SummaryAll-optical physiology (AOP) manipulates and reports neuronal activities with light, allowing for interrogation of neuronal functional connections with high spatiotemporal resolution. However, contemporary high-speed AOP platforms are limited to single-depth or discrete multi-plane recordings that are not suitable for studying functional connections among densely packed small neurons, such as neurons in Drosophila brains. Here, we constructed a 3D AOP platform by incorporating single-photon point stimulation and two-photon high-speed volumetric recordings with a tunable acoustic gradient-index (TAG) lens. We demonstrated the platform effectiveness by studying the anterior visual pathway (AVP) of Drosophila. We achieved functional observation of spatiotemporal coding and the strengths of calcium-sensitive connections between anterior optic tubercle (AOTU) sub-compartments and >70 tightly assembled 2-μm bulb (BU) microglomeruli in 3D coordinates with a single trial. Our work aids the establishment of in vivo 3D functional connectomes in neuron-dense brain areas.

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