On page 167 Harpur et al. present a fluorescence resonance energy transfer (FRET) imaging method that enables the use of bright, but previously incompatible fluorescent protein pairs, such as spectrally bright yellow/green fluorescent proteins (EYFP and EGFP), to measure FRET in individual living cells. They applied the technique to monitor caspase activity in cells during apoptosis. To do this, they inserted a caspase cleavage site between the spectrally similar EYFP/EGFP pair and measured FRET by determining the fluorescence lifetime of the combined donor/acceptor pair by fluorescence lifetime imaging microscopy. Loss of FRET upon cleavage of the site results in shorter fluorescence lifetime.
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Sinclair, M. Technical Reports. Nat Biotechnol 19, 101 (2001). https://doi.org/10.1038/84332
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DOI: https://doi.org/10.1038/84332