The development of voltage-sensitive fluorescent probes suggests fluorescence lifetime as a promising readout for electrical activity in biological systems. Existing approaches fail to achieve the speed and sensitivity required for voltage imaging in neuroscience applications. Here we demonstrate that wide-field electro-optic fluorescence lifetime imaging (EO-FLIM) allows lifetime imaging at kHz frame acquisition rates, spatially resolving action potential propagation and sub-threshold neural activity in live adult Drosophila. Lifetime resolutions of < 5 ps at 1 kHz were achieved for single cell voltage recordings. Lifetime readout is limited by photon shot noise and the method provides strong rejection of motion artifacts and technical noise sources. Recordings revealed local transmembrane depolarizations, two types of spikes with distinct fluorescence lifetimes, and phase locking of spikes to an external mechanical stimulus.

Datasets are provided for all manuscript figures along with analysis code (Matlab R2021b).