In current biological and biomedical research, quantitative endpoints have become an important factor of success. Classically, such endpoints were investigated with 2D imaging, which is usually destructive and the 3D character of tissue gets lost. 3D imaging has gained in importance as a tool for both, qualitative and quantitative assessment of biological systems. In this context synchrotron radiation based tomography has become a very effective tool for opaque 3D tissue systems. Results from a new device are presented enabling the 3D investigation of trabecular bone under mechanical load in a time-lapsed fashion. Using the highly brilliant X-rays from a synchrotron radiation source, bone microcracks and an indication for un-cracked ligament bridging are uncovered. 3D microcrack analysis proves that the classification of microcracks from 2D images is ambiguous. Fatigued bone was found to fail in burst-like fashion, whereas non-fatigued bone exhibited a distinct failure band. Additionally, a higher increase in microcrack volume was detected in fatigued in comparison to non-fatigued bone. Below the spatial resolution accessible with synchrotron radiation tomography we investigated native and fractured bone surfaces on the molecular scale with atomic force microscopy. The mineralized fibrils detected on fracture surfaces give rise to the assumption that the mineral-mineral interface is the weakest link in bone. The presented results show the power of functional micro-imaging, as well as the possibilities for AFM imaging (functional nano-imaging) in this context.