Pende, M.; Cregg, J. M.; Saghafi, S.; Broadbent, S.; Avdibasic, A.; Roeles, J.; Papadopoulos, S.-C.; Seaman, R. P.; Pende, N.; Mateos, M. S.; Jamwal, K.; Wunch, M.; Pasierbek, P.; Moreno-Cencerrado, A.; Korchynska, S.; Hauer, R.; Anderson, P.; Supper, P.; Kastriti, M. E.; Reumann, D.; Moorhead, M.; Graber, J. H. H.; Scholze, P.; Henschke, J. U.; Budinger, E.; Knoblich, J. A.; Klausberger, T.; Adameyko, I.; Harkany, T.; Kumar, V.; Joy, M. T.; Kiehn, O.; Dodt, H.-U.; Voigt, F.; Murawala, P.

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.