Deciphering the intricate cellular interactions within the bone marrow (BM) is crucial for understanding a wide range of multifactorial diseases in immunology, regenerative medicine, and BM biology. The intricate BM microenvironment, characterized by dynamic cell trafficking, production of immune cells and self-organized remodeling, constantly shapes osteoimmunological cell functions and their metabolic adaptations. This specific microenvironment is challenging to replicate in vitro or in silico. Current intravital optical imaging techniques can investigate cells within the complex BM microenvironment but are invasive or limited in observation time, depth, hindering long term investigation of bone regeneration or specific cellular niches. This dissertation presents three novel optical imaging technologies to satisfy the critical need for long-term, minimally invasive intravital microscopy of the BM: 1) a high-energy, high-repetition-rate 3-photon (3P) laser, enabling intravital visualization of plasma cell (PC) dynamics and antibody production capacity; 2) Limbostomy, a modular microendoscope for longitudinal in vivo imaging of deep femoral BM, facilitating quantification of cellular self-organization during bone healing; and 3) FLIMB, integrating microendoscopy with NAD(P)H-dependent fluorescence lifetime imaging (FLIM), enabling label-free metabolic imaging of myeloid cells in the living BM. These methods revealed an antiproportional correlation between PC motility and antibody production capacity; the chronicity of rapid vessel sprouting and subsequent reorganization into a confined network accompanied by myeloid interactions after bone injury; and metabolic heterogeneity among myeloid cells, indicating specific metabolic patterns linked to the activation of oxidative burst and phagocytic function. These innovations provide researchers with powerful tools to study complex cellular interactions in living bone marrow, develop therapeutic strategies and monitor drug responses, for example to improve bone regeneration, combat PC dysfunction and cancer, and fundamentally understand the interplay of cellular behaviour, microenvironment and disease progression in bone marrow.
