The ability to optically image cellular transmembrane voltage at millisecond-timescale resolution can offer unprecedented insight into the function of living brains in behaving animals. The chemigenetic voltage indicator Voltron is bright and photostable, making it a favorable choice for long in vivo imaging of neuronal populations at cellular resolution. Improving the voltage sensitivity of Voltron would allow better detection of spiking and subthreshold voltage signals. We performed site saturation mutagenesis at 40 positions in Voltron and screened for increased ΔF/F0 in response to action potentials (APs) in neurons. Using a fully automated patch-clamp system, we discovered a Voltron variant (Voltron.A122D) that increased the sensitivity to a single AP by 65% compared to Voltron. This variant (named Voltron2) also exhibited approximately 3-fold higher sensitivity in response to sub-threshold membrane potential changes. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, with lower baseline fluorescence. Introducing the same A122D substitution to other Ace2 opsin-based voltage sensors similarly increased their sensitivity. We show that Voltron2 enables improved sensitivity voltage imaging in mice, zebrafish and fruit flies. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Defining a complete set of cell types within the cortex requires reconciling disparate results achieved through diverging methodologies. To address this correspondence problem, multiple methodologies must be applied to the same cells in multiple single-cell experiments. Here we present an approach in which spatial transcriptomics using multiplexed fluorescence in situ hybridization (mFISH) on tissue previously interrogated through two-photon optogenetic mapping of synaptic connectivity can resolve the anatomical, transcriptomic, connectomic, electrophysiological, and morphological characteristics of single cells within the mouse cortex. MainThe parable of the Blind Men and the Elephant tells the story of a group of blind men, naïve to the nature of an elephant, presented with such a creature to try to conceptualize while observing only through touch ( Figure 1A, upper). The men are each concerned with a single area and draw a conclusion for the whole based on their limited perspective. As scientists, we are similarly a group of observers each indirectly observing one portion of a large, hidden, and complex problem. The parable's moral is that these challenging concepts may have many facets individually revealed to those with diverging perspectives. To strive towards a comprehensive description, we must be able to synthesize these unique perspectives into a consistent whole.This challenge of differing perspectives leading to seemingly conflicting outcomes is exemplified in attempts to understand the number and features of cell types within the mouse cortex (Fig 1A, lower). Recent studies report self-consistent outcomes based on anatomy 1 , transcriptomics 2,3 , electrophysiology 4 , morphology 4 , long-range 5 and local connectomics 6 , and in vivo recordings 7 . However, these studies markedly disagree on the number of underlying cell types. Even if they did agree on the number of types, how results from one study map to those of another is not obvious. To reconcile this problem of cell type correspondence, single-cells must traverse multiple modalities.Each of these modalities, save transcriptomics, explicitly or implicitly carries spatial information within the specimen at single-cell resolution. Multiplexed fluorescence in situ hybridization (mFISH) enables a transcriptomic readout with this same single-cell spatial resolution. We have extended the hairpin