Two-photon microscopy using picosecond pulses from four-wave mixing in a Yb-doped photonic crystal fiber.

Two-photon microscopy (TPM) enables deep tissue imaging but requires excitation pulses that have a large product of average and peak power, typically supplied by femtosecond solid-state lasers. However, these lasers are bulky, and femtosecond pulses require careful dispersion management to avoid pulse broadening, particularly when delivery fibers are used. Here we present a compact, fiber-based picosecond laser source operating at 790 nm for TPM using an ytterbium-doped photonic crystal fiber (Yb-doped PCF). The Yb-doped PCF simultaneously amplifies 1064 nm input pulses and efficiently converts them to 790 nm via four-wave mixing, generating pulses with a peak power of up to ∼3.8 kW. The source has a variable repetition rate (1.48 MHz-14.78 MHz), enabling the two-photon excitation fluorescence signal to be maximized in the presence of excitation saturation. We benchmark our picosecond laser source against a femtosecond Ti:Sapphire laser for TPM of stained Convallaria majalis samples and demonstrate comparable fluorescence signal when the two-photon excitation conditions are matched.