High-resolution optical imaging deep in tissues is challenging because of optical aberrations and scattering of light caused by the complex structure of living matter. Here we present an adaptive optics three-photon microscope based on analog lock-in phase detection for focus sensing and shaping (ALPHA-FSS). ALPHA-FSS accurately measures and effectively compensates for both aberrations and scattering induced by specimens and recovers subcellular resolution at depth. A conjugate adaptive optics configuration with remote focusing enables in vivo imaging of fine neuronal structures in the mouse cortex through the intact skull up to a depth of 750 µm below the pia, enabling near-non-invasive high-resolution microscopy in cortex. Functional calcium imaging with high sensitivity and high-precision laser-mediated microsurgery through the intact skull were also demonstrated. Moreover, we achieved in vivo high-resolution imaging of the deep cortex and subcortical hippocampus up to 1.1 mm below the pia within the intact brain.

由于生物体的复杂结构会导致光学像差和光散射，因此组织深处的高分辨率光学成像具有挑战性。在这里，我们提出了一种基于模拟锁定相位检测的自适应光学三光子显微镜，用于聚焦传感和整形 (ALPHA-FSS)。ALPHA-FSS 准确测量并有效补偿标本引起的像差和散射，并恢复深度的亚细胞分辨率。具有远程聚焦的共轭自适应光学配置能够通过完整的头骨对小鼠皮层中的精细神经元结构进行活体成像，深度可达软脑膜下方 750 µm，从而在皮层中实现近乎无创的高分辨率显微镜检查。还展示了通过完整颅骨进行的高灵敏度功能性钙成像和高精度激光介导显微手术。此外，我们实现了完整大脑内软脑膜下方 1.1 毫米深皮质和皮质下海马体的高分辨率体内成像。