High-speed amplitude modulation of femtosecond laser pulses for frequency-multiplexed two photon imaging

We present a frequency-multiplexed multi-site two-photon imaging method utilizing amplitude modulation of femtosecond laser pulses in the MHz range to tag each excitation beam and the corresponding fluorescence signals with specific frequencies. The frequency tags are generated with an interferometric setup employing acousto-optic deflectors (AODs) to achieve precise spatial overlap of femtosecond pulses with acoustically-shifted frequencies. Creating matching excitation beam patterns in each interferometer arm using multiple AOD driving frequencies and subsequently overlapping these matching patterns results in multiple excitation beams with unique beat frequencies available for scanning. As a proof-of-concept, we demonstrate multiplexed two-photon image acquisition using test samples, and compare the performance of this approach to conventional two-photon laser scanning microscopy.