Depth-Dependent Advantages of Three-Photon Microscopy for Imaging Through Intact Murine Cortical Bone and Into the Marrow

Bone adapts to its mechanical and physiological environment through the coordinated actions of osteocytes and marrow-resident cell populations. Because these processes depend on complex interactions within native tissue microenvironments, intravital imaging offers a unique opportunity to reveal cellular behaviors that cannot be fully captured ex vivo. However, the highly scattering nature of mineralized bone limits imaging depth and direct observation of cells within intact tissue. Two-photon (2P) microscopy has enabled important advances in bone biology, while three-photon (3P) microscopy has been proven to extend imaging depth and image quality in skeletal tissues. Here, we directly compared the performance of 2P and 3P microscopy in ex vivo mouse long bones by quantifying laser attenuation, signal-to-noise ratio, signal-to-background ratio, and spatial resolution, as well as assessed the impact of wave-front correction. We found that 2P and 3P microscopy generated comparable image quality through approximately 50 {micro}m of cortical bone. Beyond this depth, however, 3P microscopy provided superior brightness, contrast, and resolution, enabling improved visualization of structures deep within the cortex and at the cortical-marrow interface. To assess compatibility with intravital imaging, we evaluated endogenous markers of cellular stress during 3P imaging. Although prolonged continuous imaging decreased osteocyte spontaneous calcium signaling magnitude and increased autofluorescence, short intermittent imaging bouts produced negligible evidence of cellular damage compared to controls. Leveraging the enhanced penetration depth of 3P microscopy, we further show visualization of immune cell migration within the marrow cavity through intact cortical bone in both the third metatarsal (MT3) and tibia. Together, these findings affirm 3P microscopy as a powerful tool for studying cellular dynamics in living bone. By extending imaging beyond superficial cortical regions and enabling direct visualization of marrow-resident cells through intact bone, 3P microscopy expands opportunities to investigate osteocyte biology, marrow niche function, and skeletal adaptation in vivo.