A far-red hybrid voltage indicator enabled by bioorthogonal engineering of rhodopsin on live neurons

Membrane potential is a key aspect of cellular signalling and is dynamically regulated by an array of ion-selective pumps and channels. Fluorescent voltage indicators enable non-invasive optical recording of the cellular membrane potential with high spatial resolution. Here, we report a palette of bright and sensitive hybrid voltage indicators (HVIs) with fluorescence intensities sensitive to changes in membrane potential via electrochromic Forster resonance energy transfer. Enzyme-mediated site-specific incorporation of a probe, followed by an inverse-electron-demand Diels-Alder cycloaddition, was used to create enhanced voltage-sensing rhodopsins with hybrid dye-protein architectures. The most sensitive indicator, HVI-Cy3, displays high voltage sensitivity (-39% Delta F/F-0 per 100 mV) and millisecond response kinetics, enabling optical recording of action potentials at a sampling rate of 400 Hz over 10 min across a large neuronal population. The far-red indicator HVI-Cy5 could be paired with optogenetic actuators and green/red-emitting fluorescent indicators, allowing an all-optical investigation of neuronal electrophysiology.

Automated summary

A range of bright and sensitive hybrid voltage indicators (HVIs) with fluorescence intensities sensitive to changes in membrane potential via electrochromic Forster resonance energy transfer were developed. These HVIs offer the potential for optical investigation of neuronal electrophysiology, enabling the recording of action potentials with high temporal resolution and sensitivity.