Osteoclasts are derived from the monocyte-macrophage lineage of the bone marrow and are bone-resorbing cells essential for bone homeostasis. Osteoclasts contain more mitochondria and require more oxygen to support the energy demands associated with bone resorption. However, local oxygen distribution within bone marrow has not been completely understood. Intravital two-photon microscopy is a powerful tool for investigating biological processes in live animals. In this study, we performed two-photon phosphorescence lifetime imaging to characterize the oxygen tension in osteoclasts.
　Two-photon-enhanced phosphorescent probe, iridium complex (BTPDM1), was injected into knock-in mice in which EGFP is expressed in osteoclast lineage cells. The emissive triplet state of the BTPDM1 is sensitive to local oxygen tension. By measuring the phosphorescence from EGFP positive cells, oxygen tension in osteoclasts was determined. Furthermore, we quantitatively analyzed changes in oxygen tension in osteoclasts by measuring the phosphorescence decay time in EGFP positive cells. Thus, we have succeeded in detecting oxygen tension in vivo. This method is applicable to various cell types for determining local oxygen tension inside bone marrow.