Polarization induced contrast X-ray fluorescence at submicrometer resolution reveals nanometer apatite crystal orientations across entire tooth sections

Abstract

For biomedical research, successful imaging of calcified microstructures often relies on absorption differences between features, or on employing dies with selective affinity to areas of interest. When texture is concerned, e.g. for crystal orientation studies, polarization induced contrast is of particular interest. This requires sufficient interaction of the incoming radiation with the volume of interest in the sample to produce orientation-based contrast. Here we demonstrate polarization induced contrast at the calcium K-edge using submicron sized monochromatic synchrotron X-ray beams. We exploit the orientation dependent subtle absorption differences of hydroxyl-apatite crystals in teeth, with respect to the polarization field of the beam. Interaction occurs with the fully mineralized samples, such that differences in density do not contribute to the contrast. Our results show how polarization induced contrast X-ray fluorescence mapping at specific energies of the calcium K-edge reveals the micrometer and submicrometer crystal arrangements in human tooth tissues. This facilitates combining both high spatial resolution and large fields of view, achieved in relatively short acquisition times in reflection geometry. In enamel we observe the varying crystal orientations of the micron sized prisms exposed on our prepared surface. We easily reproduce crystal orientation maps, typically observed in polished thin sections. We even reveal maps of submicrometer mineralization fronts in spherulites in intertubular dentine. This Ca K-edge polarization sensitive method (XRF-PIC) does not require thin samples for transmission nor extensive sample preparation. It can be used on both fresh, moist samples as well as fossilized samples where the information of interests lies in the crystal orientations and where the crystalline domains extend several micrometers beneath the exposed surface.

The hidden structure of human enamel

“…Each rod is wrapped in a sheath of organic matrix, whereas crystals within the rod abut one another, with discontinuous, organic meshwork in between 22 . Rods run from the dentin–enamel junction (DEJ) to the surface of enamel, with their trajectories undulating in the inner enamel layer, and thus creating Hunter–Schreger bands or a decussation pattern, previously imaged with electron microscopy, x-ray tomography, or x-ray fluorescence 28–31 , which makes it 10 times more resistant than bone to crack growth 32–34 .…”

“…This effect enables the PIC mapping method used here. PIC mapping has been used extensively for carbonates 40,45–49 , bone apatite 35,50 , entire teeth 31 , parrotfish enameloid 44 , and mouse enamel 35 . In coral 48 , sea urchin teeth 46,47 , mollusk shell nacre 45 , and prismatic calcite 49 the orientations measured by PIC mapping were confirmed in precisely the same regions with x-ray diffraction.…”

Synchrotron X-ray Studies of the Structural and Functional Hierarchies in Mineralised Human Dental Enamel: A State-of-the-Art Review

“…For example, anti-correlated energies below and above the Ca L-edge (352.6 eV) were selected from each pixel imaged on tooth enamel and then divided, to create a PIC map from the different polarization angles using the Malus law [ 77 , 654 ]. For human enamel, this technique provided details of rods and inter-rod substances at different resolutions, from micro to nano scale [ 77 , 363 ]. It has been applied to the characterisation of samples based on the absorption edge of various elements present in samples, e.g., Ca, O, and C [ 77 , 372 , 654 ], on the polarization plane and c-axis, leading to the colour map encoding the details of the off-plane angle of c-axis and ‘c’-axis’ detailed in Figure 9 g. It offers high-resolution scans at or below 50 nm.…”

“…In a study involving PIC mapping with a high-resolution and Ca L-edge [ 77 ], the sample was coated with 1 nm of Pt in the region of interest and surrounded by 40 nm of Pt, to carry out PEEM analysis [ 662 ]. In another study performed at submicrometer resolution (~0.6 × 0.8 µm 2 beam size) with the Ca K-edge analysis, peritubular dentine and the rod texture in enamel were visualised, without the requirement for nanometer-polished surfaces [ 363 ], Figure 9 i. In a high-resolution PIC mapping study [ 77 ], the Hunter–Schreger bands could be seen at low magnification.…”

Research on the spectral polarization characteristics of teeth in different age groups

“…Generally, the research methods involving tooth characteristic detection include ordinary light microscopy, polarized light microscopy, microradiography, scanning electron microscopy, argon ion thinning technology and high-resolution electron microscopy, biochemical analysis, optical coherence tomography, etc [6][7][8][9][10][11][12] . Traditional detection has disadvantages such as higher cost and longer time.…”