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A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins

Nature Chemistry volume 5, pages 132–139 (2013)Cite this article

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Abstract

The ideal fluorescent probe for bioimaging is bright, absorbs at long wavelengths and can be implemented flexibly in living cells and in vivo. However, the design of synthetic fluorophores that combine all of these properties has proved to be extremely difficult. Here, we introduce a biocompatible near-infrared silicon–rhodamine probe that can be coupled specifically to proteins using different labelling techniques. Importantly, its high permeability and fluorogenic character permit the imaging of proteins in living cells and tissues, and its brightness and photostability make it ideally suited for live-cell super-resolution microscopy. The excellent spectroscopic properties of the probe combined with its ease of use in live-cell applications make it a powerful new tool for bioimaging.

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Figure 1: SiR dyes used for SNAP-, CLIP-, Halo-tag and tetrazine labelling.
Figure 2: Three-colour confocal fluorescence microscopy of the tagged proteins.
Figure 3: Ex vivo labelling of a rat brain with SiR-SNAP.
Figure 4: Live-cell GSDIM/STORM imaging of nuclear localized H2B-SNAP-SiR.
Figure 5: Confocal and STED imaging of Cep41 protein localization in living U2OS cells.
Figure 6: Site-specific labelling of genetically encoded UAAs with SiR-tetrazine.

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Acknowledgements

This work was supported by the Swiss National Science Foundation, the Chemical National Centre of Competence in Research Biology, European Research Council grant no. 243016-PALMassembly and the École Polytechnique Fédérale de Lausanne. G.L. was supported by a Federation of European Biochemical Societies long-term fellowship. K.U. was supported by a Grant-in-Aid for Postdoctoral Fellowships for Foreign Researchers Fellows. C.S. is supported by TRR83, and T.P. by the Fonds der Chemischen Industrie. E.A.L. acknowledges funding from the Emmy Noether program of the Deutsche Forschungsgemeinschaft. E.A.L. and C.S. also acknowledge funding from the SPP 1623 of the Deutsche Forschungsgemeinschaft. The authors thank A. Schena, B. Mollwitz and P. Gönczy for sharing reagents and cell lines, S. Hell and S. Jakobs (MPI Göttingen) for excellent support, Tanja Gilat (Max-Planck Institut (MPI) Göttingen) for preparation of the cells and A. Schönle (MPI Göttingen) for support with the software ImSpector.

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  1. Gražvydas Lukinavičius and Keitaro Umezawa: These authors contributed equally to this work

Authors and Affiliations

  1. Ecole Polytechnique Fédérale de Lausanne, Institute of Chemical Sciences and Engineering (ISIC), Institute of Bioengineering, National Centre of Competence in Research (NCCR) in Chemical Biology, Lausanne, 1015, Switzerland

    Gražvydas Lukinavičius, Keitaro Umezawa, Luc Reymond & Kai Johnsson

  2. Ecole Polytechnique Fédérale de Lausanne, Laboratory of Experimental Biophysics, NCCR in Chemical Biology, Lausanne, 1015, Switzerland

    Nicolas Olivier & Suliana Manley

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

    Alf Honigmann, Veronika Mueller & Christian Eggeling

  4. European Molecular Biology Laboratory, Mouse Biology Unit, via Ramarini 32, Monterotondo (RM), 00015, Italy

    Guoying Yang & Paul Heppenstall

  5. European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, 69117, Germany

    Tilman Plass, Carsten Schultz & Edward A. Lemke

  6. New England Biolabs Inc, 240 County Road, Ipswich, 01938, Massachusetts, USA

    Ivan R. Corrêa Jr

  7. Institute of Neuroscience and State Key Laboratory of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031, China

    Zhen-Ge Luo

  8. Medical Research Council Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, UK

    Christian Eggeling

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  1. Gražvydas Lukinavičius
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Contributions

All authors planned the experiments and co-wrote the paper. K.U. designed the structure of SiR-carboxyl. K.U., L.R. and I.C. performed the chemical syntheses. G.L., K.U. and L.R. characterized the dyes. G.L., A.H. and V.M. performed the confocal and STED microscopy with subsequent data analysis. N.O. and S.M. performed the GSDIM/STORM imaging and data analysis. T.P., C.S. and E.A.L performed the amber suppression experiments and analysis. G.Y., Z-G.L. and P.H. performed the labelling in brain sections.

Corresponding author

Correspondence to Kai Johnsson.

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The authors declare no competing financial interests.

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Lukinavičius, G., Umezawa, K., Olivier, N. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nature Chem 5, 132–139 (2013). https://doi.org/10.1038/nchem.1546

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