A uniform tissue-clearing framework and mesoSPIM-ultra enable cm-scale single-neuron tracing.

Tissue-clearing and light-sheet microscopy have transformed volumetric imaging of intact organs, yet limited mechanistic understanding of dehydration-based clearing continues to constrain rational protocol design and broader applicability. Here, we define the cardinal chemical and physical principles underlying dehydration-based tissue-clearing and establish a new pipeline for large-volume imaging. To maximize imaging performance, we developed the "mesoSPIM-ultra", an upgraded mesoSPIM platform with a temperature-controlled sample chamber, a large field-of-view (FoV) camera and specialized optics to achieve long-working-distance, high-resolution imaging of cleared samples. We applied this approach to investigate the projectome of Chx10 + neurons, a cell population with complex axonal morphologies along the entire mouse spinal-cord and brain, and implicated in ipsilateral orienting behaviors. By combining behavioral analysis with post-hoc single-neuron reconstructions, we revealed previously inaccessible branching architectures and long-range projections extending from the brainstem to the spinal cord. Together, our work establishes a mechanistic foundation for tissue-clearing and scalable imaging.