Optimal repetition rate and pulse duration studies for two photon imaging

Multiphoton imaging (MPI) is an important fluorescence microscopy technique that allows deep tissue and in-vivo imaging with high selectivity. According to theory, two-photon signal is proportional to the product of the peak power and the average power, allowing optimization of key imaging parameters of the excitation laser, such as average power, repetition rate and pulse duration. Recent progress in compact ultrafast lasers including femtosecond fiber lasers and optically pumped semiconductor lasers makes direct control of these parameters possible. In order to investigate the optimum laser parameters for two photon imaging we experimentally study the effects of repetition rate between 2.85 and 90 MHz and pulse duration between 336 fs and 3.5 ps on two photon signal in SYTOX Green labeled mouse intestine sections at 1030 nm. We found that the optimum repetition rate for this sample is in the range 20 – 40 MHz, depending on average power, and that the pulse duration has no effect on the MPI signal provided that the average power can be adjusted to keep the product of average and peak power constant.