Optical wavefront shaping is a powerful technique to control the distribution of light in the focus of a microscope. This ability, combined with optogenetics, holds great promise for precise manipulation of neuronal activity with light. However, a deeper understanding of complex brain circuits requires pushing light-shaping methods into a new regime: the simultaneous excitation of several tens of targets, arbitrarily distributed in the three dimensions, with single-cell resolution. To this end, we developed a new optical scheme, based on the spatio-temporal shaping of a pulsed laser beam, to project several tens of spatially confined two-photon excitation patterns in a large volume. Compatibility with several different phase-shaping strategies allows the system to be optimized towards flexibility, simplicity, or multiple independent light manipulations, thus providing new routes for precise three-dimensional optogenetics. To validate the method, we performed multi-cell volumetric excitation of photoactivatable GCaMP in the central nervous system of drosophila larvae, a challenging structure with densely arrayed neurons, and photoconversion of the fluorescent protein Kaede in zebrafish larvae.

中文翻译：

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多重时间聚焦光整形可实现高分辨率多细胞靶向

光学波前整形是一种强大的技术，可以控制显微镜焦点处的光分布。这种能力与光遗传学相结合，对于用光精确控制神经元活动具有广阔的前景。但是，对复杂的大脑回路的更深入的了解需要将光整形方法推向新的领域：以单细胞分辨率同时激发三个方向上任意分布的数十个目标。为此，我们基于脉冲激光束的时空整形开发了一种新的光学方案，以大体积投影数十个空间受限的双光子激发图案。与几种不同的相位整形策略兼容，可以优化系统的灵活性，简便性，或多个独立的光操纵，从而为精确的三维光遗传学提供了新的途径。为了验证该方法，我们在果蝇幼虫的中枢神经系统中进行了光激活GCaMP的多细胞体积激发，这是具有密集排列的神经元的具有挑战性的结构，并且在斑马鱼幼虫中对荧光蛋白Kaede进行了光转化。