The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

The signal and resolution during in vivo imaging of the mouse brain is limited by sample-induced optical aberrations. We find that, although the optical aberrations can vary across the sample and increase in magnitude with depth, they remain stable for hours. As a result, two-photon adaptive optics can recover diffraction-limited performance to depths of 450 μm and improve imaging quality over fields of view of hundreds of microns. Adaptive optical correction yielded fivefold signal enhancement for small neuronal structures and a threefold increase in axial resolution. The corrections allowed us to detect smaller neuronal structures at greater contrast and also improve the signal-to-noise ratio during functional Ca(2+) imaging in single neurons.