, C. Flors, M. Sliwa, H. Uji-i, M. B. Roeffaers, R. Ando, H. Mizuno, A. Miyawaki and J. Hofkens, 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., 2007, 129, 16132–16141

    Article  CAS  PubMed  Google Scholar 

  9. K. I. Willig, B. Harke, R. Medda, S.W. Hell, M, STED microscopy with continuous wave beams, Nat. Methods, 2007, 4, 915–918

    Article  CAS  PubMed  Google Scholar 

  10. P. Dedecker, J. Hofkens and J. Hotta, Diffraction-unlimited optical microscopy, Mater. Today, 2008, 11, 12–21

    Article  Google Scholar 

  11. M. Heilemann, S. van de Linde, M. Schüttpelz, R. Kasper, B. Seefeldt, A. Mukherjee, P. Tinnefeld and M. Sauer, Subdiffraction-resolution fluorescence imaging with conventional fluorescent probes, Angew. Chem., Int. Ed., 2008, 47, 6172–6176

    Article  CAS  Google Scholar 

  12. N. R. Conley, J.S. Biteen and W. E. Moerner, Cy3-Cy5 covalent heterodimers for single-molecule photoswitching, J. Phys. Chem. B, 2008, 112, 11878–11880

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. S. van de Linde, M. Sauer and M. Heilemann, Subdiffraction-resolution fluorescence imaging of proteins in the mitochondrial inner membrane with photoswitchable fluorophores, J. Struct. Biol., 2008, 164, 250–254

    Article  PubMed  Google Scholar 

  14. M. Heilemann, S. van de Linde, A. Mukherjee and M. Sauer, Super-resolution imaging with small organic fluorophores, Angew. Chem., Int. Ed., 2009, 48, 6903–6908

    Article  CAS  Google Scholar 

  15. S. van de Linde, U. Endesfelder, A. Mukherjee, M. Schüttpelz, G. Wiebusch, S. Wolter, M. Heilemann and M. Sauer, Multicolor photoswitching microscopy for subdiffraction-resolution fluorescence imaging, Photochem. Photobiol. Sci., 2009, 8, 465–469

    Article  PubMed  Google Scholar 

  16. J. Vogelsang, T. Cordes, C. Forthmann, C. Steinhauer and P. Tinnefeld, Controlling the fluorescence of ordinary oxazine dyes for single-molecule switching and superresolution microscopy, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 8107–8112.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. A. Miyawaki, Fluorescent proteins in a new light, Nat. Biotechnol., 2004, 22, 1374–1376

    Article  CAS  PubMed  Google Scholar 

  18. K. A. Lukyanov, D. M. Chudakov, S. Lukyanov and V. V. Verkhusha, Innovation: Photoactivatable fluorescent proteins, Nat. Rev. Mol. Cell Biol., 2005, 6, 885–891

    Article  CAS  PubMed  Google Scholar 

  19. T. J. Gould, V.V. Verkhusha and S. T. Hess, Imaging biological structures with fluorescence photoactivation localization microscopy, Nat. Protoc., 2009, 4, 291–308

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. S. A. McKinney, C. S. Murphy, K. L. Hazelwood, M.W. Davidson and L. L. Looger, A bright and photostable photoconvertible fluorescent protein, Nat. Methods, 2009, 6, 131–133.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. R. Ando, H. Hama, M. Yamamoto-Hino, H. Mizuno and A. Miyawaki, An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 12651–12656

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. J. Wiedenmann, S. Ivanchenko, F. Oswald, F. Schmitt, C. Röcker, A. Salih, K.D. Spindler and G. U. Nienhaus, EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion, Proc. Natl. Acad. Sci. U. S. A., 2004, 101, 15905–15910

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. H. Tsutsui, S. Karasawa, H. Shimizu, N. Nukina and A. Miyawaki, Semi-rational engineering of a coral fluorescent protein into an efficient highlighter, EMBO Rep., 2005, 6, 233–238

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. N. G. Gurskaya, V. V. Verkhusha, A. S. Shcheglov, D. B. Staroverov, T. V. Chepurnykh, A. F. Fradkov, S. Lukyanov and K. A. Lukyanov, Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light, Nat. Biotechnol., 2006, 24, 461–465.

    Article  CAS  PubMed  Google Scholar 

  25. G.H. Patterson, J. Lippincott-Schwartz, A photoactivatable GFP for selective photolabeling of proteins and cells, Science, 2002, 297, 1873–1877

    Article  CAS  PubMed  Google Scholar 

  26. F. V. Subach, G. H. Patterson, S. Manley, J. M. Gillette, J. Lippincott-Schwartz and V. V. Verkhusha, Photoactivatable mCherry for high-resolution two-color fluorescence microscopy, Nat. Methods, 2009, 6, 153–159.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. H. Mizuno, T. K. Mal, K. I. Tong, R. Ando, T. Furuta, M. Ikura and A. Miyawaki, Photo-induced peptide cleavage in the green-to-red conversion of a fluorescent protein, Mol. Cell, 2003, 12, 1051–1058

    Article  CAS  PubMed  Google Scholar 

  28. K. Nienhaus, G. U. Nienhaus, J. Wiedenmann and H. Nar, Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 9156–9159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. J. N. Henderson, R. Gepshtein, J. R. Heenan, K. Kallio, D. Huppert and S. J. Remington, Structure and mechanism of the photoactivatable green fluorescent protein, J. Am. Chem. Soc., 2009, 131, 4176–4177.

    Article  CAS  PubMed  Google Scholar 

  30. R. Ando, H. Mizuno and A. Miyawaki, Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting, Science, 2004, 306, 1370–1373.

    Article  CAS  PubMed  Google Scholar 

  31. H. Mizuno, T. K. Mal, M. Wälchli, A. Kikuchi, T. Fukano, R. Ando, J. Jeyakanthan, J. Taka, Y. Shiro, M. Ikura and A. Miyawaki, Light-dependent regulation of structural flexibility in a photochromic fluorescent protein, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 9227–9232.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. A. C. Stiel, M. Andresen, H. Bock, M. Hilbert, J. Schilde, A. Schönle, C. Eggeling, A. Egner, S.W. Hell and S. Jakobs, Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy, Biophys. J., 2008, 95, 2989–2997

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. V. Adam, M. Lelimousin, S. Boehme, G. Desfonds, K. Nienhaus, M. J. Field, J. Wiedenmann, S. McSweeney, G.U. Nienhaus and D. Bourgeois, Structural characterization of IrisFP, an optical highlighter undergoingmultiple photo-induced transformations, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 18343–18348.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. M. Andresen, A. C. Stiel, J. Fölling, D. Wenzel, A. Schönle, A. Egner, C. Eggeling, S.W. Hell and S. Jakobs, Photoswitchable fluorescent proteins enable monochromatic multilabel imaging and dual color fluorescence nanoscopy, Nat. Biotechnol., 2008, 26, 1035–1040

    Article  CAS  PubMed  Google Scholar 

  36. P. Dedecker, J. Hotta, R. Ando, A. Miyawaki, Y. Engelborghs and J. Hofkens, Fast and reversible photoswitching of the fluorescent protein dronpa as evidenced by fluorescence correlation spectroscopy, Biophys. J., 2006, 91, L45–47.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki and J. Hofkens, Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa, Proc. Natl. Acad. Sci. U. S. A., 2005, 102, 9511–9516

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. S. Habuchi, P. Dedecker, J. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki and J. Hofkens, Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching, Photochem. Photobiol. Sci., 2006, 5, 567–576.

    Article  CAS  PubMed  Google Scholar 

  39. H. Shroff, C. G. Galbraith, J. A. Galbraith, H. White, J. Gillette, S. Olenych, M.W. Davidson and E. Betzig, Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes, Proc. Natl. Acad. Sci. U. S. A., 2007, 104, 20308–20813

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. A. Vaziri, J. Tang, H. Shroff and C. V. Shank, Multilayer three-dimensional super resolution imaging of thick biological samples, Proc. Natl. Acad. Sci. U. S. A., 2008, 105, 20221–20226.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. A. Sawano and A. Miyawaki, Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis, Nucleic Acids Res., 2000, 28, 78e

    Article  Google Scholar 

  42. N. C. Shaner, M. Z. Lin, M. R. McKeown, P. A. Steinbach, K. L. Hazelwood, M.W. Davidson and R. Y. Tsien, Improving the photostability of bright monomeric orange and red fluorescent proteins, Nat. Methods, 2008, 5, 545–551.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Cell Function and Dynamics, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan

    Hideaki Mizuno, Ryoko Ando, Takashi Fukano & Atsushi Miyawaki

  2. Department of Chemistry and INPAC, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001, Heverlee, Belgium

    Hideaki Mizuno, Peter Dedecker & Johan Hofkens

Authors
  1. Hideaki Mizuno
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Peter Dedecker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ryoko Ando
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Takashi Fukano
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Johan Hofkens
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Atsushi Miyawaki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Hideaki Mizuno.

Additional information

This paper is part of a themed issue on synthetic and natural photoswitches.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mizuno, H., Dedecker, P., Ando, R. et al. Higher resolution in localizationmicroscopy by slower switching of a photochromic protein. Photochem Photobiol Sci 9, 239–248 (2010). https://doi.org/10.1039/b9pp00124g

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1039/b9pp00124g

Search

Navigation