Recently, we presented a new approach to create high-speed amplitude modulation of femtosecond laser pulses and tag multiple excitation beams with specific modulation frequencies. In this work, we discuss the utility of this method to record calcium signals in brain tissue with two-photon frequency-division multiplexing (2P-FDM) microscopy. While frequency-multiplexed imaging appears slightly inferior in terms of image quality as compared to conventional two-photon laser scanning microscopy due to shot noise-induced cross-talk between frequency channels, applying this technique to record average signals from regions of interest (ROI) such as neuronal cell bodies was found to be promising. We use phase information associated with each pixel or waveform within a selected ROI to phase-align and recombine the signals into one extended amplitude-modulated waveform. This procedure narrows the frequency detection window, effectively decreasing noise contributions from other frequency channels. Using theoretical analysis, numerical simulations, and in vitro imaging we demonstrate a reduction of cross-talk by more than an order of magnitude and predict the usefulness of 2P-FDM for functional studies of brain activity.

Two-photon frequency division multiplexing for functional in vivo imaging: a feasibility study.