Whole-brain mesoscale mapping in primates has been hindered by large brain sizes and the relatively low throughput of available microscopy methods. Here, we present an approach that combines primate-optimized tissue sectioning and clearing with ultrahigh-speed fluorescence microscopy implementing improved volumetric imaging with synchronized on-the-fly-scan and readout technique, and is capable of completing whole-brain imaging of a rhesus monkey at 1 × 1 × 2.5 µm³ voxel resolution within 100 h. We also developed a highly efficient method for long-range tracing of sparse axonal fibers in datasets numbering hundreds of terabytes. This pipeline, which we call serial sectioning and clearing, three-dimensional microscopy with semiautomated reconstruction and tracing (SMART), enables effective connectome-scale mapping of large primate brains. With SMART, we were able to construct a cortical projection map of the mediodorsal nucleus of the thalamus and identify distinct turning and routing patterns of individual axons in the cortical folds while approaching their arborization destinations.

灵长类动物的全脑中尺度映射受到大脑容量和可用显微镜方法相对较低吞吐量的阻碍。在这里，我们提出了一种将灵长类动物优化的组织切片和清除与超高速荧光显微镜相结合的方法，通过同步的飞行扫描和读出技术实现改进的体积成像，并且能够完成恒河猴的全脑成像猴子在 1 × 1 × 2.5 µm ³100 小时内的体素分辨率。我们还开发了一种高效方法，用于在数百 TB 的数据集中远程追踪稀疏轴突纤维。我们称之为连续切片和清除、具有半自动重建和追踪 (SMART) 的三维显微术的这条管道能够实现大型灵长类动物大脑的有效连接组尺度映射。借助 SMART，我们能够构建丘脑背侧核的皮质投影图，并在接近其树枝化目的地时识别皮质褶皱中单个轴突的不同转向和路由模式。