ABSTRACT We have developed new imaging and computational workflows to produce accurately aligned multimodal 3D images of the mouse brain that exploit high resolution magnetic resonance histology (MRH) and light sheet microscopy (LSM) with fully rendered 3D reference delineations of brain structures. The suite of methods starts with the acquisition of geometrically accurate (in-skull) brain MRIs using multi-gradient echo (MGRE) and new diffusion tensor imaging (DTI) at an isotropic spatial resolution of 15 μm. Whole brain connectomes are generated using over 100 diffusion weighted images acquired with gradients at uniformly spaced angles. Track density images are generated at a super-resolution of 5 μm. Brains are dissected from the cranium, cleared with SHIELD, stained by immunohistochemistry, and imaged by LSM at 1.8 μm/pixel. LSM channels are registered into the reference MRH space along with the Allen Brain Atlas (ABA) Common Coordinate Framework version 3 (CCFv3). The result is a hi gh- d imensional i ntegrated v olum e with r egistration ( HiDiver ) that has a global alignment accuracy of 10–50 μm. HiDiver enables 3D quantitative and global analyses of cells, circuits, connectomes, and CNS regions of interest (ROIs). Throughput is sufficiently high that HiDiver is now being used in comprehensive quantitative studies of the impact of gene variants and aging on rodent brain cytoarchitecture.

HiDiver: A Suite of Methods to Merge Magnetic Resonance Histology, Light Sheet Microscopy, and Complete Brain Delineations