Abstract:
Recently, we presented a new approach to create high-speed amplitude modulation of femtosecond laser pulses in the MHz range to tag multiple excitation beams and their corresponding fluorescence signals with specific frequencies. In this work, we discuss the sensitivity of this method and its utility for in vivo recordings of Ca2+signals in brain tissue compared to conventional two-photon laser scanning microscopy. While frequency- multiplexed imaging appears slightly inferior in terms of image quality due to shot noise- induced cross-talk between individual frequency channels, the specific application of the technique for recording average signals from selected regions of interest such as neuronal cell bodies is found to be exceptionally promising. We used phase information contained within each pixel of the selected area represented by a short waveform to phase-align and recombine them into one extended waveform, thereby effectively increasing the waveform measurement time. This procedure narrows the detection band of a digital lock-in filter and thus decreases noise contributions from other frequency channels by more than an order of magnitude.