A new study published in Cell on July 10 has reported the world’s fastest high-resolution three-dimensional (3D) imaging technology for the entire body of small animals at subcellular resolution, enabling efficient mapping of the fine architecture of the peripheral nervous system (PNS).

This study was conducted by a team led by Prof. BI Guoqiang and Prof. LIU Beiming from the University of Science and Technology of China (USTC) of the Chinese Academy of Sciences (CAS), along with researchers from the Institute of Artificial Intelligence, Hefei Comprehensive National Science Center, and the Shenzhen Institute of Advanced Technology of CAS.

The PNS serves as the body's "internet of things" by mediating bidirectional communication and modulation between the brain and organs, which enables efficient physiological coordination among various tissues and organs. Mapping the intricate connections of the PNS throughout the whole body is essential for fundamentally understanding its functional mechanisms and the pathogenesis of related diseases.

The knowledge of PNS architecture has long relied on millimeter-resolution anatomical studies. Advances in 3D optical microscopy have since propelled micron-resolution whole-brain mesoscopic connectomic mapping. Current imaging techniques struggle to balance high imaging resolution and high speed. Using them to resolve nerve routes of the PNS at the whole-body scale remains challenging.

The researchers have developed Volumetric Imaging with Synchronized on-the-fly scan and Readout (VISoR) technology for 3D imaging of cleared thick-sectioned brain samples. This technology has the advantage of high imaging speed, high resolution, and scalability. However, this approach is not suitable for the whole mouse body as the mammalian body is much larger and highly heterogeneous, containing irregular structures and diverse tissue types.

To address this challenge, the researchers proposed an "in situ sectioning + 3D blockface imaging" strategy for this study. They developed a blockface-VISoR imaging system integrating a precision vibratome and the ARCHmap protocol for whole-body clearing and hydrogel embedding. 

The blockface-VISoR imaging system captures ~600 μm-depth 3D surface images and then automatically removes a 400-μm-thick tissue layer in each imaging cycle, repeating until the entire sample is processed. Automated inter-section stitching algorithms then perform seamless 3D alignment using ~200-μm overlapping regions between adjacent sections. The shallow imaging depth minimizes light scattering in cleared tissue, enabling high resolution.

Based on this strategy, the researchers established an optimized technical pipeline for whole-body clearing and imaging and achieved 3D imaging of an entire adult mouse body at a uniform subcellular resolution within 40 hours, which generated ~70 terabytes of data per fluorescence channel. Over four petabytes of raw data from dozens of mice were collected.

Due to the excellent fluorescent signal preservation afforded by the sample processing method, the researchers verified that the technology is compatible with commonly used labeling methods in neuroscience, such as transgenic labeling, virus labeling, and immunostaining. 

Combining various labeling methods with imaging, the researchers analyzed the fine structure and single-fiber projection paths of different types of peripheral nerves throughout the mouse body. They revealed the cross-segmental projection characteristics of individual thoracic spinal neurons for the first time, mapped the organ-specific vascular distribution pattern of sympathetic nerves throughout the body, and resolved the overall projection architecture of the vagus nerve and the complex single-fiber routes.

This technology helps establish a new paradigm for connectivity mapping of the PNS and resolving fundamental questions in neural regulation. The future steps for the optimization of this technology involve using two or more cameras for more efficient multi-channel imaging, and exploring its application in imaging other larger-scale biological samples.

This study represents a major breakthrough in 3D imaging of large-scale biological tissues, which provides insights into research areas such as developmental biology, comparative anatomy, and biomedical research in general. "These analyses produced strikingly detailed data at both the population and single-cell levels. Importantly, new insights emerged from this initial exploration," said the reviewer from Cell.

Link to experimental techniques and imaging datasets: https://mesoanatomy.org/mesomouse/

3D Visualization of Peripheral Nerves Throughout the Mouse Body (Video by Prof. BI's team)