Time-Resolved Fluorescence Intensity and Anisotropy Decays of 2,5-Diphenyloxazole by Two-Photon Excitation and Frequency-Domain Fluorometry.

We report the first time-resolved fluorescence measurements of the intensity and anisotropy decays resulting from two-photon excitation. A 10-GHz frequency-domain fluorometer (Rev. Sci. Instrum 1990, 61, 2331), equipped with two focal lenses and an emission monochromator, was used for steady-state and time-resolved measurements of PPO fluorescence. The emission spectra and the intensity decays observed with single- and two-photon excitation were essentially identical. The steady-state limiting anisotropy r 0 of PPO in glycerol at -5 °C measured for two-photon excitation is significantly higher than that observed for one-photon excitation. The r 0 value of 0.54 for two-photon excitation is well in excess of the theoretical maximum of 0.4 for single-photon excitation. A similar value of r 0 ≃ 0.50 was obtained from the frequency-domain anisotropy data with two-photon excitation of PPO in methanol, butanol, and propylene glycol at 20 °C. These higher values of r 0 indicate that two-photon excitation results in a more highly oriented photoselected population, which can increase the resolution of rotational correlation times and/or complex anisotropy decays. The anisotropy resolution can still be increased by using global analysis of anisotropy decays measured with single- and two-photon excitation.