Real‐Time Live Imaging of Osteoclast Activation via Cathepsin K Activity in Bone Diseases

Abstract Intravital fluorescence imaging of functional osteoclasts within their intact disease context provides valuable insights into the intricate biology at the microscopic level, facilitating the development of therapeutic approaches for osteoclast‐associated bone diseases. However, there is a lack of studies investigating osteoclast activity within deep‐seated bone lesions using appropriate fluorescent probes, despite the advantages offered by the multi‐photon excitation system in enhancing deep tissue imaging resolution. In this study, we report on the intravital tracking of osteoclast activity in three distinct murine bone disease models. We utilized a cathepsin K (CatK)‐responsive two‐photon fluorogenic probe ( CatKP1 ), which exhibited a notable fluorescence turn‐on response in the presence of active CatK. By utilizing CatKP1 , we successfully monitored a significant increase in osteoclast activity in hindlimb long bones and its attenuation through pharmacological intervention without sacrificing mice. Thus, our findings highlight the efficacy of CatKP1 as a valuable tool for unraveling pathological osteoclast behavior and exploring novel therapeutic strategies.