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Live-cell super-resolution imaging with trimethoprim conjugates

Nature Methods volume 7, pages 717–719 (2010)Cite this article

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Abstract

The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chemical tag that allows proteins to be labeled with an organic fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at ∼20 nm resolution.

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Figure 1: dSTORM of core histones using the TMP tag.
Figure 2: Dynamic dSTORM imaging of the movement of histone H2B core proteins in the nucleus of living HeLa cells.

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Acknowledgements

This work was supported by the Biophotonics and the Systems Biology Initiative (Forschungseinheiten der Systembiologie) of the Bundesministerium für Bildung und Forschung and by the US National Institutes of Health (RO1GM54469 and RC1GM091804 to V.W.C. and M.P.S.). R.W. was supported by a Deutscher Akademischer Austausch Dienst fellowship.

Author information

Author notes

  1. Richard Wombacher

    Present address: Present address: Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany.,

  2. Richard Wombacher, Meike Heidbreder and Sebastian van de Linde: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, Columbia University, New York, New York, USA

    Richard Wombacher & Virginia W Cornish

  2. Applied Laser Physics and Laser Spectroscopy, Bielefeld University, Bielefeld, Germany

    Meike Heidbreder & Mike Heilemann

  3. Department of Biotechnology & Biophysics, Julius Maximilians University Wuerzburg, Wuerzburg, Germany

    Sebastian van de Linde & Markus Sauer

  4. Department of Biological Sciences, Columbia University, New York, New York, USA

    Michael P Sheetz

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  1. Richard Wombacher
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  4. Michael P Sheetz
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  5. Mike Heilemann
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  6. Virginia W Cornish
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  7. Markus Sauer
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Contributions

R.W., M.H., S.v.d.L., M.P.S., M.H., V.W.C. and M.S. conceived and designed the experiments. R.W., M.H. and S.v.d.L. performed the experiments. M.H., S.v.d.L. and M.S. analyzed the data. R.W., V.W.C. and M.S. wrote the paper.

Corresponding authors

Correspondence to Mike Heilemann, Virginia W Cornish or Markus Sauer.

Ethics declarations

Competing interests

V.W.C. and M.P.S. are inventors of the TMP-tag technology. The TMP-tag technology is licensed and commercialized by Active Motif.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Note 1 (PDF 286 kb)

Supplementary Video 1

H2B labeled HeLa cells shown in Figure 1 excited at 647 nm with 5 kW cm−2 at a frame rate of 50 Hz. (MOV 1441 kb)

Supplementary Video 2

H2B labeled HeLa cell excited at 647 nm with 0.5 kW cm−2 at a frame rate of 10 Hz. (MOV 2624 kb)

Supplementary Video 3

H2B labeled HeLa cell shown in Figure 2 excited at 647 nm with 5 kW cm−2 at a frame rate of 50 Hz. (MOV 2826 kb)

Supplementary Video 4

Sequence constructed from Supplementary Video 3 using 500 subsequent frames shifted by 50 frames, respectively. A movie is generated with an apparent time resolution of 1 Hz. However, one should be aware that the experimental time resolution is 10 s corresponding to a frame rate of 50 Hz. (MOV 1311 kb)

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Wombacher, R., Heidbreder, M., van de Linde, S. et al. Live-cell super-resolution imaging with trimethoprim conjugates. Nat Methods 7, 717–719 (2010). https://doi.org/10.1038/nmeth.1489

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