Ultrastructural features of dentinoenamel junction revealed by focused gallium ion beam milling

To take full advantage of focused ion beam (FIB) in preparation of ultrathin sections of biological tissues, we have used a cryo-milling process. In this study, extracted human teeth were scanned by optical coherence tomography to inspect the samples for intactness and to determine the area of interest. Then, the selected area of interest was cross-sectioned for examination under a confocal laser scanning microscope to determine the target location of the dentinoenamel junction (DEJ) that was later milled by cryo-FIB at preset parameters, followed by transmission electron microscope examination of the final sliced specimens for ultrastructural characterization. The proposed technique was able to outline the DEJ and to identify the different tooth layers in a single section, without artefacts or tissue damage. The DEJ was outlined as fine longitudinal projections intermingling between the solid electron-dense enamel and intricate electron-lucent hollow dentin. In conclusion, this study has shown the great potential of cryo-FIB in handling different biological tissues having different physical properties, with great precision and accuracy and minimum artefacts.