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Proceedings Paper

Thermal bleaching in single fluorescent molecules under two-photon excitation regime
Author(s): Giuseppe Chirico; Fabio Cannone; Giancarlo Baldini; Alberto Diaspro
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Paper Abstract

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.

Paper Details

Date Published: 10 July 2003
PDF: 7 pages
Proc. SPIE 4963, Multiphoton Microscopy in the Biomedical Sciences III, (10 July 2003); doi: 10.1117/12.478031
Show Author Affiliations
Giuseppe Chirico, INFM, Univ. degli Studi di Milano-Bicocca (Italy)
Fabio Cannone, INFM, Univ. degli Studi di Milano-Bicocca (Italy)
Giancarlo Baldini, INFM, Univ. degli Studi di Milano-Bicocca (Italy)
Alberto Diaspro, Univ. degli Studi di Genova (Italy)

Published in SPIE Proceedings Vol. 4963:
Multiphoton Microscopy in the Biomedical Sciences III
Ammasi Periasamy; Peter T. C. So, Editor(s)

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