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Diffraction-unlimited all-optical imaging and writing with a photochromic GFP

Abstract

Lens-based optical microscopy failed to discern fluorescent features closer than 200 nm for decades, but the recent breaking of the diffraction resolution barrier by sequentially switching the fluorescence capability of adjacent features on and off is making nanoscale imaging routine. Reported fluorescence nanoscopy variants switch these features either with intense beams at defined positions or randomly, molecule by molecule. Here we demonstrate an optical nanoscopy that records raw data images from living cells and tissues with low levels of light. This advance has been facilitated by the generation of reversibly switchable enhanced green fluorescent protein (rsEGFP), a fluorescent protein that can be reversibly photoswitched more than a thousand times. Distributions of functional rsEGFP-fusion proteins in living bacteria and mammalian cells are imaged at <40-nanometre resolution. Dendritic spines in living brain slices are super-resolved with about a million times lower light intensities than before. The reversible switching also enables all-optical writing of features with subdiffraction size and spacings, which can be used for data storage.

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Figure 1: Properties of rsEGFP.
Figure 2: Rewritable data storage.
Figure 3: RESOLFT nanoscopy of living cells.
Figure 4: Subdiffraction-resolution writing and reading using rsEGFP and visible light.

References

  1. Hell, S. W. & Wichmann, J. Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt. Lett. 19, 780–782 (1994)

    Article  ADS  CAS  Google Scholar 

  2. Hell, S. W. & Kroug, M. Ground-state depletion fluorescence microscopy, a concept for breaking the diffraction resolution limit. Appl. Phys. B 60, 495–497 (1995)

    Article  ADS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  4. Klar, T. A., Jakobs, S., Dyba, M., Egner, A. & Hell, S. W. Fluorescence microscopy with diffraction resolution barrier broken by stimulated emission. Proc. Natl Acad. Sci. USA 97, 8206–8210 (2000)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Hell, S. W. Microscopy and its focal switch. Nature Methods 6, 24–32 (2009)

    Article  CAS  Google Scholar 

  7. Westphal, V. et al. Video-rate far-field optical nanoscopy dissects synaptic vesicle movement. Science 320, 246–249 (2008)

    Article  ADS  CAS  Google Scholar 

  8. Hein, B., Willig, K. I. & Hell, S. W. Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell. Proc. Natl Acad. Sci. USA 105, 14271–14276 (2008)

    Article  ADS  CAS  Google Scholar 

  9. Eggeling, C. et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2009)

    Article  ADS  CAS  Google Scholar 

  10. Nägerl, U. V., Willig, K. I., Hein, B., Hell, S. W. & Bonhoeffer, T. Live-cell imaging of dendritic spines by STED microscopy. Proc. Natl Acad. Sci. USA 105, 18982–18987 (2008)

    Article  ADS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  12. Hell, S. W., Dyba, M. & Jakobs, S. Concepts for nanoscale resolution in fluorescence microscopy. Curr. Opin. Neurobiol. 14, 599–609 (2004)

    Article  CAS  Google Scholar 

  13. Hell, S. W. Strategy for far-field optical imaging and writing without diffraction limit. Phys. Lett. 326, 140–145 (2004)

    Article  CAS  Google Scholar 

  14. Gustafsson, M. G. L. Nonlinear structured-illumination microscopy: wide-field fluorescence imaging with theoretically unlimited resolution. Proc. Natl Acad. Sci. USA 102, 13081–13086 (2005)

    Article  ADS  CAS  Google Scholar 

  15. Hell, S. W. in Topics in Fluorescence Spectroscopy Vol. 5 (ed. Lakowicz, J. R.) 361–422 (Plenum, 1997)

    Google Scholar 

  16. 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  ADS  CAS  Google Scholar 

  17. Lukyanov, K. A. et al. Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J. Biol. Chem. 275, 25879–25882 (2000)

    Article  CAS  Google Scholar 

  18. Schwentker, M. A. et al. Wide-field subdiffraction RESOLFT microscopy using fluorescent protein photoswitching. Microsc. Res. Tech. 70, 269–280 (2007)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  20. 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 

  21. Huang, B., Babcock, H. & Zhuang, X. Breaking the diffraction barrier: super-resolution imaging of cells. Cell 143, 1047–1058 (2010)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  24. Rust, M. J., Bates, M. & Zhuang, X. Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nature Methods 3, 793–796 (2006)

    Article  CAS  Google Scholar 

  25. Dickson, R. M., Cubitt, A. B., Tsien, R. Y. & Moerner, W. E. On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature 388, 355–358 (1997)

    Article  ADS  CAS  Google Scholar 

  26. Bossi, M., Foelling, J., Dyba, M., Westphal, V. & Hell, S. W. Breaking the diffraction resolution barrier in far-field microscopy by molecular optical bistability. N. J. Phys. 8, 275 (2006)

    Article  Google Scholar 

  27. Scott, T. F., Kowalski, B. A., Sullivan, A. C., Bowman, C. N. & McLeod, R. R. Two-color single-photon photoinitiation and photoinhibition for subdiffraction photolithography. Science 324, 913–917 (2009)

    Article  ADS  CAS  Google Scholar 

  28. Li, L., Gattass, R. R., Gershgoren, E., Hwang, H. & Fourkas, J. T. Achieving l/20 resolution by one-color initiation and deactivation of polymerization. Science 324, 910–913 (2009)

    Article  ADS  CAS  Google Scholar 

  29. Andrew, T. L., Tsai, H. Y. & Menon, R. Confining light to deep subwavelength dimensions to enable optical nanopatterning. Science 324, 917–921 (2009)

    Article  ADS  CAS  Google Scholar 

  30. Fischer, J., Freymann, G. & Wegener, M. The materials challenge in diffraction-unlimited direct-laser-writing optical lithography. Adv. Mater. 22, 3578–3582 (2010)

    Article  CAS  Google Scholar 

  31. Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996)

    Article  ADS  Google Scholar 

  32. 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  ADS  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  34. 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  ADS  CAS  Google Scholar 

  35. Adam, V. et al. Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations. Proc. Natl Acad. Sci. USA 105, 18343–18348 (2008)

    Article  ADS  CAS  Google Scholar 

  36. Brakemann, T. et al. Molecular basis of the light-driven switching of the photochromic fluorescent protein Padron. J. Biol. Chem. 285, 14603–14609 (2010)

    Article  CAS  Google Scholar 

  37. 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 

  38. Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002)

    Article  ADS  CAS  Google Scholar 

  39. Bizzarri, R. et al. Single amino acid replacement makes Aequorea victoria fluorescent proteins reversibly photoswitchable. J. Am. Chem. Soc. 132, 85–95 (2010)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. Adam, V. et al. Data storage based on photochromic and photoconvertible fluorescent proteins. J. Biotechnol. 149, 289–298 (2010)

    Article  CAS  Google Scholar 

  42. Vats, P. & Rothfield, L. Duplication and segregation of the actin (MreB) cytoskeleton during the prokaryotic cell cycle. Proc. Natl Acad. Sci. USA 104, 17795–17800 (2007)

    Article  ADS  CAS  Google Scholar 

  43. Riedl, J. et al. Lifeact: a versatile marker to visualize F-actin. Nature Methods 5, 605–607 (2008)

    Article  CAS  Google Scholar 

  44. Harris, K. M. & Kater, S. B. Dendritic spines: cellular specializations imparting both stability and flexibility to synaptic function. Annu. Rev. Neurosci. 17, 341–371 (1994)

    Article  CAS  Google Scholar 

  45. Hopt, A. & Neher, E. Highly nonlinear photodamage in two-photon fluorescence microscopy. Biophys. J. 80, 2029–2036 (2001)

    Article  ADS  CAS  Google Scholar 

  46. Heintzmann, R., Jovin, T. M. & Cremer, C. Saturated patterned excitation microscopy—a concept for optical resolution improvement. JOSA A 19, 1599–1609 (2002)

    Article  ADS  Google Scholar 

  47. Hell, S. W., Schmidt, R. & Egner, A. Diffraction-unlimited three-dimensional optical nanoscopy with opposing lenses. Nature Photon. 3, 381–387 (2009)

    Article  ADS  CAS  Google Scholar 

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Acknowledgements

We thank J. Jethwa for careful reading and M. Andresen, T. Brakemann, S. Löbermann, R. Schmitz-Salue and A. C. Stiel for discussions and support, as well as T. Gilat and F. Voss (MPI of Neurobiology, Munich) for help with the slice culture preparation and A. Schönle for adapting the software Imspector. We thank The Project Gutenberg for making Grimm’s Fairy Tales available in electronic format, L. Rothfield (University of Connecticut Health Center) for providing the plasmid pLE7, R. Wedlich-Söldner (MPI of Biochemistry, Munich) for the lifeact–YFP construct and V. Stein (MPI of Neurobiology, Munich) for the virus protocol. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the DFG-Research Center for Molecular Physiology of the Brain (to S.J.) and by a Gottfried-Wilhelm-Leibniz prize of the DFG (to S.W.H.).

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Contributions

T.G., I.T., M.L., H.B., F.L.-C. performed research, I.T., M.L., T.G., H.B., C.E. set up the microscopes, N.T.U., K.I.W. prepared samples, M.L., T.G., I.T., K.I.W., S.J., S.W.H. analysed data, S.J., C.E., S.W.H. designed research. S.J., M.L., S.W.H. wrote the paper. All authors discussed the data and commented on the manuscript.

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Correspondence to Stefan Jakobs or Stefan W. Hell.

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Grotjohann, T., Testa, I., Leutenegger, M. et al. Diffraction-unlimited all-optical imaging and writing with a photochromic GFP. Nature 478, 204–208 (2011). https://doi.org/10.1038/nature10497

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