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Single-molecule evaluation of fluorescent protein photoactivation efficiency using an in vivo nanotemplate

Nature Methods volume 11, pages 156162 (2014) | Download Citation

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Abstract

Photoswitchable fluorescent probes are central to localization-based super-resolution microscopy. Among these probes, fluorescent proteins are appealing because they are genetically encoded. Moreover, the ability to achieve a 1:1 labeling ratio between the fluorescent protein and the protein of interest makes these probes attractive for quantitative single-molecule counting. The percentage of fluorescent protein that is photoactivated into a fluorescently detectable form (i.e., the photoactivation efficiency) plays a crucial part in properly interpreting the quantitative information. It is important to characterize the photoactivation efficiency at the single-molecule level under the conditions used in super-resolution imaging. Here, we used the human glycine receptor expressed in Xenopus oocytes and stepwise photobleaching or single-molecule counting photoactivated localization microcopy (PALM) to determine the photoactivation efficiency of fluorescent proteins mEos2, mEos3.1, mEos3.2, Dendra2, mClavGR2, mMaple, PA-GFP and PA-mCherry. This analysis provides important information that must be considered when using these fluorescent proteins in quantitative super-resolution microscopy.

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Change history

  • 13 January 2014

    In the version of this article initially published online, the institution name "ICFO" was incorrectly written as "IFCO" in the author affiliations. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank J. Dent (McGill University) for the GlyR-α–VFP and GlyR-β–VFP; X. Zhuang (Harvard University), for mEos2; T. Xu and P. Xu (Chinese Academy of Sciences) for mEos3.1 and mEos3.2; A. McEvoy (University of California, Berkeley) for Dendra2, mClavGR2, mMaple and PA-GFP; T. Misgeld (Technical University of Munich) for PAmCherry; and M. Ulbrich (University Freiburg) for pGEMHE-α1E-Ca2+-mEGFP. We thank C.D. Heyes (University of Arkansas) for critical reading of the manuscript, and I. Vernos and J. Cela-Gallego (Center for Genomic Regulation) for the oocytes. This work was supported in part by Fundació Cellex Barcelona and in part by a Marie Curie International Reintegration grant to M.L. (FP7-PEOPLE-2010-RG). N.D. is a NEST postdoctoral fellow partially supported by the Marie Curie Co-funding of Regional, National and International Programs (COFUND) action of the European Commission.

Author information

Affiliations

  1. ICFO—Institut de Ciencies Fotoniques, Mediterranean Technology Park, Castelldefels, Spain.

    • Nela Durisic
    • , Lara Laparra-Cuervo
    • , Ángel Sandoval-Álvarez
    • , Joseph Steven Borbely
    •  & Melike Lakadamyali

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Contributions

M.L. and N.D. designed the experiments. N.D. and L.L.-C. performed the experiments and the data analysis. Á.S.-Á. provided reagents and performed the cloning of the plasmids. J.S.B. carried out simulations and wrote software. M.L. and N.D. wrote the manuscript. M.L. supervised the project.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Melike Lakadamyali.

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DOI

https://doi.org/10.1038/nmeth.2784

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