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Imaging FRET between spectrally similar GFP molecules in single cells

Nature Biotechnology volume 19, pages 167–169 (2001)Cite this article

Abstract

Fluorescence resonance energy transfer (FRET)1 detection in fusion constructs consisting of green fluorescent protein (GFP)2 variants linked by a sequence that changes conformation upon modification by enzymes or binding of ligands has enabled detection of physiological processes such as Ca2+ ion release3, and protease4,5,6,7 and kinase activity8. Current FRET microscopy techniques are limited to the use of spectrally distinct GFPs such as blue or cyan donors in combination with green or yellow acceptors. The blue or cyan GFPs have the disadvantages of less brightness and of autofluorescence. Here a FRET imaging method is presented that circumvents the need for spectral separation of the GFPs by determination of the fluorescence lifetime of the combined donor/acceptor emission by fluorescence lifetime imaging microscopy (FLIM)9,10,11,12. This technique gives a sensitive, reproducible, and intrinsically calibrated FRET measurement that can be used with the spectrally similar and bright yellow and green fluorescent proteins (EYFP/EGFP), a pair previously unusable for FRET applications. We demonstrate the benefits of this approach in the analysis of single-cell signaling by monitoring caspase activity in individual cells during apoptosis.

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Figure 1: In vitro characterization of recombinant His-tagged EYFP–DEVD–EGFP.
Figure 2: Predicted and experimental demonstration of acceptor in-growth measured by frequency domain FLIM.
Figure 3: FLIM measurements of Rat-1/c-mycER fibroblasts transiently transfected with pEYFP–DEVD–EGFP.

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Acknowledgements

The authors would like to thank Gerard Evan and David Hancock of the Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, for the Rat-1/c-mycER fibroblasts.

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  1. Philippe I.H. Bastiaens

    Present address: Cell Biology and Cell Biophysics Program, European Molecular Biology Laboratory, Meyerhofstraβe 1, 69117, Heidelberg, Germany

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  1. Cell Biophysics Laboratory, Imperial Cancer Research Fund, Lincoln's Inn Fields, London, UK

    Ailsa G. Harpur, Fred S. Wouters & Philippe I.H. Bastiaens

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Correspondence to Philippe I.H. Bastiaens.

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Harpur, A., Wouters, F. & Bastiaens, P. Imaging FRET between spectrally similar GFP molecules in single cells. Nat Biotechnol 19, 167–169 (2001). https://doi.org/10.1038/84443

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