Analysis of in vitro demineralised human enamel using multi-scale correlative optical and scanning electron microscopy, and high-resolution synchrotron wide-angle X-ray scattering

Cyril Besnard, Robert A. Harper, Thomas E. J. Moxham, Jonathan D. James, Malte Storm, Enrico Salvati, Gabriel Landini, Richard M. Shelton, Alexander M.Korsunsky - MBLEM, Department of Engineering Science, University of Oxford ; School of Dentistry, University of Birmingham ; Diamond Light Source Ltd., Didcot, Oxfordshire

Enamel caries is a highly prevalent worldwide disease that involves the demineralisation of the outer tooth structure. In this study, we report the analysis of artificially demineralised human enamel sections (‘slices’) etched using lactic acid (2% v/v) in comparison with healthy enamel using correlative techniques of optical and electron microscopy, as well as scanning diffraction. Demineralisation of the enamel was characterised at the micron to sub-micron scale. The structure of the healthy enamel was investigated using Focused Ion Beam – Scanning Electron Microscopy (FIB-SEM) and compared with an etched sample to reveal their structural differences.

Additional chemical analysis using energy-dispersive X-ray spectroscopy (EDS) was performed and a decrease in the Ca/P atomic % ratio was found in etched samples in comparison with healthy enamel, suggesting greater loss of calcium compared with phosphorus. Synchrotron wide-angle X-ray scattering (WAXS) was performed on the samples to reveal the differences in the diffraction patterns before and after etching in terms of lattice structure and preferred orientation (texture). Texture maps were extracted from diffraction analysis at 500 nm spatial resolution. These maps were correlated with the dimension of the enamel structure. The multi-scale correlative approach provided insights into the demineralisation-induced enamel structure alteration at a resolution approaching 500 nm.

How Amira-Avizo Software is used

The three-dimensional (3D) reconstruction was done using Avizo Software, the image slices were filtered with a median filter, aligned and manually segmented to extract the rod using mainly the presumed inter-rod structure. The voxel size was ̴̃29 nm. The elemental analysis was acquired using an Xmax 150 EDS detector and Aztec Oxford Instruments software (Oxford Instruments, U.K.) with a voltage of 10 keV using the same time of acquisition, process and number of channels to standardise the measurements. Quantification of the ratio of calcium/phosphorus (Ca/P) atomic (%) was done using Aztec, using Ca (emission line Kα1 = 3691.68 eV) and P (Kα1 = 2013.7 eV).