Bone homeostasis is regulated by communication between bone-forming mature osteoblasts (mOBs) and bone-resorptive mature osteoclasts (mOCs). However, the spatial–temporal relationship and mode of interaction in vivo remain elusive. Here we show, by using an intravital imaging technique, that mOB and mOC functions are regulated via direct cell–cell contact between these cell types. The mOBs and mOCs mainly occupy discrete territories in the steady state, although direct cell–cell contact is detected in spatiotemporally limited areas. In addition, a pH-sensing fluorescence probe reveals that mOCs secrete protons for bone resorption when they are not in contact with mOBs, whereas mOCs contacting mOBs are non-resorptive, suggesting that mOBs can inhibit bone resorption by direct contact. Intermittent administration of parathyroid hormone causes bone anabolic effects, which lead to a mixed distribution of mOBs and mOCs, and increase cell–cell contact. This study reveals spatiotemporal intercellular interactions between mOBs and mOCs affecting bone homeostasis in vivo.

Intravital imaging by two-photon excitation microscopy (TPEM) has been widely used to visualize cell functions. However, small molecular probes (SMPs), commonly used for cell imaging, cannot be simply applied to intravital imaging because of the challenge of delivering them into target tissues, as well as their undesirable physicochemical properties for TPEM imaging. Here, we designed and developed a functional SMP with an active-targeting moiety, higher photostability, and a fluorescence switch and then imaged target cell activity by injecting the SMP into living mice. The combination of the rationally designed SMP with a fluorescent protein as a reporter of cell localization enabled quantitation of osteoclast activity and time-lapse imaging of its in vivo function associated with changes in cell deformation and membrane fluctuations. Real-time imaging revealed heterogenic behaviors of osteoclasts in vivo and provided insights into the mechanism of bone resorption.

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