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Bright cyan fluorescent protein variants identified by fluorescence lifetime screening

Nature Methods volume 7, pages 137–139 (2010)Cite this article

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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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Figure 1: Fluorescence lifetime screening and spectral properties of cyan fluorescent protein variants.
Figure 2: Three-component lifetime unmixing of cyan fluorescent protein variants from a single (frequency) FLIM experiment.

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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).

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Author notes

  1. Joachim Goedhart and Laura van Weeren: These authors contributed equally to this work.

Authors and 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

Authors
  1. Joachim Goedhart
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  2. Laura van Weeren
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  3. Mark A Hink
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  4. Norbert O E Vischer
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  5. Theodorus W J Gadella Jr
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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.

Corresponding author

Correspondence to Theodorus W J Gadella Jr.

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The authors declare no competing financial interests.

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Supplementary Figures 1–6, Supplementary Table 1, Supplementary Note and Supplementary Data (PDF 4861 kb)

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Goedhart, J., van Weeren, L., Hink, M. et al. Bright cyan fluorescent protein variants identified by fluorescence lifetime screening. Nat Methods 7, 137–139 (2010). https://doi.org/10.1038/nmeth.1415

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