Unbiased quantitative analysis of macroscopic biological samples demands fast imaging systems capable of maintaining high resolution across large volumes. Here we introduce RAPID (rapid autofocusing via pupil-split image phase detection), a real-time autofocus method applicable in every widefield-based microscope. RAPID-enabled light-sheet microscopy reliably reconstructs intact, cleared mouse brains with subcellular resolution, and allowed us to characterize the three-dimensional (3D) spatial clustering of somatostatin-positive neurons in the whole encephalon, including densely labeled areas. Furthermore, it enabled 3D morphological analysis of microglia across the entire brain. Beyond light-sheet microscopy, we demonstrate that RAPID maintains high image quality in various settings, from in vivo fluorescence imaging to 3D tracking of fast-moving organisms. RAPID thus provides a flexible autofocus solution that is suitable for traditional automated microscopy tasks as well as for quantitative analysis of large biological specimens.

宏观生物样品的无偏定量分析需要能够在大体积内保持高分辨率的快速成像系统。在这里，我们介绍了 RAPID（通过瞳孔分割图像相位检测进行快速自动对焦），这是一种适用于每个宽场显微镜的实时自动对焦方法。启用 RAPID 的光片显微镜可靠地重建具有亚细胞分辨率的完整、清晰的小鼠大脑，并允许我们表征整个脑中生长抑素阳性神经元的三维 (3D) 空间聚类，包括密集标记的区域。此外，它还可以对整个大脑的小胶质细胞进行 3D 形态分析。除了光片显微镜外，我们还证明了 RAPID 在各种环境下都能保持高图像质量，从体内荧光成像到快速移动生物体的 3D 跟踪。因此，RAPID 提供了一种灵活的自动对焦解决方案，适用于传统的自动显微镜任务以及大型生物标本的定量分析。