Intravital imaging of the lacunar-canalicular network in mouse calvaria using third harmonic generation microscopy

Third harmonic generation (THG) microscopy was used as a noninvasive technique for in vivo investigations of the osteocytic lacunar-canalicular network (LCN) in the mouse calvarium. The origin of THG contrast was investigated by simultaneous imaging with multiphoton excitation fluorescence. The THG signal in lacuna originates from the bone-interstitial fluid boundary and the interstitial fluid-osteocyte cell boundary. Canaliculi also emit intense THG allowing 3D investigations of the LCN network. Subsequently, THG microscopy was used to study the structural variations in the LCN in live wild type and transgenic mouse models. 2D and 3D analyses were performed to obtain the lacunar area, volume and surface area as well as the number of canaliculi per lacuna area and volume, differentiating the transgenic mice. THG microscopy of bone in live animals is a promising technique for noninvasive quantitative analysis of the LCN network.