Time-resolved detection of the one- and two-photon excited fluorescence of single molecules of a folding enhanced green fluorescent protein

Mircea Cotlet; Peter M. Goodwin; Geoffrey S. Waldo; James H. Werner

doi:10.1117/12.646697

27 February 2006 Time-resolved detection of the one- and two-photon excited fluorescence of single molecules of a folding enhanced green fluorescent protein

Mircea Cotlet, Peter M. Goodwin, Geoffrey S. Waldo, James H. Werner

Author Affiliations +

Proceedings Volume 6092, Ultrasensitive and Single-Molecule Detection Technologies; 609204 (2006) https://doi.org/10.1117/12.646697
Event: SPIE BiOS, 2006, San Jose, California, United States

Abstract

We use time-resolved single molecule fluorescence detection (MSMD) to investigate the fluorescence dynamics of a mutant of the wild-type Green Fluorescent Protein (GFP) from Aequorea victoria, the folding enhanced GFP (FEGFP). The folding enhanced GFP is a novel and robust variant designed for in vivo high-throughput screening of protein expression levels. This variant shows increased thermal stability and the ability to retain its fluorescence when fused to poorly folding proteins. Here we apply one- (OPE) and two- (TPE) photon excitation on freely diffusing FEGFP molecules. Under OPE, single FEGFP molecules undergo fluorescence flickering in the time scale of μs and tens of μs due to triplet formation and ground-state protonation-deprotonation, respectively. OPE fluorescence lifetimes of single FEGFP molecules show evidence for the presence of different emitting species, the I and B forms of FEGFP chromophore. TPE single FEGFP molecules flicker in fluorescence in the time scale of μs due to singlet-triplet transitions of the chromophore. Two-photon excitation of single FEGFP molecules results in the creation of a photoconverted species with a fluorescence lifetime of 2.5 ns, a species which is bright enough to be detected at the single molecule level. Our results indicate FEGFP is a promising fusion reporter for intracellular applications when using OPE and TPE microscopy with single molecule sensitivity.

Citation Download Citation

Mircea Cotlet, Peter M. Goodwin, Geoffrey S. Waldo, and James H. Werner "Time-resolved detection of the one- and two-photon excited fluorescence of single molecules of a folding enhanced green fluorescent protein", Proc. SPIE 6092, Ultrasensitive and Single-Molecule Detection Technologies, 609204 (27 February 2006); https://doi.org/10.1117/12.646697

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