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Coherent control of mutiphoton excitation process for biological fluorescence imaging

The two-photon excitation fluorescence (TPEF) process of an enhanced green fluorescent protein (EGFP) for fluorescence signals was adaptively controlled by the phase-modulation of femtosecond pulses. After the iteration of pulse shaping, a twofold increase in the ratio of the fluorescence signal to the laser peak power was achieved. Compared with conventional pulses optimized for peak power, phase-optimized laser pulses reduced the bleaching rate of EGFP by a factor of 4 while maintaining the same intensity of the fluorescence signal. The ratio of two-photon (2P) fluorescence from EGFP and three-photon (3P) fluorescence from the essential amino acid L-Tryptophan was also enhanced by using the adaptive pulse shaping technique. To achieve a trade-off between the 2P/3P fluorescence ratio and the 2P fluorescence intensity, we then engineered the cost function in the self-learning algorithm. These methods have much potential for immediate application to various important biological and medical studies.