Observations of ultrastructures in human-tooth by Ultra-High Voltage Electron Tomography

Objectives: 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 (TEM) observation without demineralizing them. We present a novel method for creating three-dimensional ultrastructural images of human enamel and dentin using Focus ion beam (FIB) method and ultra-high voltage electron microscope tomography.

Methods: Thin specimens (less than 2 ìm) of enamel and dentin were prepared using the FIB method. FIB is appropriate for nano-fabrication of thin specimens for brittle materials such as enamel and dentin. This allowed an ultra-high voltage electron beam of 3000 kV maximal acceleration voltage to penetrate, enabling projections of specimens to be taken.

Results: These methods facilitated tomography of the enamel rods, sheaths, dentinal tubules, and collagen fibrils with a high resolution of 2 nm. The component percentages in ultrastructures of dentin can be expressed numerically by using three-dimensional reconstruction images of tomograms. The matrix of peritubular dentin (PTD) differed from that of intertubular dentin (ITD) by having relatively fewer collagen fibrils.

Conclusions: The major advantage of this method is its ability to visualize ultrastructural tomograms of highly calcified specimens without demineralization.