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The development of voltage-sensitive fluorescent probes suggests fluorescencelifetime as a promising readout for electrical activity in biological systems.Existing approaches fail to achieve the speed and sensitivity required forvoltage imaging in neuroscience applications. Here we demonstrate thatwide-field electro-optic fluorescence lifetime imaging (EO-FLIM) allowslifetime imaging at kHz frame acquisition rates, spatially resolving actionpotential propagation and sub-threshold neural activity in live adultDrosophila. Lifetime resolutions of $< 5$ ps at 1 kHz were achieved for singlecell voltage recordings. Lifetime readout is limited by photon shot noise andthe method provides strong rejection of motion artifacts and technical noisesources. Recordings revealed local transmembrane depolarizations, two types ofspikes with distinct fluorescence lifetimes, and phase locking of spikes to anexternal mechanical stimulus.

Wide-field fluorescence lifetime imaging of neuron spiking and sub-threshold activity in vivo