Efficient multi-site two-photon functional imaging of neuronal circuits

Two-photon imaging using high-speed multi-channel detectors is a promising approach for optical recording of cellular membrane dynamics at multiple sites. A main bottleneck of this technique is the limited number of photons captured within a short exposure time (~1ms). Here, we implement temporal gating to improve the two-photon fluorescence yield from holographically projected multiple foci whilst maintaining a biologically safe incident average power. We observed up to 6x improvement in the signal-to-noise ratio (SNR) in Fluorescein and cultured hippocampal neurons showing evoked calcium transients. With improved SNR, we could pave the way to achieving multi-site optical recording of fluorogenic probes with response times in the order of ~1ms.