# Advances in Engineering and Application of Optogenetic Indicators for Neuroscience
## 4 Voltage Sensors  
Voltage changes are the primary means of millisecond-scale computation in neurons because they enable sophisticated fast dendritic computations and rapid transmission of signals across long distances [78]. Voltage changes are the most direct measure of a neuron's electrical potential [79]. Therefore, optical voltage sensors are an ideal method to noninvasively measure electrical activity at single cell resolution. However, despite recent progress in development of new voltage sensitive probes [60; 65; 80; 81; 82], voltage imaging still remains technically more challenging than calcium imaging. One of the major challenges is associated with the sub-millisecond timescale of membrane potential changes during neuronal activity. Subsequently, voltage imaging requires kHz acquisition rates to resolve brief voltage changes upon action potentials, while calcium imaging is typically performed at 1 to 10 Hz. Although currently available scientific-grade cameras can perform imaging at kHz rates, these high speed result in pixel counts reduced by an order of magnitude than those commonly used for calcium imaging. Furthermore, compared to the GECIs expressed in the bulk cytosol, the maximum number of voltage sensor molecules that can be localized on plasma membrane is several orders of magnitude smaller. These factors significantly reduce the number of photons integrated during each acquired frame making fluorescence signal increasingly difficult to distinguish cells from background fluorescence in brain tissue. The first proof of principle of a fully genetically-encoded voltage sensor was reported in 1997 [83], followed by almost two decades of intensive protein engineering efforts to develop a fluorescence sensor capable of reporting neuronal activity with single cell, single spike resolution in awake mice [84]. To date a number of improved and newly engineered genetically-encoded voltage indicators (GEVIs) with diverse spectral and biophysical characteristics reached the level of performance sufficient for in vivo imaging with high temporal precision at single-cell resolution in awake mice surpassingbeyond \"proof of principle\" applications (Table 2). Currently, the majority of developed GEVIs are based on two types of voltage-sensitive moieties: microbial rhodopsins and voltage-sensing domains (VSDs) from voltage-sensitive phosphatases. Naturally-occurring and engineered microbial rhodopsins have already found widespread application in neuroscience due to their ability to optically control electrical activity of genetically targeted neurons [85]. Photoactivated rhodopsin translocates ions across the plasma membrane thus modulating membrane potentials, usually in a predictable manner [86; 87; 88]. Several years ago, [PERSON]'s group made a discovery that paved the way for the utilization of opsins not only for manipulating membrane potential but also for observing changes in voltage across the plasma membrane. His group showed that one class of opsins, light-driven proton pumps, can work in reverse, i.e., emitting light in response to membrane voltage alternations [89; 90]. Indeed, spectroscopic studies of the proton pumping mechanism in rhodopsins, cloned from archaea and bacteria, revealed a complex spectroscopic photocycle involving multiple intermediates with diverse absorption spectra [89; 90; 91]. Certain transitions between intermediates appeared to be voltage dependent while occurring at submillisecond time scales [92]. Furthermore, a single point mutation in the chromophore vicinity of proton pumps was enough to almost completely abolish light-driven currents while retaining their voltage dependent spectral changes. Altogether, these features confer optical recording of membrane potential dynamics at biologically-relevant temporal resolution via probing opsin absorption spectrum. Voltage modulated spectral changes, or the so called electrochromic effect, can be read-out via nonradiative quenching of an attached fluorescent moiety, a form of FRET [93; 94].