Ultra-structural observation of human enamel and dentin by ultra-high-voltage electron tomography and the focus ion beam technique

Human enamel and dentin are hard and brittle mineralized tissues. It is difficult to prepare thin specimens (under 200 nm) of these tissues for transmission electron microscope observation without demineralizing them. We present a novel method of creating three-dimensional ultra-structural images of human enamel and dentin, using the focus ion beam (FIB) method and ultra-high-voltage electron microscope tomography. Thin specimens (less than 2 μm) of enamel and dentin were prepared using the FIB method. This method is appropriate for nano-fabrication of thin specimens for brittle materials such as enamel and dentin. It allows penetration of an ultra-high-voltage electron beam of a 3000 kV maximal acceleration voltage into a specimen, enabling projections of the specimen to be taken. It facilitates tomography of the enamel rods, sheaths, dentinal tubules and collagen fibrils with a high resolution of 2 nm. The component percentages in ultra-structures of dentin can be expressed numerically by using three-dimensional reconstruction images of tomograms. The matrix of peritubular dentin differed from that of intertubular dentin by having relatively fewer collagen fibrils. The major advantage of this method is its ability to visualize ultra-structural tomograms of highly calcified specimens without demineralization.