Uterine collagen deposition fluctuates throughout the estrous cycle and provides a scaffold for gland visualization via the SHG-casting method.

Uterine extracellular matrix (ECM) and uterine glands are essential parts of the endometrium, with important roles in remodeling associated with the estrous cycle, fertility, and pregnancy. Using second harmonic generation (SHG) microscopy, enabling the label-free imaging of fibrillar collagen, we analyzed mouse endometrial tissue across estrous cycle phases. We observed quantifiable 2D and 3D changes in fibrillar collagen accumulation, especially around uterine glands in the estrus phase. We also validate that the 3D SHG fibrillar collagen scans can serve as a template to recreate 3D uterine gland morphology by a method we call SHG-casting, enabling simultaneous label-free studies of endometrial fibrillar collagen and glands. Our custom deep learning-based segmentation model enables rapid, accurate gland segmentation. SHG-casting reveals variable gland structures, including lobular morphology in estrus and involution in later phases. SHG- casting offers a powerful tool for the 3D imaging of epithelial structures supported by collagen-rich ECM, with broad research and diagnostic potential.