High-throughput 3D mapping of large mammalian brains via integration of precision tissue sectioning and light sheet imaging

Whole-brain mapping at the cellular and subcellular levels is crucial to systematically understand brain functions and disorders. Recent developments in tissue transformation techniques, such as CLARITY, SHIELD, MAP, ExM, CUBIC, and DISCO-based methods, have made significant progress towards whole-organ molecular labeling and microscopic imaging by rendering intact tissue chemically permeable and optically transparent. Alternative approaches to generate 3D whole brain datasets are based on integration of block-face imaging and mechanical tissue sectioning, such as the STP, FAST, and WVT methods. Though these approaches have successfully enabled volumetric imaging of the mouse brain and contributed substantially to neuroscience research, they are not realistically applicable to larger scale mammalian brains. Currently, the only way to image large intact mammalian brain samples is to dissect the sample into small blocks, followed by the processing and imaging of each block individually. Not only is this process laborious and low-throughput but it causes excessive tissue damage, making it nearly impossible to piece together a complete picture of a large sample without information loss. Furthermore, large-scale studies that require using considerable amounts of rodent brains are extremely laborious and time consuming based on current technologies. As a high-throughput solution is urgently needed, LifeCanvas Technologies proposes to pioneer new developments for efficient, automated processing and high-resolution microscopic imaging of large scale mammalian brains. Specifically, our new technological developments will focus on 1) an advanced vibrating microtome for automatic precision slicing of samples as large as an intact human brain hemisphere. The vibrating microtome will be optimized for slicing highly complex samples with minimal tissue damage, uniform thickness and extremely even surface profile; and 2) an integrated volumetric imaging system consisting of the vibrating microtome and a custom-developed, high-resolution light sheet microscope. The system will perform serial tissue sectioning and imaging at unprecedented speed for a wide range of samples, including rodent brains, expanded rodent brains, intact non-human primate brains, and whole human brain coronal slabs. In this phase, we will build prototypes of the instruments and demonstrate the functionality on expanded, intact mouse brains and a large array of mouse brains. In the next phase, we will build user-friendly, ready-to-commercialize instruments tested on human and non-human primate brains. We anticipate the new developments will expand LifeCanvas’ current tissue clearing, labeling, imaging and quantitative analysis pipeline, and offer the neuroscience community a complete solution for mammalian brain high-throughput processing and volumetric imaging.