Visualizing the "sandwich" structure of osteocytes in their native environment deep in bone in vivo

Osteocytes are the most abundant cells in bone and always the focus of bone research. They are embedded in the highly scattering mineralized bone matrix. Consequently, visualizing osteocytes deep in bone with subcellular resolution poses a major challenge for in vivo bone research. Here we overcome this challenge by demonstrating 3-photon imaging of osteocytes through the intact mouse skull in vivo. Through broadband transmittance characterization, we establish that the excitation at the 1700-nm window enables the highest optical transmittance through the skull. Using label-free third-harmonic generation (THG) imaging excited at this window, we visualize osteocytes through the whole 140-μm mouse skull and 155 μm into the brain in vivo. By developing selective labeling technique for the interstitial space, we visualize the "sandwich" structure of osteocytes in their native environment. Our work provides novel imaging methodology for bone research in vivo.