# Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
## 4 Voltage Sensors
Using electrochromic FRET (eFRET) approach, a palette of GEVIs with fluorescence ranging from green to red were engineered by appending various fluorescent proteins to the C-terminus of the proton pumping microbial opsins [80; 84; 95; 96] (Figure 2a). The first generation of eFRET-opsin voltage sensors, incorporating Mac and Arch proton pumps [97] as voltage-sensing scaffolds, can report single action potentials in cultured neurons with a moderate signal-to-noise ratio (7-9 at 1 kHz acquisition rate) [95; 96]. However, these sensors suffer from poor membrane localization and relatively slow kinetics, on the order of few milliseconds, that precluded their adaptation by the neuroscience community. Substantial improvement in overall performance was achieved by utilizing the blue-shifted _Acetabuluria acetabulum_ rhodopopsin (Ace) [98] in fusion with the very bright green fluorescent protein mNeonGreen. The resulting group of GEVIs, usually collectively called Ace2N-mNeon (2N stands for the point mutation abolishing proton pumping), is characterized by high brightness, moderate photostability, and submillisecond kinetics, providing high spike detection fidelity under wide-field microscopy in both ex vivo and in vivo preparations [84]. For example, the Ace2N-4 aa-mNeon variant, containing four amino acids in the linker between Ace and mNeonGreen, was demonstrated to report single action potentials in individual neurons in the primary visual cortex of awake mice (Table 1). In turn, a variant with a shorter linker, Ace2N-2 aa-mNeon, was able to detect odor-evoked voltage signals in single olfactory projection neurons and their dendritic arbors and axonal buttons in live fruit flies. \begin{table}
\begin{tabular}{l l l l l l l l} \hline \hline
**Sensor** & **E/Em** & **A/IF/EP** & **SNR** & **On kinetics *** & **Off kinetics *** & **Number of Simultaneously** & **Ref** \\  & **(nm)** & **(\%)** & & **(ms)** & **(ms)** & **Imaged Neurons In Vivo** & \\ \hline
**Ace2N-4 aa-mNeon** & 506/517 & \(-6.5\) & ND & 0.37 (58)/5.5 & 0.5 (60)/5.9 & 2 in primary visual cortex L2/3 & [84] \\
**ASAP3** & 485/510 & \(-10\,^{\text{b}}\) & \(19\,^{\text{b}}\) & 0.94 (72)/7.2 & 3.8 (76)/16 & 1 in primary visual cortex L1 & [81] \\  & & & & & and L2/3 & & \\
**Voltrong\({}_{\text{ES}}\)** & 525/549 & \(-6.5\,^{\text{b}}\) & \(27\,^{\text{b}}\) & 0.64 (61)/4.1 & 0.78 (55)/3.9 & 46 in primary visual cortex L1 & [82] \\
**VARNAM** & 558/592 & \(-4.8\) & 12 & 0.9/5.2 & 0.8/4.7 & Aggregated population signal & [80] \\
**QuasAr2** & 590/715 & 15 & 8.5 & 0.3 (62)/3.2 & 0.3/(73) 4.0 & 14 in mouse nodog ganglia & [99] \\
**pad0 asAr3** & 590/715 & \(23\,^{\text{b}}\) & \(28\,^{\text{b}}\) & 0.9 (57)/15.9 & 0.93 (79)/15 & 6 in hippocampus & [60] \\
**Archon1** & 590/715 & \(53\,^{\text{b}}\) & \(37\,^{\text{b}}\) & 0.61 (68)/8.1\({}^{\text{c}}\) & \(1.1 (88)/13\,^{\text{c}}\) & 18 in CA1 hippocampus & [65] \\ \hline \hline \end{tabular}  
* Ex–excitation wavelength; Em—emission wavelength; SNR—signal-to-noise ratio per single action potential measured in intact mouse brain slice at 22 \({}^{\circ}\)C under wide-field microscopy at 500 Hz acquisition rate; ND—not determined. \({}^{\text{a}}\) voltage kinetics evaluated by bi-exponential fitting (in the format fast/slow), where the value in parentheses represents the % of current magnitude in the fast r component, measured in HEK cells at 34 \({}^{\circ}\)C; \({}^{\text{b}}\) values measured for soma localized version of the sensor; \({}^{\text{c}}\) measured in cultured neurons.  
\end{table} 
Table 2: Performance of selected genetically-encoded and hybrid fluorescent voltage sensors. Coupling Ace2N with the bright fluorescent protein mRuby3 led to the development of a red-shifted GEVI named VARNAM [80], which inherited the fast kinetics of Ace2N and high brightness and photostability of mRuby3.