Origin and effect of high-order dispersion in ultrashort pulse multiphoton microscopy in the 10 fs regime

Short pulses can induce high nonlinear excitation, and thus they should be favorable for use in multiphoton microscopy. However, the large spectral dispersion can easily destroy the advantages of the ultrashort pulse if there is no compensation. The group delay dispersion (GDD), third-order dispersion, and their effects on the intensity and bandwidth of second-harmonic generation (SHG) signal were analyzed. We found that the prism pair used for compensating the GDD of the two-photon microscope actually introduces significant negative high-order dispersion (HOD), which dramatically narrowed down the two-photon absorption probability for ultrashort pulses. We also investigated the SHG signal after GDD and HOD compensation for different pulse durations. Without HOD compensation, the SHG efficiency dropped significantly for a pulse duration below 20 fs. We experimentally compared the SHG and two-photon excited fluorescence (TPEF) signal intensity for 11 fs versus 50 fs pulses, a pulse duration close to that commonly used in conventional multiphoton microscopy. The result suggested that after adaptive phase compensation, the 11fs pulse can yield a 3.2- to 6.0-fold TPEF intensity and a 5.1-fold SHG intensity, compared to 50 fs pulses.