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.

中文翻译：

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电生理学，未插电：使用荧光指示剂成像膜电位。

膜电位是地球上每个细胞维持的基本生物物理特性。在像神经元这样的特殊细胞中，膜电位的快速变化驱动化学神经递质的释放。跨越大量相互连接的神经元的神经元膜电位的协调快速变化，构成了人类所有认知，感觉和记忆的基础。尽管这种高度协调和分散的活动非常重要，但记录膜电位的传统方法是使用高度侵入性的单细胞电极，该电极仅能提供系统中总活动的一小部分。响应于生物电压的变化而改变其光学性质的荧光染料有可能为传统的基于电极的研究方法提供有力的补充。电压敏感型荧光指示剂可以直接观察膜电位的变化，从而极大地扩展了我们监测生物系统中膜电位动态的能力。为此，我们启动了一个程序来设计，合成和应用对电压敏感的荧光团，以高灵敏度和高速度报告膜电位动态。这种光学电压感测的基础是膜电位依赖性光致电子转移（PeT）。压敏荧光团或VoltageFluors具有荧光团，共轭分子线和苯胺供体。在静止电位下，其中细胞相对于细胞外部具有超极化或负电位，来自苯胺供体的PeT增强，荧光减弱。在去极化电势下，膜电位降低了PeT的速率，从而增加了荧光强度。我们表明，可以使用许多不同的荧光团，分子线和苯胺供体来产生快速而灵敏的VoltageFluor染料。多种证据表明，基于PeT的电压感测机制具有快速的响应动力学（〜25 ns），良好的灵敏度（> 60％ΔF/ F），与双光子照明兼容，出色的信噪比，以及在单个试验中检测神经元和心脏动作电位的能力。在此帐户中，我们概述了荧光电压指示器设计面临的挑战。我们追踪了我们小组中基于分子线的荧光电压指示器的发展，从基于荧光素的VoltageFluor到使用现代荧光团（如若丹明和碳荧光素）的长波长指示器。我们研究了基于PeT的电压指示器的设计原理，展示了我们最新的指示器在完整大脑中的双光子电压成像中的应用，并探索了可以定位到遗传定义的细胞的混合指示器的发展。最后，我们重点介绍了电压成像的巨大挑战和机遇。