Increasing efficiency of two-photon excited fluorescence and second harmonic generation using ultrashort pulses

Multiphoton microscopy (MPM) has become an important tool for high-resolution and non-invasive imaging in biological tissues. However, the efficiencies of two-photon excited fluorescence (TPEF) and second harmonic generation (SHG) are relatively low because of their nonlinear nature. Therefore, it is critical to optimize laser parameters for most efficient excitation of MPM. Reducing the pulse duration can increase the peak intensity of excitation and thus potentially increase the excitation efficiency. In this paper, a multiphoton microscopy system using a 12 fs Ti:Sapphire laser is reported. With adjustable dispersion pre-compensation, the pulse duration at the sample location can be varied from 400 fs to sub-20 fs. The efficiencies of TPEF and SHG are studied for the various pulse durations, respectively. Both TPEF and SHG are found to increase proportionally to the inverse of the pulse duration for the entire tested range. To transmit most of the SHG and TPEF signals, the spectral transmission widow of the detection optics needs to be carefully considered. Limitation from phase-matching in SHG generation is not significant because the effective interaction length for SHG is less than 10 μm at the focal depth of the objectives. These results are important in improving MPM excitation efficiency using ultrashort pulses. MPM images from human artery wall are also demonstrated.