Bright GFP with subnanosecond fluorescence lifetime

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.

Figure 1

Fluorescence decay curves of the purified EGFP (a) and its mutants (b,c) recorded using two-photon excitation in aqueous solution, and their single-exponential fits. Experimental decay curves are shown in black, exponential fits – in red. Lifetimes (τ) are shown under the protein names.

Figure 2

Fluorescence and fluorescence lifetime imaging microscopy of live HeLa cells expressing EGFP-actin (mainly in cytoplasm; Tm ~2.2 ns), EGFP-T65G-histone 2B (in nucleus; Tm ~1.1 ns), BrUSLEE-mito (in mitochondria; Tm ~0.8 ns). (a) Fluorescence intensity image in grayscale, (b) Color-coded combined intensity + lifetime image, brightness represents photon counts, color represents fluorescence lifetime, (c) Color legend for the fluorescence lifetime image with a histogram of lifetime distribution (legend range is 780–2350 ps). Single-photon fluorescence excitation at 488 nm was used to acquire these images. Fluorescence decay data and analysis are shown in Supplementary Fig. 2.