CONCENTRATION DEPENDENT FLUORESCENCE SPECTROSCOPY OF ACRIDINE ORANGE

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Two-photon excitation cross-section (?(2)) measurements of mutant Green Fluorescent Protein (EGFP) has been carried out in the wavelength range of 810-980 nm using ~80 fs laser pulses at 82 MHz repetition rate. At each wavelength, power-dependent measurements were conducted to confirm the order (e.g. 2.02 q 0.02 for pH11) of the nonlinear process that leads to the observed fluorescence. The dependence of ?(2) on pH-value (4.9, 5.8, 8, and 11) of the buffer was also investigated. ?(2) = 97 q 5 x 10-50 cm4.s/photon.molecule was measured at 950 nm for pH11. This value was reduced by ~76% in pH5 buffer. A broad band centered at ~950 nm suggests that the energy of S2<---- S0 transition is ~ 21053 cm-1 and seems to be independent of the pH of the buffer. The intensity of a weak shoulder at ~840 nm slightly increases as the pH-value decreases. Comparison between the one- and two-photon excitation excitation cross-section and insights on the involved spectroscopy are drawn.