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基于光物理的两光子电压成像使用FRET-opsin电压指示器

F Phil Brooks¹, Hunter C Davis¹, Pojeong Park¹

¹Department of Chemistry and Chemical Biology, Harvard University.

bioRxiv : the preprint server for biology

|April 15, 2024

概括

遗传编码电压指示器 (GEVIs) 显示来自光循环中间体的电压灵敏度,而不是基本状态. 优化的协议使得在活小鼠中实现高速的两光子电压成像.

科学领域:

神经科学是一个神经科学.
生物物理学的生物物理.
分子成像学分子成像学

背景情况:

来自微生物罗多素的遗传编码电压指示器 (GEVIs) 对于绘制生物电活动的地图至关重要.
福斯特共振能量转移 (FRET) - - 奥普辛GEVI提供高亮度,速度和电压灵敏度,但在双光子 (2P) 激发下往往会失去灵敏度.

研究的目的:

为了研究FRET-opsin GEVIs Voltron1和Voltron2.2的光物理.
了解电压灵敏度机制,并优化GEVI用于2P激发.

主要方法:

沃尔特朗1和沃尔特朗2GEVI的光物理特征.
分析电压灵敏度作为照明强度的函数.
开发光循环优化的2P照明协议.

主要成果:

在FRET-opsin GEVIs中的电压灵敏性源自光循环中间体,而不是opsin基态.
灵敏度是照明强度的非线性函数,在低强度下可能出现信号反转.
通过使用Voltron2与优化的协议,成功地在小鼠皮质中展示了2P电压成像.

结论:

在老鼠的皮质中证明了Voltron2的成功2P电压成像.
这些发现为FRET-opsin GEVIs in vivo的高速2P电压成像铺平了道路.
了解光物理是克服GEVIs在2P显微镜下的局限性的关键.

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