Three-dimensional visualization of osteoclasts in embryonic mouse mandibles using SEM array tomography

Objective: Three-dimensional (3-D) images of osteoclasts in vivo have been elusive, due to their large size and intricate morphology. The present study was designed to reconstruct the 3-D morphology of whole osteoclasts in developing mouse mandibles using scanning electron microscopy (SEM) array tomography. Methods: Mandibles of 16 days post coitum mouse embryos were fixed and embedded in epoxy resin after decalcification. Epoxy blocks were trimmed, and serial sections of 1 μm in thickness were cut with an ultramicrotome and mounted on glass microscope slides. Consecutive images of every fourth or fifth serial section were obtained by SEM after electron staining and platinum coating. Three dimensional reconstruction of osteoclasts was performed using these consecutive images. Results: Multinucleated osteoclasts were observed to cluster around developing bone in the embryonic mouse mandible. The outlines of osteoclasts and their sealing zones were identified in the serial sections. The reconstructed 3-D image revealed whole osteoclast morphology with the sealing zone. Osteoclasts were adherent to bone with the anchoring structure between the osteoclast and the bone. Conclusions: SEM array tomography with our modification revealed 3-D imagery of a whole osteoclast and its sealing zone in vivo for the first time. This methodology could provide useful information on in vivo structures and dynamics of large cells, such as osteoclasts.