3D Visualization of Osteogenic Capillaries in Mineralized Bone Tissue by Integrating Micro-CT with Light Sheet Fluorescence Microscopy Imaging

Abstract

Bone microstructure morphology can be visualized in three dimensions by X-ray computed tomography (CT) using high resolution X-ray microscopes.Light sheet fluorescence microscopy (LSFM), or multi-angle fluorescence microscopy, also allows whole-mount three-dimensional imaging of fluorescently-labeled molecules rather than X-rays. Integration of spatial information based on both X-rays and fluorescence would allow one to visualize the location of fluorescent molecules within bone. Here, we focused on morphology of the malleus, the auditory ossicle that attaches to the tympanic membrane. In mice, the malleus contains abundant “osteogenic capillaries” during postnatal endochondral ossification. To visualize those capillaries, we perfused mice with fluorescence-labeled tomato-lectin to mark vessels and then performed micro-CT scanning, decalcification, and LSFM imaging. We then compared canal structures observed using micro-CT with the network of blood capillaries visualized using tomato-lectin. By integrating images obtained by applying two different principles of three-dimensional reconstruction, we showed that intra-osseous blood capillaries can be superimposed within regions lacking bone in the malleus. These methods will be discussed here with future perspectives.