A pipeline for mouse brain registration to an atlas after in vivo neurophysiological recordings.

Neurophysiological recordings and histological examinations, along with behavioral observations, are interconnected methodological dimensions of systems neuroscience. Current progresses in the neurophysiological data acquisition and machine learning-based data-driven behavioral analysis emphasize the need for precise anatomical localization of recorded neurons. Here, we describe an integrated pipeline for mapping mouse brain regions expressing genetically encoded calcium indicator imaged with two-photon microscopy, and high-density multichannel electrode positions marked with lipophilic dye, to standardized anatomical coordinates. This protocol consists of three parts. First, we present a step-by-step procedure of the Fast 3D Clear method applied to mouse brains. Second, we describe the configuration and acquisition of the three-dimensional whole-brain imaging system using descSPIM, a custom-made light-sheet fluorescence microscope. Finally, we provide a detailed explanation and practical guide for image analysis for whole-brain image volume, including stitching, alignment, and registration to the Allen Common Coordinate Framework. Our workflow successfully localized a region of interest from two-photon imaging and a Neuropixel probe trajectory in the coordinate system. Our scalable, affordable, and accessible protocol allows researchers to replicate and adapt it to align with their objectives, including application to other species.