Protocol for using serial two-photon tomography to map cell types and cerebrovasculature at single-cell resolution in the whole adult mouse brain

Here, we present a protocol using serial two-photon tomography (STPT) to quantitatively map genetically defined cell types and cerebrovasculature at single-cell resolution across the entire adult mouse brain. We describe the preparation of brain tissue and sample embedding for cell type and vascular STPT imaging and image processing using MATLAB codes. We detail the computational analyses for cell signal detection, vascular tracing, and three-dimensional image registration to anatomical atlases, which can be implemented for brain-wide mapping of different cell types. For complete details on the use and execution of this protocol, please refer to Wu et al. (2022),¹ Son et al. (2022),² Newmaster et al. (2020),³ Kim et al. (2017),⁴ and Ragan et al. (2012).⁵