Two‐photon (2P) optogenetic stimulation is currently the only method for precise, fast, and non‐invasive cellular excitation deep inside brain tissue; it is typically combined with holographic wavefront‐shaping techniques to generate distributed light patterns and target them to multiple specific cells in the brain. During propagation in the brain, these light patterns undergo severe distortion, mainly due to scattering, which leads to a discrepancy between the desired and actual light distribution. However, despite its importance, measurement of these tissue‐induced distortions and their effects on the light patterns has yet to be demonstrated in situ. To this end, holographic optogenetics confocally unraveled sculpting (HOCUS), a system for real‐time in situ evaluation of holographic light patterns, based on confocally descanning the stimulation light's reflection from the brain, is developed. HOCUS measures both tissue and wave propagation properties and enables the real‐time measurement and correction of the dimensions and positions of holographic spots relative to neurons targeted for stimulation. It can also be used to measure tissue attenuation length, and thus should facilitate future attempts to optimize the generated hologram to pre‐compensate for tissue‐induced distortions, thereby improving the reliability of 2P holographic stimulation experiments.

中文翻译：

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实时原位全息光遗传学共聚焦解开雕刻显微镜

目前，双光子（2P）光遗传学刺激是在脑组织内部进行精确，快速且无创性细胞激发的唯一方法。它通常与全息波前成形技术结合使用，以生成分布式的光模式，并将其定向到大脑中的多个特定细胞。在大脑中传播期间，这些光模式主要由于散射而遭受严重的畸变，从而导致所需的光分布与实际的光分布之间存在差异。然而，尽管它很重要，但这些组织诱发的畸变及其对光模式的影响的测量方法尚未在现场得到证实。为此，全息光遗传学共同解决了雕刻问题（HOCUS）的问题，这是一种用于全息光图案实时原位评估的系统，基于共焦消扫描来自大脑的刺激光的反射，就得到了发展。HOCUS可测量组织和波的传播特性，并可实时测量和校正全息斑点相对于刺激目标神经元的尺寸和位置。它也可以用于测量组织衰减的长度，因此应该有助于将来尝试优化生成的全息图，以预先补偿组织引起的畸变，从而提高2P全息刺激实验的可靠性。