Corral-Bolanos, M.; Swami, P.; Vila-Merkle, H.; Lima, J.; Vange, S. P.; Destrieux, C.; Poupon, C.; Pingel, J.; Kaalund, S. S.; Bardia, L.; Kjer, H. M.; Pizzolato, M.; Frisvad, J. R.; Colombelli, J.; Dyrby, T. B.

Mapping the three-dimensional directional organization of biological tissues is essential for understanding their structure and function. However, existing methods cannot resolve micrometer-scale orientation in volumetric samples. We introduce tensor light-sheet scattering microscopy (tLSSM), a label-free method that reconstructs 3D fiber orientations at micrometer resolution in optically cleared tissues, enabling whole-organ imaging. We discovered that even in transparent samples, organized structures such as neural fibers scatter light in directional patterns, consistent with models of light scattering by cylinders. We compared tLSSM against diffusion MRI in the mouse brain, demonstrating strong agreement and orders of magnitude superior spatial resolution. Furthermore, we showcase tLSSM's versatility across diverse contexts, including whole-brain label-free tracing, pathological demyelination lesions, heart tissue, peripheral nerves, and human white matter. By enabling whole-organ fiber orientation mapping compatible with standard light-sheet microscopes, tLSSM establishes a new standard for mesoscopic connectivity studies by mapping tissue architecture beyond the limits of traditional sectioning.