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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, Daozheng Gong¹, Hunter C Davis¹

¹Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA, USA.

Science advances

|January 8, 2025

概括

基因编码的电压指示器 (GEVIs) 在双光子激发下显示电压敏感性问题. 优化的协议使得使用FRET-opsin GEVIs (如Voltron2.2) 的高速度体内电压成像成为可能.

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.
分子生物学分子生物学

背景情况:

来自微生物罗多素的遗传编码电压指标 (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) -opsin GEVI 提供高亮度,速度和电压灵敏度.
一个显著的局限性是FRET-opsin GEVIs在两光子 (2P) 激发下的降低电压灵敏度.

研究的目的:

为了研究FRET-opsin GEVIs Voltron1和Voltron2.2的光物理,
了解2P激发下电压敏感性下降的原因.
为有效的2P电压成像 in vivo开发优化的协议.

主要方法:

沃尔特朗1和沃尔特朗2的光物理特征.
分析电压灵敏度作为照明强度的函数.
开发和应用光循环优化的2P照明协议.
在活体中2P电压成像在一个活的小鼠模型.

主要成果:

在FRET-opsin GEVIs中观察到的电压灵敏度来源于光循环中间体,而不是基态.
电压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.
成功开发了光循环优化的2P照明协议.
高速2P电压成像在体内使用Voltron2在小鼠大脑中进行了证明.

结论:

在2P激发下FRET-opsin GEVIs的光物理是复杂的,受到光循环中间体和照明强度的影响.
优化的2P照明协议可以克服以前的局限性,从而实现有效的体内电压成像.
这些发现为神经科学中先进的高速2P电压成像应用铺平了道路.

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