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Bright cyan fluorescent protein variants identified by fluorescence lifetime screening

Nature Methods volume 7, pages 137139 (2010) | Download Citation

Abstract

Optimization of autofluorescent proteins by intensity-based screening of bacteria does not necessarily identify the brightest variant for eukaryotes. We report a strategy to screen excited state lifetimes, which identified cyan fluorescent proteins with long fluorescence lifetimes (>3.7 ns) and high quantum yields (>0.8). One variant, mTurquoise, was 1.5-fold brighter than mCerulean in mammalian cells and decayed mono-exponentially, making it an excellent fluorescence resonance energy transfer (FRET) donor.

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Acknowledgements

We thank I. Elzenaar and M. Adjobo-Hermans for pilot experiments on the 2A co-expression assay and the members of our laboratory for encouraging discussions, J.E.M. Vermeer (University of Amsterdam) for providing plasmid encoding peroxi-YFP with a C-terminal SKL peroxisomal targeting sequence and E.L. Snapp (Albert Einstein College of Medicine) for providing plasmid encoding ER-tdTomato. Part of this work is supported by the EU integrated project on 'Molecular Imaging' (LSHG-CT-2003-503259).

Author information

Author notes

    • Joachim Goedhart
    •  & Laura van Weeren

    These authors contributed equally to this work.

Affiliations

  1. Swammerdam Institute for Life Sciences, Section of Molecular Cytology, Centre for Advanced Microscopy, University of Amsterdam, Amsterdam, The Netherlands.

    • Joachim Goedhart
    • , Laura van Weeren
    • , Mark A Hink
    • , Norbert O E Vischer
    •  & Theodorus W J Gadella Jr
  2. Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.

    • Kees Jalink

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Contributions

J.G. and T.W.J.G. designed research; J.G., L.v.W., M.A.H., K.J. and T.W.J.G. conducted experiments; J.G., L.v.W., M.A.H., N.O.E.V. and T.W.J.G. analyzed data; and J.G. and T.W.J.G. wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Theodorus W J Gadella Jr.

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DOI

https://doi.org/10.1038/nmeth.1415

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