A versatile platform for two-photon neuronal population voltage imaging across cortical depths

Genetically encoded voltage indicators have emerged as a tool for resolving neuronal spiking activity with high spatiotemporal resolution within genetically specific populations; however, their fast temporal dynamics, low signal-to-noise ratio (SNR) and fast photobleaching have posed substantial challenges limiting their broader utility and, together with suboptimal optical acquisition schemes, preventing their efficient scale-up to larger neuronal populations. Here we introduce a versatile, scalable, spatiotemporally and energetically efficient two-photon optical imaging system scheme based on a flexible lateral-temporal multiplexing (FlatMux) platform. We demonstrate FlatMux's capability and its flexible reconfigurability for meeting different recording requirements. This includes a large field-of-view mode, a 2-kHz high-speed mode, a deep-tissue imaging mode allowing recordings of cortical spiking activity at up to 500-µm depth, a dual-plane imaging mode allowing for simultaneous recording of population spiking activity of neurons in cortical L2/3 and L4, and a high-SNR imaging mode for recording of subthreshold neuronal activity and high-SNR spiking activity, all while minimizing pixel crosstalk and bleaching. Thus, FlatMux meets the challenging demands of multiphoton voltage imaging across the mammalian cortex and can be expected to enable a range of studies of complex brain functions at single-spike and single-trial level for large neuronal populations.