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Live-cell dSTORM with SNAP-tag fusion proteins

Nature Methods volume 8, pages 7–9 (2011)Cite this article

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To the Editor:

In the September 2010 issue of Nature Methods we demonstrated live-cell direct stochastic optical reconstruction microscopy (dSTORM) of histone H2B proteins using a trimethoprim chemical tag (TMP tag) for genetic encoding with photostable standard fluorophores1. The method takes advantage of the fact that cells contain the reducing thiol glutathione—a cysteine-containing tripeptide—at millimolar concentrations, which enables reversible photoswitching of synthetic organic fluorophores2,3. The generality of the method can be easily understood considering that most Alexa Fluors (Invitrogen) and Atto fluorophores (ATTO-TEC) belong to the class of rhodamine and oxazine derivatives that have similar redox properties, that is, the triplet state of rhodamine and oxazine fluorophores is reduced by thiols such as glutathione3.

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Figure 1: Live-cell dSTORM with SNAP tags, and impact of direct excitation of radical anions at 405 nm.

References

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Acknowledgements

We thank B. Vogel and T. Holm for assistance with experiments. This work was supported by the Biophotonics Initiative of the Bundesministerium für Bildung und Forschung.

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  1. Department of Biotechnology and Biophysics, Julius Maximilians University Wuerzburg, Wuerzburg, Germany

    Teresa Klein, Anna Löschberger, Sven Proppert, Steve Wolter, Sebastian van de Linde & Markus Sauer

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  1. Teresa Klein
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  2. Anna Löschberger
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  3. Sven Proppert
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  4. Steve Wolter
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  5. Sebastian van de Linde
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  6. Markus Sauer
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Correspondence to Markus Sauer.

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The authors declare no competing financial interests.

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Supplementary Figures 1–4, Supplementary Methods (PDF 650 kb)

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Klein, T., Löschberger, A., Proppert, S. et al. Live-cell dSTORM with SNAP-tag fusion proteins. Nat Methods 8, 7–9 (2011). https://doi.org/10.1038/nmeth0111-7b

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