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10 July 2003 Thermal bleaching in single fluorescent molecules under two-photon excitation regime
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Single molecule spectroscopy often requires the immobilization of the molecules onto solid or quasi-solid substrates and the use of relatively high excitation intensity. We have studied the fluorescence emission of four common dyes used for bio-imaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level under two-photon excitation regime. We focus on two-photon excitation thermal effects on the stability of the single molecules, influencing the internal photo-dynamics and the total duration of the fluorescent emission. Single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output till a sudden transition to a dark state. The bleaching time varies in the series pyrene, indo-1, fluorescein and rhodamine 6g from the fastest to the slowest one respectively, has a gaussian distribution suggesting that bleaching is not due to photo-bleaching. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon excitation process and the thermal bleaching is correlated to the amount of absorbed power that is not re-irradiated as fluorescence.
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Giuseppe Chirico, Fabio Cannone, Giancarlo Baldini, and Alberto Diaspro "Thermal bleaching in single fluorescent molecules under two-photon excitation regime", Proc. SPIE 4963, Multiphoton Microscopy in the Biomedical Sciences III, (10 July 2003);

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