Flexible and Tunable Nonlinear Microscopy Platform

In nonlinear microscopy sample damage threshold depends on multiple parameters such as average photon flux, energy, pulse duration, and repetition rate of the laser used. Moreover, the optimal set of these parameters depends on the order of the nonlinear imaging process. In this study, we performed two and three-photon imaging with varying pulse duration, repetition rate and average power and systematically investigated the fluorescence signal strength depending on those parameters. Using a repetition rate tunable laser (TruMicro Serie 2000, Trumpf) in combination with a multipass spectral broadening [1] and compression module (MIKS, n2-Photonics) we could vary the repetition rate between 1 to 20 MHz and the pulse durations between 50–250 fs. This highly flexible platform allows us to choose the optimal parameter range and stay below the damage threshold of the sample. We demonstrate the different optimal parameter sets for a binary system sufficiently simple to verify the theoretically expected signal intensity dependencies experimentally. Independently from each other, the pulse duration, repetition rate, and average power were changed during simultaneous two-photon and three-photon imaging.