Mitochondrial membrane potential (MMP) plays a crucial role in the function of cells and organelles, involving various cellular physiological processes, including energy production, formation of reactive oxygen species (ROS), unfolded protein stress, and cell survival. Currently, there is a lack of genetically encoded fluorescence indicators (GEVIs) for MMP. In our screening of various GEVIs for their potential monitoring MMP, the Accelerated Sensor of Action Potentials (ASAP) demonstrated optimal performance in targeting mitochondria and sensitivity to depolarization in multiple cell types. However, mitochondrial ASAPs also displayed sensitivity to ROS in cardiomyocytes. Therefore, two ASAP mutants resistant to ROS were generated. A double mutant ASAP3-ST exhibited the highest voltage sensitivity but weaker fluorescence. Overall, four GEVIs capable of targeting mitochondria were obtained and named mitochondrial potential indicators 1-4 (MPI-1-4). In vivo, fiber photometry experiments utilizing MPI-2 revealed a mitochondrial depolarization during isoflurane-induced narcosis in the M2 cortex.

Most animal cell types are classified as non-excitable because they do not generate action potentials observed in excitable cells, such as neurons and muscle cells. Thus, resolving voltage signals in non-excitable cells demands sensors with exceptionally high voltage sensitivity. In this study, the ultrabright, ultrasensitive, and calibratable genetically encoded voltage sensor rEstus is developed using structure-guided engineering. rEstus is most sensitive in the resting voltage range of non-excitable cells and offers a 3.6-fold improvement in brightness change for fast voltage spikes over its precursor ASAP3. Using rEstus, it is uncovered that the membrane voltage in several non-excitable cell lines (A375, HEK293T, MCF7) undergoes spontaneous endogenous alterations on a second to millisecond timescale. Correlation analysis of these optically recorded voltage alterations provides a direct, real-time readout of electrical cell-cell coupling, showing that visually connected A375 and HEK293T cells are also largely electrically connected, while MCF7 cells are only weakly coupled. The presented work provides enhanced tools and methods for non-invasive voltage imaging in living cells and demonstrates that spontaneous endogenous membrane voltage alterations are not limited to excitable cells but also occur in a variety of non-excitable cell types.

Scalable and high-throughput electrophysiological measurement systems are necessary to accelerate the elucidation of cardiac diseases in drug development. Optical mapping is the primary method of simultaneously measuring several key electrophysiological parameters, such as action potentials, intracellular free calcium and conduction velocity, at high spatiotemporal resolution. This tool has been applied to isolated whole-hearts, whole-hearts in-vivo, tissue-slices and cardiac monolayers/tissue-constructs. Although optical mapping of all of these substrates have contributed to our understanding of ion-channels and fibrillation dynamics, cardiac monolayers/tissue-constructs are scalable macroscopic substrates that are particularly amenable to high-throughput interrogation. Here, we describe and validate a scalable and fully-automated monolayer optical mapping robot that requires no human intervention and with reasonable costs. As a proof-of-principle demonstration, we performed parallelized macroscopic optical mapping of calcium dynamics in the well-established neonatal-rat-ventricular-myocyte monolayer plated on standard 35 mm dishes. Given the advancements in regenerative and personalized medicine, we also performed parallelized macroscopic optical mapping of voltage dynamics in human pluripotent stem cell-derived cardiomyocyte monolayers using a genetically encoded voltage indictor and a commonly-used voltage sensitive dye to demonstrate the versatility of our system.

Genetically encoded voltage indicators (GEVIs) expressed pan-neuronally were able to optically resolve bicuculline induced spontaneous oscillations in brain slices of the mouse motor cortex. Three GEVIs were used that differ in their timing of response to voltage transients as well as in their voltage ranges. The duration, number of cycles, and frequency of the recorded oscillations reflected the characteristics of each GEVI used. Multiple oscillations imaged in the same slice never originated at the same location, indicating the lack of a \"hot spot\" for induction of the voltage changes. Comparison of pan-neuronal, Ca2+/calmodulin-dependent protein kinase II α restricted, and parvalbumin restricted GEVI expression revealed distinct profiles for the excitatory and inhibitory cells in the spontaneous oscillations of the motor cortex. Resolving voltage fluctuations across space, time, and cell types with GEVIs represent a powerful approach to dissecting neuronal circuit activity.

The CA1 area in the mammalian hippocampus is essential for spatial learning. Pyramidal cells are the hippocampus output neurons and their activities are regulated by inhibition exerted by a diversified population of interneurons. Lateral inhibition has been suggested as the mechanism enabling the reconfiguration of pyramidal cell assembly activity observed during spatial learning tasks in rodents. However, lateral inhibition in the CA1 lacks the overwhelming evidence reported in other hippocampal areas such as the CA3 and the dentate gyrus. The use of genetically encoded voltage indicators and fast optical recordings permits the construction of cell-type specific response maps of neuronal activity. Here, we labelled mouse CA1 pyramidal neurons with the genetically encoded voltage indicator ArcLight and optically recorded their response to Schaffer Collaterals stimulation in vitro. By undertaking a manifold learning approach, we report a hyperpolarization-dominated area focused in the perisomatic region of pyramidal cells receiving late excitatory synaptic input. Functional network organization metrics revealed that information transfer was higher in this area. The localized hyperpolarization disappeared when GABAA receptors were pharmacologically blocked. This is the first report where the spatiotemporal pattern of lateral inhibition is visualized in the CA1 by expressing a genetically encoded voltage indicator selectively in principal neurons. Our analysis suggests a fundamental role of lateral inhibition in CA1 information processing.

Optogenetic approaches combine the power to allocate optogenetic tools (proteins) to specific cell populations (defined genetically or functionally) and the use of light-based interfaces between biological wetware (cells and tissues) and hardware (controllers and recorders). The optogenetic toolbox contains two main compartments: tools to interfere with cellular processes and tools to monitor cellular events. Among the latter are genetically encoded voltage indicators (GEVIs). This chapter outlines the development, current state of the art and prospects of emerging optical GEVI imaging technologies.

In yeast, adaptation to varying conditions often requires proper regulation of the plasma membrane potential. To determine yeast membrane potential change, optical methods involving potentiometric dyes have been supplemental to the direct electrode-based method. However, the hydrophobic nature of the dyes and their slow distribution across the membrane still limits their utilization. Genetically encoded voltage indicator (GEVI) proteins employed in neuroscience offer a tantalizing alternative for monitoring yeast membrane potential change. In this work, several widely used GEVI proteins were assessed in Saccharomyces cerevisiae for their expression and function as a voltage reporter. Among them, only ArcLight and Accelerated Sensor of Action Potential (ASAP) proteins could be expressed and transported to the plasma membrane. While the voltage-sensing capability was demonstrated for both ArcLight and ASAP, ArcLight fluorescence was sensitive to the intracellular pH change concurrently with the voltage change. Therefore, we established that ASAP is the more suitable GEVI protein for reporting yeast membrane potential change. This voltage-sensing reporter for yeast based on ASAP offers a new effective strategy for real-time optical detection of yeast membrane potential change, which potentially facilitates many areas of yeast research including optimizing growth conditions for industrial use and investigating yeast ion transport system.

The neural membrane potential of nerve cells is the basis of neural activity production, which controls advanced brain activities such as memory, emotion, and learning. In the past decades, optical voltage indicator has emerged as a promising tool to decode neural activities with high-fidelity and excellent spatiotemporal resolution. In particular, the hybrid optical probes can combine the advantageous photophysical properties of different components such as voltage-sensitive molecules, highly fluorescent fluorophores, membrane-targeting tags, and optogenetic materials, thus showing numerous advantages in improving the photoluminescence intensity, voltage sensitivity, photostability, and cell specificity of probes. In this review, the current state-of-the-art hybrid probes are highlighted, that are designed by using fluorescent proteins, organic dyes, and fluorescent nanoprobes as the fluorophores, respectively. Then, the design strategies, voltage-sensing mechanisms and the in vitro and in vivo neural activity imaging applications of the hybrid probes are summarized. Finally, based on the current achievements of voltage imaging studies, the challenges and prospects for design and application of hybrid optical probes in the future are presented.

Fluorescent probes that indicate biologically important quantities are widely used for many different types of biological experiments across life sciences. During recent years, limitations of small molecule-based indicators have been overcome by the development of genetically encoded indicators. Here we focus on fluorescent calcium and voltage indicators and point to their applications mainly in neurosciences.

Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation. Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltage-sensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in terms of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.