Fluorescent voltage indicators are an attractive alternative for studying the electrical activity of excitable cells; however, the development of indicators that are both highly sensitive and low in toxicity over long-term experiments remains a challenge. Previously, we reported a fluorene-based voltage-sensitive fluorophore that exhibits much lower phototoxicity than previous voltage indicators in cardiomyocyte monolayers, but suffers from low sensitivity to membrane potential changes. Here, we report that the addition of a single vinyl spacer in the fluorene molecular wire scaffold improves the voltage sensitivity 1.5- to 3.5-fold over fluorene-based voltage probes. Furthermore, we demonstrate the improved ability of the new vinyl-fluorene VoltageFluors to monitor action potential kinetics in both mammalian neurons and human-induced pluripotent stem cell-derived cardiomyocytes. Addition of the vinyl spacer between the aniline donor and fluorene monomer results in indicators that are significantly less phototoxic in cardiomyocyte monolayers. These results demonstrate how structural modification to the voltage sensing domain have a large effect on improving the overall properties of molecular wire-based voltage indicators.

中文翻译：

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用于电压成像的乙烯基芴分子线，具有增强的灵敏度和降低的光毒性

荧光电压指示器是研究可兴奋细胞电活动的有吸引力的替代方案；然而，在长期实验中开发既高度敏感又低毒性的指标仍然是一个挑战。以前，我们报道了一种基于芴的电压敏感荧光团，它在心肌细胞单层中表现出比以前的电压指标低得多的光毒性，但对膜电位变化的敏感性较低。在这里，我们报告说，在芴分子线支架中添加单个乙烯基间隔物将电压灵敏度提高了 1.5 到 3.5 倍，比基于芴的电压探针。此外，我们展示了新的乙烯基芴VoltageFluors 提高了监测哺乳动物神经元和人类诱导的多能干细胞衍生心肌细胞动作电位动力学的能力。在苯胺供体和芴单体之间添加乙烯基间隔导致指示剂在心肌细胞单层中的光毒性显着降低。这些结果证明了电压传感域的结构修改如何对改善基于分子线的电压指示器的整体性能产生很大影响。