Sorbonne Université, INSERM, CNRS, Institut de la Vision, F-75012, Paris, France.
Nat Commun. 2023 Apr 5;14(1):1888. doi: 10.1038/s41467-023-37416-w.
Two-photon, single-cell resolution optogenetics based on holographic light-targeting approaches enables the generation of precise spatiotemporal neuronal activity patterns and thus a broad range of experimental applications, such as high throughput connectivity mapping and probing neural codes for perception. Yet, current holographic approaches limit the resolution for tuning the relative spiking time of distinct cells to a few milliseconds, and the achievable number of targets to 100-200, depending on the working depth. To overcome these limitations and expand the capabilities of single-cell optogenetics, we introduce an ultra-fast sequential light targeting (FLiT) optical configuration based on the rapid switching of a temporally focused beam between holograms at kHz rates. We used FLiT to demonstrate two illumination protocols, termed hybrid- and cyclic-illumination, and achieve sub-millisecond control of sequential neuronal activation and high throughput multicell illumination in vitro (mouse organotypic and acute brain slices) and in vivo (zebrafish larvae and mice), while minimizing light-induced thermal rise. These approaches will be important for experiments that require rapid and precise cell stimulation with defined spatio-temporal activity patterns and optical control of large neuronal ensembles.
基于全息光靶向方法的双光子、单细胞分辨率光遗传学能够产生精确的时空神经元活动模式，从而实现广泛的实验应用，如高通量连接映射和探测感知神经码。然而，目前的全息方法将调节不同细胞相对尖峰时间的分辨率限制在几毫秒，并且根据工作深度，可实现的目标数量限制在 100-200 个。为了克服这些限制并扩展单细胞光遗传学的功能，我们引入了一种超快顺序光靶向 (FLiT) 光学配置，该配置基于以 kHz 速率在全息图之间快速切换时间聚焦光束。我们使用 FLiT 演示了两种照明方案，称为混合照明和循环照明，并实现了亚毫秒级的顺序神经元激活控制和高通量多细胞体外（鼠器官型和急性脑切片）和体内（斑马鱼幼虫和小鼠）照明，同时最大限度地减少光诱导的热上升。这些方法对于需要具有定义的时空活动模式的快速和精确细胞刺激以及对大型神经元集合的光学控制的实验将非常重要。
