# Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
## 4 Voltage Sensors
However, despite a single point mutation that presumably improved eFRET efficiency by red-shifting absorbance spectrum of Ace2N, VARNAM exhibits lower voltage sensitivity than other advanced GENIs suitable for in vivo imaging (Table 1). For example, averaging up to 10 optical trails was required to resolve evoked excitatory and inhibitory postsynaptic potentials using VARNAM in intact brain tissue [80]. In live mice, VARNAM was able to track cell-type specific oscillatory dynamics for subsets of neurons in somatosensory and frontal cortex and CA1 of the hippocampus using the fiberoptic imaging. In addition, VARNAM reported spontaneous neuronal activity with single spike precision in multiple cell types in live fruit flies. Another recently developed fluorescent voltage sensor utilizing Ace2N as a voltage sensing domain is Voltron [82]. While Voltron is not a fully genetically-encoded probe, but rather a chemogenetic or hybrid voltage sensor, we discuss it here in comparison with other GEVIs due to its high performance in multiple species in vivo, including mice, zebrafish, and fruit flies. Voltron combines Ace2N with a self-labeling protein tag domain that covalently binds improved rhodamine dyes such as the Janelia Fluor(r) (JF) dyes [100] (Figure 2b). While a palette of JF dyes with emission maxima ranging from 505 to 635 nm were shown to work well with Voltron in cultured neurons, the Voltron/JF525 combination exhibited the highest voltage sensitivity and, thus, used for all further in vivo applications. To perform voltage imaging in vivo in mice, Voltron was fused to the soma-localization motif from the Kv2.1 potassium channel [101] to reduce neuropil fluorescence contamination thus improving single cell resolution, similarly to the strategy previously used to enable single-cell precision optogenetics [102; 103]. Using a simple wide-field imaging set-up, soma-localized Voltron reported single action potentials in hippocampal parvalbumin neurons and visual cortex pyramidal neurons in awake mice. Particularly impressive was the ability to perform single spike detection in up to 46 GABAergic neurons in visual cortex layer I imaged simultaneously for over 15 minutes (Table 2). Voltron also retained its high performance in live zebrafish and fruit flies, reliably reporting single action potentials. Voltron relies on bright synthetic dyes delivered exogenously. While dye administration is straightforward, even for mice, easily passing through the blood brain barrier, quick internalization and accumulation of the JF dyes in the cytoplasm of the cells create high background fluorescence, significantly reducing imaging quality. Development of new dyes that fluoresce only upon binding to the HaloTag will significantly improve Voltron class of GEVIs. Among all tested light-driven proton pumps, Arch uniquely possesses dim near-infrared fluorescence under strong red laser excitation [90]. Furthermore, Arch fluorescence intensifies as membrane potential increases. The mechanism of this voltage sensitive fluorescence is still poorly studied, but might involve chromophore protonation [92]. [PERSON] et al. subjected Arch to directed molecular evolution to improve its brightness and voltage sensitivity [59]. The result, dubbed QuasAr2, is capable of reporting single action potentials with high temporal resolution in intact brain tissue and even in mouse nodose ganglia in vivo [99] (Table 2). Near-infrared fluorescence and high voltage sensitivity of QuasAr2 enabled the recording of optogenetically-triggered synaptic inputs in single trials to probe synaptic strength in intact brain tissue [27]. However, due to its extremely low brightness, requiring up to three orders of magnitude higher light intensity than for GFP imaging, QuasAr2 did not become a widely used tool. Subsequent enhancement of QuasAr2 brightness generated several variants with distinct biochemical and photophysical characteristics (Figure 2c). For example, [PERSON]'s group developed a blue light activated version of QuasAr, named NovArch [104] and paQuasAr3 [60].