Volumetric voltage imaging of neuronal populations in the mouse brain by confocal light-field microscopy

Voltage imaging measures neuronal activity directly and holds promise for understanding information processing within individual neurons and across populations. However, imaging voltage over large neuronal populations has been challenging owing to the simultaneous requirements of high imaging speed and signal-to-noise ratio, large volume coverage and low photobleaching rate. Here, to overcome this challenge, we developed a confocal light-field microscope that surpassed the traditional limits in speed and noise performance by incorporating a speed-enhanced camera, a fast and robust scanning mechanism, laser-speckle-noise elimination and optimized light efficiency. With this method, we achieved simultaneous recording from more than 300 spiking neurons within an 800-µm-diameter and 180-µm-thick volume in the mouse cortex, for more than 20 min. By integrating the spatial and voltage activity profiles, we have mapped three-dimensional neural coordination patterns in awake mouse brains. Our method is robust for routine application in volumetric voltage imaging. Confocal light-field microscopy allows volumetric voltage imaging in the mouse brain at speeds sufficiently high to extract the spiking activity of hundreds of neurons.