Intravital two-photon microscopy enables deep tissue imaging at high temporospatial resolution in live animals. However, the endosteal bone compartment and underlying bone marrow pose unique challenges to optical imaging as light is absorbed, scattered and dispersed by thick mineralised bone matrix and the adipose-rich bone marrow. Early bone intravital imaging methods exploited gaps in the cranial sutures to bypass the need to penetrate through cortical bone. More recently, investigators have developed invasive methods to thin the cortical bone or implant imaging windows to image cellular dynamics in weight-bearing long bones. Here we provide a step-by-step protocol for the preparation of animals for minimally-invasive, non-destructive longitudinal intravital imaging of the murine tibia. This method involves the use of mixed bone marrow radiation chimeras to unambiguously double-label osteoclasts and osteomorphs. The tibia is exposed by a simple skin incision and an imaging chamber constructed using thermoconductive T-putty. Imaging sessions up to 12h long can be repeated over multiple timepoints to provide a longitudinal time window into the endosteal and marrow niches. The protocol can be used to investigate cellular dynamics in bone remodelling, cancer cell life cycle, haematopoiesis and long-lived humoral and cellular immunity.