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Fluorogenic probes for live-cell imaging of the cytoskeleton

Nature Methods volume 11, pages 731–733 (2014)Cite this article

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Abstract

We introduce far-red, fluorogenic probes that combine minimal cytotoxicity with excellent brightness and photostability for fluorescence imaging of actin and tubulin in living cells. Applied in stimulated emission depletion (STED) microscopy, they reveal the ninefold symmetry of the centrosome and the spatial organization of actin in the axon of cultured rat neurons with a resolution unprecedented for imaging cytoskeletal structures in living cells.

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Figure 1: SiR-tubulin and SiR-actin.
Figure 2: Live-cell STED microscopy with SiR-tubulin and SiR-actin.

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Acknowledgements

The authors are grateful to J.E. Bear (University of North Carolina at Chapel Hill), P. Gönczy (École Polytechnique Fédérale de Lausanne (EPFL)), R. Jahn (University of Göttingen), D. Abankwa (Åbo Akademi University), U. Ruegg (University of Geneva) and A. Seitz (EPFL) for sharing reagents and for technical assistance. K.J. acknowledges support from the Swiss National Science Foundation and the National Centre of Competence of Research (NCCR) Chemical Biology. Support from the Körber foundation was received through the European Science Prize to S.W.H. D.W.G. acknowledges support from the European Community's Seventh Framework Programme FP7/2007-2013 under grant agreements nos. 241548 (MitoSys) and 258068 (Systems Microscopy) and from an European Research Council (ERC) Starting Grant (agreement no. 281198).

Author information

Author notes

  1. Gražvydas Lukinavičius and Luc Reymond: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Chemical Sciences and Engineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    Gražvydas Lukinavičius, Luc Reymond, Anastasiya Masharina & Kai Johnsson

  2. National Centre of Competence of Research in Chemical Biology, Lausanne, Switzerland

    Luc Reymond & Kai Johnsson

  3. Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Elisa D'Este, Fabian Göttfert, Haisen Ta & Stefan W Hell

  4. Institute of Organic Chemistry and Macromolecular Chemistry, Friedrich Schiller University, Jena, Germany

    Angelika Güther & Hans-Dieter Arndt

  5. Bioimaging and Optics Platform, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland

    Mathias Fournier

  6. Max Planck Institute of Molecular Physiology, Dortmund, Germany

    Stefano Rizzo & Herbert Waldmann

  7. Institute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria

    Claudia Blaukopf, Christoph Sommer & Daniel W Gerlich

Authors
  1. Gražvydas Lukinavičius
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Contributions

G.L., L.R. and K.J. devised this study. All authors except S.R. and A.G. contributed to manuscript writing. G.L., E.D., A.M., C.B., C.S. and D.W.G. characterized the probes. L.R., A.G. and S.R. performed synthesis of probes, supervised by K.J., H.W. and H.-D.A.; G.L., E.D., F.G. and H.T. performed STED microscopy, guided by S.W.H.; G.L. and M.F. performed SIM microscopy.

Corresponding authors

Correspondence to Hans-Dieter Arndt, Stefan W Hell or Kai Johnsson.

Ethics declarations

Competing interests

G.L. and K.J. have filed a patent application on SiR derivatives (PCT/EP2011/064750). S.R., H.-D.A. and H.W. have filed a patent application on the use of jasplakinolide derivatives (PCT/EP2012/073002).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14, Supplementary Tables 1–3 and Supplementary Notes 1–3 (PDF 7301 kb)

Long-term live-cell microscopy of untreated control HeLa Kyoto cells stably expressing H2B-mRFP and MyrPalm-mEGFP.

The movie shows a region-of-interest of 1/16th of the full image frame and the full movie (0 – 23 h). (MOV 4300 kb)

Long-term live-cell microscopy of HeLa Kyoto cells stably expressing H2B-mRFP and MyrPalm-mEGFP, treated with 100 nM SiR-tubulin.

The movie shows a region-of-interest of 1/16th of the full image frame and the full movie (0 – 23 h), starting 0.5 h after addition of SiR-tubulin. (MOV 10798 kb)

Long-term live-cell microscopy of HeLa Kyoto cells stably expressing H2B-mRFP and MyrPalm-mEGFP, treated with 100 nM SiR-actin.

The movie shows a region-of-interest of 1/16th of the full image frame and the full movie (0 – 23 h), starting 0.5 h after addition of SiR-actin. (MOV 6974 kb)

Long-term live-cell microscopy of HeLa Kyoto cells stably expressing H2B-mRFP and MyrPalm-mEGFP, treated with 1 nM taxol.

The movie shows a region-of-interest of 1/16th of the full image frame and the full movie (0 – 23 h), starting 0.5 h after addition of taxol. (MOV 4344 kb)

Long-term live-cell microscopy of HeLa Kyoto cells stably expressing H2B-mRFP and MyrPalm-mEGFP, treated with 100 nM jasplakinolide.

The movie shows a region-of-interest of 1/16th of the full image frame and the full movie (0 – 23 h), starting 0.5 h after addition of jasplakinolide. (MOV 3735 kb)

2D SIM time series of microtubule dynamics in human primary dermal fibroblasts.

Cells were stained with 2 μM SiR-tubulin in complete DMEM medium for 60 min at 37°C and washed before imaging on a Nikon Eclipse Ti microscope. Time is indicated in min : s format. (AVI 10440 kb)

3D SIM time series of actin dynamics in lamellipodia of human primary dermal fibroblasts.

Cells were stained with 2 μM SiR-actin in complete DMEM medium for 120 min and imaged directly without washing on Nikon Eclipse Ti microscope. Time is indicated in min : s format. (AVI 12885 kb)

STED time series imaging of microtubule dynamics in human primary dermal fibroblasts.

Cells were stained with 2 μM SiR-tubulin in complete DMEM medium for 60 min at 37°C and washed before imaging on a STED microscope. Time is indicated in min : s format. (AVI 5124 kb)

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Lukinavičius, G., Reymond, L., D'Este, E. et al. Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat Methods 11, 731–733 (2014). https://doi.org/10.1038/nmeth.2972

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