Electrophysiology, Unplugged: Imaging Membrane Potential with Fluorescent Indicators.

Membrane potential is a fundamental biophysical property maintained by every cell on earth. In specialized cells like neurons, rapid changes in membrane potential drive the release of chemical neurotransmitters. Coordinated, rapid changes in neuronal membrane potential across large numbers of interconnected neurons form the basis for all of human cognition, sensory perception, and memory. Despite the importance of this highly orchestrated and distributed activity, the traditional method for recording membrane potential is through the use of highly invasive single-cell electrodes that offer only a small glimpse of the total activity within a system. Fluorescent dyes that change their optical properties in response to changes in biological voltage have the potential to provide a powerful complement to traditional electrode-based methods of inquiry. Voltage-sensitive fluorescent indicators would allow the direct observation of membrane potential changes, significantly expanding our ability to monitor membrane potential dynamics in living systems. Toward this end, we have initiated a program to design, synthesize, and apply voltage-sensitive fluorophores that report on membrane potential dynamics with high sensitivity and speed. The basis for this optical voltage sensing is membrane potential-dependent photoinduced electron transfer (PeT). Voltage-sensitive fluorophores, or VoltageFluors, possess a fluorophore, a conjugated molecular wire, and an aniline donor. At resting potentials, in which the cell has a hyperpolarized or negative potential relative to the outside of the cell, PeT from the aniline donor is enhanced and fluorescence is diminished. At depolarized potentials, the membrane potential decreases the rate of PeT, allowing an increase in fluorescence. We show that a number of different fluorophores, molecular wires, and aniline donors can be employed to generate fast and sensitive VoltageFluor dyes. Multiple lines of evidence point to a PeT-based mechanism for voltage sensing, delivering fast response kinetics (∼25 ns), good sensitivity (>60% ΔF/F), compatibility with two-photon illumination, excellent signal-to-noise, and the ability to detect neuronal and cardiac action potentials in single trials. In this Account, we provide an overview of the challenges facing the design of fluorescent voltage indicators. We trace the development of molecular wire-based fluorescent voltage indicators within our group, beginning from fluorescein-based VoltageFluor to long-wavelength indicators that use modern fluorophores like silicon rhodamine and carbofluorescein. We examine design principles for PeT-based voltage indicators, showcase the use of our recent indicators for two-photon voltage imaging in intact brains, and explore the development of hybrid indicators that can localize to genetically defined cells. Finally, we highlight outstanding challenges to and opportunities for voltage imaging.