Bright GFP with subnanosecond fluorescence lifetime

Anastasia V. Mamontova, Ilya D. Solovyev, Alexander P. Savitsky, Alexander Shakhov, Konstantin A. Lukyanov, Alexey M. Bogdanov

Research output: Contribution to journalArticlepeer-review

19 Citations (Scopus)

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.

Original languageEnglish
Article number13224
JournalScientific Reports
Volume8
Issue number1
DOIs
Publication statusPublished - 1 Dec 2018
Externally publishedYes

Fingerprint

Dive into the research topics of 'Bright GFP with subnanosecond fluorescence lifetime'. Together they form a unique fingerprint.

Cite this