Gest Anneliese M M, Lazzari-Dean Julia R, Ortiz Gloria, Yaeger-Weiss Susanna K, Boggess Steven C, Miller Evan W
Department of Chemistry, University of California, Berkeley.
Department of Molecular & Cell Biology, University of California, Berkeley.
bioRxiv. 2023 Oct 10:2023.10.06.561080. doi: 10.1101/2023.10.06.561080.
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 new fluorescent indicator which can address both. We describe the synthesis of a new 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 with greater signal-to-noise than state-of-the-art BeRST (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 action potentials 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.
生物膜电位或电压是细胞生命的核心方面。用荧光指示剂可视化细胞膜电压的光学方法是传统基于电极方法的一种有吸引力的补充，因为成像方法可以高通量、侵入性较小，并且比电极提供更高的空间分辨率。最近开发的用于电压的荧光指示剂主要通过监测荧光强度中依赖电压的波动来报告膜电压的变化。然而，不仅能够监测变化，还能够测量膜电位的值将是很有用的。本研究公开了一种能够解决这两个问题的新型荧光指示剂。我们描述了一种新型磺化四甲基碳硼罗丹明荧光团的合成。当这种碳硼罗丹明与富含电子、含有甲氧基（-OMe）的亚苯基亚乙烯基分子线共轭时，所得分子CRhOMe是一种具有红/远红荧光的电压敏感荧光团。使用CRhOMe，可以通过荧光强度或寿命读出细胞膜电位的变化。在荧光强度模式下，CRhOMe跟踪快速发放动作电位时的信噪比高于目前最先进的BeRST（另一种电压敏感荧光团）。CRhOMe还可以测量膜电位的值。CRhOMe的荧光寿命遵循单指数衰减，显著改善了使用荧光寿命成像显微镜（FLIM）对膜电位值的定量。红移激发和发射、单指数衰减以及高电压敏感性的结合使得能够对心肌细胞中的动作电位进行快速FLIM记录。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一种重要方法。
