Radium-223 (223Ra) is a bone-targeting, alpha particle-emitting radionuclide approved for the treatment of patients with metastatic prostate cancer that is currently being tested in a variety of clinical trials for a diverse set of bone-related disease. 223Ra has been shown to accumulate on mineralized bone tissue due to its calcium-mimetic properties and is highly enriched in areas with high bone turnover like the growth plates of long bones. Recent clinical studies have shown a significant fracture rate increase associated with the use of 223Ra, predominantly in tumor-free bones (68% of fractures). However, the biological mechanisms underlying this bone fragility are still unclear and generate a great concern in the clinical setting. Therefore, preclinical studies addressing the role of 223Ra-mediated modulation of the bone stromal components and the consequences on bone mechanical properties are much needed. In this thesis, we combined mechanical testing, micro-CT, and conventional endpoint analysis with fluorescent reporter mice for ex vivo 3D spatial biology microscopy analysis to clarify the effects of 223Ra on bone stromal components including osteoblasts, osteoclasts, adipocytes, and blood vessels. This approach assumes relevance when studying the response to 223Ra, which is spatially confined within 100 μm of the bone interface, due to the short penetration of alpha particles. Additionally, a novel tool for bone tissue image analysis was developed and validated. Overall, this thesis advances our understanding in bone biology and the unexplored impact of alpha-particle radiation on the bone microenvironment.