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Journal of Biomedical Optics

Three-photon excitation of 2,5-bis(4-biphenyl)oxazole: steady-state and time-resolved intensities and anisotropies
Author(s): Ignacy Gryczynski; Henryk M. Malak; Stefan W. Hell; Joseph R. Lakowicz
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Paper Abstract

Three-photon excitation of 2,5-bis(4-biphenyl) oxazole (BBO) was observed when it was excited with the fundamental output of a femtosecond Ti:sapphire laser above 820 nm. The emission spectrum of BBO was identical for one-, two-, and three-photon excitation at 320, 640, and 960 nm, respectively. In toluene and triacetin, the emission intensity of BBO depended on the square of the laser power for wavelengths below 820 nm and displayed a sharp transition to a cubic dependence at longer wavelengths. The spatial distribution of the emission of BBO with three-photon excitation was more strongly localized than for two-photon excitation of a coumarin fluorophore at the same wavelength. The same single exponential intensity decay was observed for one-, two-, and three-photon excitation. However, the frequency domain anisotropy decay with threephoton excitation at 960 nm revealed a larger time-zero anisotropy, larger differential polarized phase angle, and larger modulated anisotropy than is possible for two-photon excitation with colinear oscillators. In triacetin, the anisotropy is not constant for three-photon excitation at different wavelengths. Surprisingly, the fluorescence intensities for three-photon excitation were only about 100-fold less than for two-photon excitation. The increasing availability of Ti:sapphire lasers suggests that multiphoton excitation can become a common tool in fluorescence spectroscopy.

Paper Details

Date Published: 1 October 1996
PDF: 8 pages
J. Biomed. Opt. 1(4) doi: 10.1117/12.250670
Published in: Journal of Biomedical Optics Volume 1, Issue 4
Show Author Affiliations
Ignacy Gryczynski, Univ. of Maryland School of Medicine (United States)
Henryk M. Malak, Univ. of Maryland/Baltimore School of Medicine (United States)
Stefan W. Hell, Univ. of Turku (Germany)
Joseph R. Lakowicz, Univ. of Maryland/Baltimore (United States)

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