# Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
## 4 Voltage Sensors
Near-infrared fluorescence of NovArch and paQuasAr3 can be reversibly enhanced by blue illumination without affecting voltage sensitivity (Table 2). This blue light enhanced brightness of paQuasAr3 is sufficient to perform population voltage imaging with single cell resolution in hippocampal cells of awake mice. At the same time, it complicates combining voltage imaging with optogenetic control by channelrhodopsins. In another study, significant improvement of near-infrared brightness was achieved by implementation of a robotic multidimensional directed evolution approach [65]. Large libraries, up to tens of millions of independent clones, expressed in mammalian cells were screened for improved brightness and localization using robotic cell picker. Selected variant Archon1 exhibited several fold higher fluorescence and nearly ideal membrane localization in neurons when expressed in vivo in mice, zebrafish, and worms. The soma-localized version of Archon1 enabled population voltage imaging in striatum, cortex, and hippocampus of behaving mice under a cheap and simple wide-field imaging set up (personal communication). In addition, Archon1 was the first reported GEVI to report single action potentials in zebrafish larvae. Despite several generations of improved Arch-derived voltage sensors, the sensors still suffer from very low fluorescence brightness in comparison to other classes of GEVIs. In the past few years the field of GEVIs was strongly dominated by opsin-based sensors, however, opsin-based sensors all share one disappointing limitation: they do not exhibit voltage sensitivity under two-photon excitation. Although fundamentally and technically more challenging than wide-field imaging, two-photon microscopy offers increased imaging depth with higher spatial resolution in scattering tissue. For example, two-photon microscopy has been used for subcellular voltage imaging in live fruit flies using GEVIs [105; 106; 107] as well as dendritic [108] and somatic [109] voltage using chemical dyes. The development of the improved version of the ASAP sensor [110], named ASAP3, enabled two-photon voltage imaging in awake mice using a fully genetically-encoded probe [81]. The ASAP family of GEVIs is based on the insertion of a circularly permuted EGFP into the voltage-sensing domain from _Gallus gallus_ of voltage-sensitive phosphatases between the third and fourth transmembrane domains (Figure 2d). ASAP3 development involved random mutagenesis of the linkers between circularly permuted EGFP and voltage-sensing domain to optimize coupling of four transmembrane helix movement during depolarization to chromophore protonation. Using the soma-localized version of ASAP3, [PERSON] et al. performed reliable voltage imaging with single cell single spike resolution in visual cortex at a depth up to \(\sim\)130 \(\upmu\)m. Improved performance of ASAP3 compared to its precursors can be partially attributed to its kinetics. ASAP3 has a submillisecond on rate, but several-fold slower off kinetics, unlike opsin-based sensors (Table 2). This combination of kinetic parameters results in significant boarding of optical action potential waveforms thus increasing the effective number of photons emitted during spikes. However, fast two-photon imaging is still limited to a small imaging area and recording of more than one cell at a time still represent significant technical challenge. Additionally, wide-field imaging of opsin-based sensor is more cost efficient.