23 February 2006 Increasing two-photon fluorescence signals by coherent control
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Abstract
Two-photon excitation fluorescence of complex solvated molecules (Rhodamine590, Fluorescein, and G5-dendrimer conjugated Fluorescein) was successfully controlled using adaptive pulse shaping. We were able to maximize and minimize the ratio of fluorescent yield to average incident power or second-harmonic generation (SHG) in a thin optical crystal. The optimal excitation pulse shape was found experimentally using a genetic learning algorithm and no a priori knowledge. Pulses were shaped with an acousto-optic programmable dispersive filter (Dazzler AOPDF) controlling phase and amplitude of 20 individual frequency components. Convergence occurred over the order of 100 generations of experiments from an original set of 50 random individual pulses. Femtosecond laser pulses (~75 fs, 76 MHz repetition, 800 nm center wavelength, 3nJ without shaping) selected to maximize fluorescence yield / SHG were found to be complementary to those minimizing this ratio when visualized with a SHG-frequency resolved optical gating (SHG-FROG) device. At these powers, linear chirp of the pulse was far less significant in establishing coherent control than the more complex pulse shape. Regeneratively amplified pulses (~150 fs, 20 kHz repetition, 795 nm center wavelength, 2 μJ before shaping) were selected for maximum efficiency of fluorescent yield relative to incident power. The peak intensity, as determined by SHG, did not change significantly for optimal pulses when compared to early generations. This indicates that the improved two-photon fluorescent signal was not the result of simple convergence to a transform limited pulse, and suggests that the dye molecule excited state population is being coherently controlled. We are currently investigating the application of this result to enhancing signal in flow-cytometry and improved discrimination for multi-photon microscopy.
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Eric R. Tkaczyk, Augustin Mignot, Jing Yong Ye, Istvan Majoros, James R Baker, Theodore B. Norris, "Increasing two-photon fluorescence signals by coherent control", Proc. SPIE 6089, Multiphoton Microscopy in the Biomedical Sciences VI, 608910 (23 February 2006); doi: 10.1117/12.644979; https://doi.org/10.1117/12.644979
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