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Abstract：
Biological membrane potentials, or voltages, are a central facet of cellular life. Optical methods to visualize cellular membrane voltages with fluorescent indicators are an attractive complement to traditional electrode-based approaches, since imaging methods can be high throughput, less invasive, and provide more spatial resolution than electrodes. Recently developed fluorescent indicators for voltage largely report changes in membrane voltage by monitoring voltage-dependent fluctuations in fluorescence intensity. However, it would be useful to be able to not only monitor changes but also measure values of membrane potentials. This study discloses a fluorescent indicator which can address both. We describe the synthesis of a sulfonated tetramethyl carborhodamine fluorophore. When this carborhodamine is conjugated with an electron-rich, methoxy (-OMe) containing phenylenevinylene molecular wire, the resulting molecule, CRhOMe, is a voltage-sensitive fluorophore with red/far-red fluorescence. Using CRhOMe, changes in cellular membrane potential can be read out using fluorescence intensity or lifetime. In fluorescence intensity mode, CRhOMe tracks fast-spiking neuronal action potentials (APs) with greater signal-to-noise than state-of-the-art BeRST 1 (another voltage-sensitive fluorophore). CRhOMe can also measure values of membrane potential. The fluorescence lifetime of CRhOMe follows a single exponential decay, substantially improving the quantification of membrane potential values using fluorescence lifetime imaging microscopy (FLIM). The combination of red-shifted excitation and emission, mono-exponential decay, and high voltage sensitivity enable fast FLIM recording of APs in cardiomyocytes. The ability to both monitor and measure membrane potentials with red light using CRhOMe makes it an important approach for studying biological voltages.
摘要：
生物膜电位，或电压，是细胞生命的核心方面。使用荧光指示剂可视化细胞膜电压的光学方法是对传统的基于电极的方法的有吸引力的补充。由于成像方法可以是高通量的，侵入性较小，并提供比电极更多的空间分辨率。最近开发的用于电压的荧光指示剂通过监测荧光强度的电压依赖性波动来报告膜电压的变化。然而,这将是有用的，不仅能够监测变化，而且测量值的膜电位。该研究公开了可以解决这两者的荧光指示剂。我们描述了磺化的四甲基卡罗丹明荧光团的合成。当这种卡洛丹明与富电子共轭时，含甲氧基(-OMe)亚苯基乙烯分子丝,产生的分子，CRhOMe,是具有红色/远红色荧光的电压敏感荧光团。使用CRhOMe,细胞膜电位的变化可以使用荧光强度或寿命来读出。在荧光强度模式下，CRhOMe以比现有技术的BeRST1（另一种电压敏感荧光团）更大的信噪比跟踪快速尖峰神经元动作电位（AP）。CRhOMe还可以测量膜电位的值。CRhOMe的荧光寿命遵循单指数衰减，使用荧光寿命成像显微镜（FLIM）大大改善了膜电位值的定量。红移激发和发射的组合，单指数衰减，和高电压灵敏度能够快速FLIM记录心肌细胞中的AP。使用CRhOMe用红光监测和测量膜电位的能力使其成为研究生物电压的重要方法。
