Imaging neuronal voltage beyond the scattering limit

Voltage imaging is a promising technique for high-speed recording of neuronal population activity. However, tissue scattering severely limits its application in dense neuronal populations. Here we adopt the principle of localization microscopy, a technique that enables super-resolution imaging of single molecules, to resolve dense neuronal activities in vivo. Leveraging the sparse activation of neurons during action potentials (APs), we precisely localize the fluorescence changes associated with each AP, creating a super-resolution image of neuronal activity. This approach, termed activity localization imaging (ALI), identifies overlapping neurons and separates their activities with over tenfold greater precision than what tissue scattering permits. We applied ALI to widefield, targeted illumination and light sheet microscopy data, resolving neurons that cannot be distinguished by existing signal extraction pipelines. In the mouse hippocampus, ALI generates a cellular resolution map of theta oscillations, revealing the diversity of neuronal phase locking within a dense local network.