Abstract
Fluorescence lifetime imaging microscopy (FLIM) measures fluorescence decay rate at every pixel of an image. FLIM can separate probes of the same color but different fluorescence lifetimes (FL), thus it is a promising approach for multiparameter imaging. However, available GFP-like fluorescent proteins (FP) possess a narrow range of FLs (commonly, 2.3–3.5 ns) which limits their applicability for multiparameter FLIM. Here we report a new FP probe showing both subnanosecond fluorescence lifetime and exceptional fluorescence brightness (80% of EGFP). To design this probe we applied semi-rational amino acid substitutions selection. Critical positions (Thr65, Tyr145, Phe165) were altered based on previously reported effect on FL or excited state electron transfer. The resulting EGFP triple mutant, BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter), allows for both reliable detection of the probe and recording FL signal clearly distinguishable from that of the spectrally similar commonly used GFPs. We demonstrated high performance of this probe in multiparameter FLIM experiment. We suggest that amino acid substitutions described here lead to a significant shift in radiative and non-radiative excited state processes equilibrium.
Highlights
Since the cloning of the avGFP gene in the early 1990s, fluorescent proteins (FP) have become an indispensable instrument in biology[1]
The main reason is that fluorescence lifetime shortening is normally correlated with a proportional decrease of fluorescence quantum yield
MGarnet[2] and TagRFP675 – FPs with the shortest fluorescence lifetimes reported to date (760 and 900 ps, respectively) and red emission – have quantum yield of less than 10%9,10
Summary
Since the cloning of the avGFP gene in the early 1990s, fluorescent proteins (FP) have become an indispensable instrument in biology[1]. MGarnet[2] and TagRFP675 – FPs with the shortest fluorescence lifetimes reported to date (760 and 900 ps, respectively) and red emission – have quantum yield of less than 10%9,10. EGFP-T65G possessed shorter lifetime and lower quantum yield (QY) compared to the parental EGFP (1.3 ns vs 2.8 ns in EGFP, Table 1 and Fig. 1a,b).
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