Voltage imaging with genetically encoded indicators

Abstract

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.

Figure 1. The family of GEVIs

A) and B) Approximate lineages of the major classes of GEVIs. The GEVIs highlighted in bold have shown the greatest sensitivity for use in vivo and are colored with their approximate excitation wavelengths. A) GEVIs based on voltage-sensor domains. B) GEVIs based on microbial rhodopsins. C)–G) Voltage sensing mechanisms in the major classes of GEVIs.

Figure 2. Applications of GEVI imaging

A) All-optical electrophysiology (‘Optopatch’) in neurons co-expressing a blue-shifted channelrhodopsin, CheRiff, and a red-shifted NIR GEVI, QuasAr2. Left: Optical stimuli evoke electrical spikes and closely corresponding fluorescence transients. Right: Patterned optogenetic stimulation on a dendritic region evokes action potentials whose sub-cellular propagation initiates at the axon initial segment, marked by an Ankyrin G immunostain. Figures from Ref. [32]. B) Ace2N-mNeon reports neuronal spikes in mouse visual cortex in vivo. Figure from Ref. [36]. C) Two-photon imaging of ASAP2f in Drosophila visual neurons reports sub-cellular details of stimulus-evoked electrical responses. Figure from Ref. [20]. D) Wide-field optical voltage mapping in mouse cortex in vivo. The optical signal correlates with the EEG from the ipsilateral but not the contralateral hemisphere. Figure from Ref. [16].

Figure 3. GEVI screening pipeline

A) A hierarchical approach screens large libraries for the most easily measured parameters (brightness), and then characterizes hits for voltage sensitivity and speed. Trafficking must ultimately be tested by GEVI expression in vivo and imaging in acute slice. Figure modified from Ref. [56]. B) Bacterial screens for brightness can be performed in a pooled library assay where highly fluorescent colonies are manually selected for propagation. To distinguish brightness from colony size, it is important to have a spectrally distinct reference fluorophore expressed at a constant level. C) Tests for voltage sensitivity and kinetics can be performed in cultured mammalian cells, using either (left) spiking HEK cells, (middle) induced transmembrane voltage, or (right) manual patch-clamp electrophysiology. D) Tests in cultured neurons probe trafficking and high-speed kinetics. Neural activity can be induced either optogenetically or via field-stimulation electrodes.