Representative single-molecule traces for Cy5 in (a) air saturated buffer, (b) deoxygenated buffer, and (c) deoxygenated buffer plus 1 mM COT, NBA, and Trolox.
(a) A framework for understanding the nature of fluorophore instabilities. (b) Kinetic regimes that lead to different behaviors of a fluorophore.
Accurate interpretation of FRET states often requires fluorophores that do not easily photobleach and are resistant to blinking. Without such conditions, poor signal-to-noise can confound data interpretation and reproducibility. To address these short comings, we have recently developed self-healing fluorophores which result in longer-lived fluorescence with minimal blinking. These fluorophores allow smFRET data to be acquired over long time courses at millisecond time resolution thereby affording the ability to capture rare conformational dynamics that occur on fast timescales.
The bright future of single-molecule fluorescence imaging. Juette MF, Terry DS, Wasserman MR, Zhou Z, Altman RB, Zheng Q, Blanchard SC. CURR. OPIN. CHEM. BIOL. (2014), 20:103-111.
The contribution of reactive oxygen species to the photobleaching of organic fluorophores. Zheng Q, Jockusch S, ZhouZ, Blanchard SC. PHOTOCHEM PHOTOBIOL (2014), 90(2):448-454.
Ultra-stable organic fluorophores for single-molecule research. Zheng Q, Juette MF, Jockusch S, Wasserman MR, Zhou Z, Altman RB, Blanchard SC, CHEM SOC REV. (2014), 43(4):1044-1056.
Small-molecule photostabilizing agents are modifiers of lipid bilayer properties. Alejo JL, Blanchard SC, Andersen OS. BIOPHYS J (2013) 104(11)2410:2418.
Enhanced Photostability of Cyanine Fluorophores across the Visible Spectrum. Altman RB., Zheng Q., Zhou Z., Terry DS., Warren JD., Blanchard SC., NATURE METHODS (2012): Vol.9(1):68-71. PMID:22543373.
Cyanine Fluorophore Derivatives with Enhanced Photostability. Altman RB, Terry DS, Zhou Z, Zheng Q, Geggier P, Kolster RA, Zhao Y, Javitch JA, Warren JD, Blanchard SC, NATURE METHODS (2011) 9(1):68-71.