In bone tissue, multiscale interfaces provide the structural basis of essential bone functions and contribute to its macroscopic mechanical properties. The lacuno-canalicular network (LCN) hosting the osteocytes in the bone matrix, in particular, represents a biological signature of the mechanotransduction activity in response to external biomechanical loading. We have demonstrated that label-free third-harmonic generation (THG) microscopy reveals the structure of the LCN in 3D with submicron precision over millimetric fields of view compatible with histology and can be coupled to second-harmonic generation (SHG) signals relating to the collagen organization in the bone matrix. Taking advantage of these label-free imaging methods, we investigate the impact of microgravity on the LCN structure in mice following a 1- month space flight. We show that our current lack of understanding of the extent of the LCN heterogeneity at the organ level hinders the interpretation of such investigations based on a limited number of samples and we discuss the implications for future biomedical studies.