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Spiropyrans as molecular optical switches

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Abstract

Optical microscopes use visible light and an arrangement of lenses to provide us with magnified images of small samples. Combined with efficient fluorescent probes and highly sensitive fluorescence detection techniques they allow the non-invasive 3D study of subcellular structures even in living cells or tissue. However, optical microscopes are subject to diffraction of light which limits optical resolution to approximately 200 nm in the imaging plane. In the recent past, powerful methods emerged that enable fluorescence microscopy with subdiffraction optical resolution. Since most of these methods are based on the temporal control of fluorescence emission of fluorophores, photochromic molecules that can be switched reversibly between a fluorescent on- and a non-fluorescent off-state are the key for super-resolution imaging methods. Here, we present our approach to use spiropyran-fluorophore conjugates as efficient molecular optical switches (photoswitches). In these photochromic conjugates fluorescence emission of the fluorophore is controlled by the state of the spiropyran, which can be switched reversibly between a colorless spiropyran and a colored merocyanine form upon irradiation with light. Thus, the efficiency of energy transfer from the fluorophore to the spiropyran can be modulated by the irradiation conditions. We present ensemble data of the switching process of various spiropyrans and spiropyran-fluorophore conjugates and demonstrate photoswitching at the single-molecule level. Our data suggest that spiropyrans have to be immobilized in polymers to stabilize the merocyanine form in order to be useful for super-resolution fluorescence imaging based on precise localization of individual emitters. Special emphasis is put on photobleaching of donor fluorophores due to UV irradiation, i.e. photoswitching of the photochromic acceptor. Furthermore, we present a water soluble switchable spiropyran derivative and demonstrate the first intermolecular single-molecule photoswitching experiments in polymers.

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References

  1. E. Abbe, Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung, Arch. Mikrosk. Anat., 1873, 9, 413–420.

    Article  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  3. N. Ji, H. Shroff, H. Zhong and E. Betzig, Advances in the speed and resolution of light microscopy, Curr. Opin. Neurobiol., 2008, 18, 605–616.

    Article  CAS  PubMed  Google Scholar 

  4. M. Heilemann, P. Dedecker, J. Hofkens and M. Sauer, Photoswitches: Key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification, Laser & Photonics Rev., 2009, 3, 180–202.

    Article  CAS  Google Scholar 

  5. S. W. Hell and J. Wichmann, Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy, Opt. Lett., 1994, 19, 780–783.

    Article  CAS  PubMed  Google Scholar 

  6. T. A. Klar, S. Jakobs, M. Dyba, A. Egner and S. W. Hell, Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission, Proc. Natl. Acad. Sci. U. S. A., 2000, 97, 8206–8210.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. C. Flors, J. Hotta, H. Uji-i, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki and J. Hofkens, Superresolution microscopy on the basis of engineered dark states, J. Am. Chem. Soc., 2007, 129, 13970–13977.

    Article  CAS  PubMed  Google Scholar 

  8. 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, Development and use of fluorescent protein markers in living cells, Science, 2006, 313, 1642–1645.

    Article  CAS  PubMed  Google Scholar 

  9. 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 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. J. Fölling, M. Bossi, H. Bock, R. Medda, C. A. Wurm, B. Hein, S. Jakobs, C. Eggeling and S. W. Hell, Fluorescence nanoscopy by ground-state depletion and single-molecule return, Nat. Methods, 2008, 5, 943–945.

    Article  PubMed  CAS  Google Scholar 

  12. M. Heilemann, S. van de Linde, M. Schuttpelz, 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 

  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  CAS  Google Scholar 

  14. C. Steinhauer, C. Forthmann, J. Vogelsang and P. Tinnefeld, Super-resolution microscopy on the basis of engineered dark states, J. Am. Chem. Soc., 2008, 130, 16840–16841.

    Article  CAS  PubMed  Google Scholar 

  15. S. van de Linde, R. Kasper, M. Heilemann and M. Sauer, Photoswitching microscopy with standard fluorophores, Appl. Phys. B: Lasers Opt., 2008, 93, 725–731.

    Article  CAS  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. 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  CAS  Google Scholar 

  18. S. Wolter, M. Schüttpelz, M. Tscherepanow, S. van de Linde, M. Heilemann and M. Sauer, Real-time computation of subdiffraction-resolution fluorescence images, J. Microsc., 2010, 237, 12–22.

    Article  CAS  PubMed  Google Scholar 

  19. 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 

  20. R. E. Thompson, D. R. Larson and W. W. Webb, Precise Nanometer Localization Analysis for Individual Fluorescent Probes, Biophys. J., 2002, 82, 2775–2783.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. A. Yildiz, M. Tomishige, R. D. Vale and P. R. Selvin, Kinesin walks hand-over-hand, Science, 2004, 303, 676–678.

    Article  CAS  PubMed  Google Scholar 

  22. 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 

  23. 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 

  24. 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 

  25. 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 

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

    Article  CAS  PubMed  Google Scholar 

  27. G. Berkovic, V. Krongauz and V. Weiss, Spiropyrans and Spirooxazines for Memories and Switches, Chem. Rev., 2000, 100, 1741–1754.

    Article  CAS  PubMed  Google Scholar 

  28. M. Irie, Diarylethenes for memories and switches, Chem. Rev., 2000, 100, 1685–1716.

    Article  CAS  PubMed  Google Scholar 

  29. Y. Yokoyama, Fulgides for memories and switches, Chem. Rev., 2000, 100, 1717–1740.

    Article  CAS  PubMed  Google Scholar 

  30. M. Irie, T. Fukaminato, T. Sasaki, N. Tamai and T. Kawai, A digital fluorescent molecular photoswitch, Nature, 2002, 420, 759–760.

    Article  CAS  PubMed  Google Scholar 

  31. T. Fukaminato, T. Sasaki, T. Kawai, N. Tamai and M. Irie, Digital photoswitching of fluorescence based on the photochromism of diarylethene derivatives at a single-molecule level, J. Am. Chem. Soc., 2004, 126, 14843–14849.

    Article  CAS  PubMed  Google Scholar 

  32. X. Sheng, A. Peng, H. Fu, Y. Liu, Y. Zhao, Y. Ma and J. Yao, Modulation of a fluorescence switch based on photochromic spirooxazine in composite organic nanoparticles, Nanotechnology, 2007, 18, 145707–145714.

    Article  CAS  Google Scholar 

  33. Z. Tian, W. Wu and A. D. Q. Li, Photoswitchable fluorescent nanoparticles: preparation, properties and applications, ChemPhysChem, 2009, 10, 2577–2591.

    Article  CAS  PubMed  Google Scholar 

  34. S. Yamamoto, K. Matsuda and M. Irie, Absolute asymmetric photocyclization of a potochromic diarylethene derivative in single crystals, Angew. Chem., Int. Ed., 2003, 42, 1636–1639.

    Article  CAS  Google Scholar 

  35. T. Fukaminato, T. Umemoto, Y. Iwata, S. Yokojima, M. Yo-Neyama, S. Nakamura and M. Irie, Photochromism of diarylethene single molecules in polymer matrices, J. Am. Chem. Soc., 2007, 129, 5932–5938.

    Article  CAS  PubMed  Google Scholar 

  36. S. F. Yan, V. Belov, M. Bossi and S. W. Hell, Switchable fluorescent and solvatochromic molecular probes based on 4-amino-N-methylphthalimide and a photochromic diarylethene, Eur. J. Org.Chem., 2008, 2531–2538.

    Google Scholar 

  37. Photochromism, ed. G. H. Brown, Wiley-Interscience, New York, 1971.

    Google Scholar 

  38. Photochromism: Molecules and Systems, ed. H. Dürr and H. Bouas-Laurent, Elsevier, Amsterdam, 1990.

    Google Scholar 

  39. H. Duerr, Organische Photochromie, Angew. Chem., 2004, 116, 3404–4318.

    Article  Google Scholar 

  40. A. Menju, K. Hayashi and M. Irie, Photoresponsive polymers. 3. Reversible solution viscosity change of poly(methacrylic acid) having spirobenzopyran pendant groups in methanol, Macromolecules, 1981, 14, 755–758.

    Article  CAS  Google Scholar 

  41. V. I. Minkin, Photo-, thermo-, solvato- and electrochromic spiroheterocyclic compounds, Chem. Rev., 2004, 104, 2751–2776.

    Article  CAS  PubMed  Google Scholar 

  42. T. Minami, N. Tamai, T. Yamazaki and L. Yamazaki, Picosecond time-resolved fluorescence spectroscopy of the photochromic reaction of spiropyran in Langmuir-Blodgett films, J. Phys. Chem., 1991, 95, 3988–3993.

    Article  CAS  Google Scholar 

  43. J. L. Bahr, G. Kodis, L. de la Garza, S. Lin, A. L. Moore, T. A. Moore and D. Gust, Photoswitched singlet energy transfer in a porphyrin−spiropyran dyad, J. Am. Chem. Soc., 2001, 123, 7124–7133.

    Article  CAS  PubMed  Google Scholar 

  44. M. Q. Zhu, L. Zhu, J. J. Han, W. Wu, J. K. Hurst and A. D. Q. Li, Spiropyran-based photochromic polymer nanoparticles with optically switchable luminescence, J. Am. Chem. Soc., 2006, 128, 4303–4309.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. L. Zhu, W. Wu, M. Q. Zhu, J. J. Han, J. K. Hurst and A. D. Q. Li, Reversibly photoswitchable dual-color fluorescent nanoparticles as new tools for live-cell imaging, J. Am. Chem. Soc., 2007, 129, 3524–3526.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. M. Bossi, V. Belov, S. Polyakova and S. W. Hell, Reversible red fluorescent molecular switch, Angew. Chem., Int. Ed., 2006, 45, 7462–7465.

    Article  CAS  Google Scholar 

  47. J. Fölling, S. Polyakova, V. Belov and A. van Blaaderen, et al., Synthesis and characterization of photoswitchable fluorescent silica nanoparticles, Small, 2008, 4, 134–142.

    Article  CAS  PubMed  Google Scholar 

  48. A. S. Dvornikov and P. M. Rentzepis, Photochromism: non-linear picosecond kinetics and 3D computer memory, Mol. Cryst. Liq. Cryst., 1994, 246, 379–388.

    Article  CAS  Google Scholar 

  49. M. Heilemann, E. Margeat, R. Kasper, M. Sauer and P. Tinnefeld, Carbocyanine dyes as efficient reversible single-molecule optical switch, J. Am. Chem. Soc., 2005, 127, 3801–3806.

    Article  CAS  PubMed  Google Scholar 

  50. J. Vogelsang, T. Cordes and P. Tinnefeld, Single-molecule photophysics of oxazines on DNA and its application in a FRET switch, Photochem. Photobiol. Sci., 2009, 8, 486–492.

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Applied Laser Physics and Laser Spectroscopy and Bielefeld Institute for Biophysics and Nanoscience, Bielefeld University, Universitätsstrasse 25, 33615, Bielefeld, Germany

    Britta Seefeldt, Robert Kasper & Mike Heilemann

  2. Organic Chemistry I, Bielefeld University, Universitätsstrasse 25, 33615, Bielefeld, Germany

    Mirco Beining & Jochen Mattay

  3. Physical Chemistry, Siegen University, Adolf-Reichwein-Str. 2, 57068, Siegen, Germany

    Jutta Arden-Jacob, Norbert Kemnitzer & Karl Heinz Drexhage

  4. Biotechnology & Biophysics, Julius-Maximilians-University, Am Hubland, 97074, Wuerzburg, Germany

    Markus Sauer

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  1. Britta Seefeldt
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Correspondence to Markus Sauer.

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

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Seefeldt, B., Kasper, R., Beining, M. et al. Spiropyrans as molecular optical switches. Photochem Photobiol Sci 9, 213–220 (2010). https://doi.org/10.1039/b9pp00118b

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