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

Nature volume 478, pages 204–208 (2011)Cite this article

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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.

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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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Author notes

  1. Tim Grotjohann, Ilaria Testa and Marcel Leutenegger: These authors contributed equally to this work.

Authors and Affiliations

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

    Tim Grotjohann, Ilaria Testa, Marcel Leutenegger, Hannes Bock, Nicolai T. Urban, Flavie Lavoie-Cardinal, Katrin I. Willig, Christian Eggeling, Stefan Jakobs & Stefan W. Hell

  2. University of Göttingen Medical School, Robert-Koch-Str. 40, 37075 Göttingen, Germany ,

    Stefan Jakobs

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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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The authors declare no competing financial interests.

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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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