•Recent advances in genetically encoded voltage indicators (GEVIs) have brought neural voltage imaging in vivo within reach.•Recently introduced tools such as electrically spiking HEK cells facilitate rapid testing of new GEVIs.•Light-gated voltage integrators and reporters of absolute voltage reporters implement new forms of molecular logic.•Many potentially useful GEVI scaffolds and designs remain to be tested.•Improvements in microscopy and software will be needed to attain full benefit from the newest GEVIs.Membrane voltages are ubiquitous throughout cell biology. Voltage is most commonly associated with excitable cells such as neurons and cardiomyocytes, although many other cell types and organelles also support electrical signaling. Voltage imaging in vivo would offer unique capabilities in reporting the spatial pattern and temporal dynamics of electrical signaling at the cellular and circuit levels. Voltage is not directly visible, and so a longstanding challenge has been to develop genetically encoded fluorescent voltage indicator proteins. Recent advances have led to a profusion of new voltage indicators, based on different scaffolds and with different tradeoffs between voltage sensitivity, speed, brightness, and spectrum. In this review, we describe recent advances in design and applications of genetically-encoded voltage indicators (GEVIs). We also highlight the protein engineering strategies employed to improve the dynamic range and kinetics of GEVIs and opportunities for future advances.Download high-res image (180KB)Download full-size image

A holy grail in neuroscience is to understand how brain functions arise from neural network-level electrical activities. Voltage imaging allows for the direct visualization of electrical signaling at high spatial and temporal resolutions across a large neuronal population. Central to this technique is a palette of genetically-encoded fluorescent probes with fast and sensitive voltage responses. In this review, we chronicle the development and applications of genetically-encoded voltage indicators (GEVIs) over the past two decades, with a primary focus on the structural design that harness the power of fluctuating transmembrane electric fields. We hope this article will inform chemical biologists and protein engineers of the GEVI history and inspire novel design ideas.Download high-res image (86KB)Download full-size imageVoltage imaging with genetically-encoded sensors has allowed for the direct visualization of electrical signaling at high spatial resolutions. Over the history of voltage indicator development, variousdesign strategies have been employed toharness the power of the fluctuating transmembrane electric field.