Bright cyan fluorescent protein variants identified by fluorescence lifetime screening


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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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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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.

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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).

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