The characterisation of dental enamel using transmission Kikuchi diffraction in the scanning electron microscope combined with dynamic template matching.

The remarkable physical properties of dental enamel can be largely attributed to the structure of the hydroxyapatite (HAp) crystallites on the sub-micrometre scale. Characterising the HAp microstructure is challenging, due to the nanoscale of individual crystallites and practical challenges associated with HAp examination using electron microscopy techniques. Conventional methods for enamel characterisation include imaging using transmission electron microscopy (TEM) or specialised beamline techniques, such as polarisation-dependent imaging contrast (PIC). These provide useful information at the necessary spatial resolution but are not able to measure the full crystallographic orientation of the HAp crystallites. Here we demonstrate the effectiveness of enamel analyses using transmission Kikuchi diffraction (TKD) in the scanning electron microscope, coupled with newly-developed pattern matching methods. The pattern matching approach, using dynamic template matching coupled with subsequent orientation refinement, enables robust indexing of even poor-quality TKD patterns, resulting in significantly improved data quality compared to conventional diffraction pattern indexing methods. The potential of this method for the analysis of nanocrystalline enamel structures is demonstrated by the characterisation of a human enamel TEM sample and the subsequent comparison of the results to high resolution TEM imaging. The TKD - pattern matching approach measures the full HAp crystallographic orientation enabling a quantitative measurement of not just the c-axis orientations, but also the extent of any rotation of the crystal lattice about the c-axis, between and within grains. Results presented here show how this additional information highlights potentially significant aspects of the HAp crystallite structure, including intra-crystallite distortion and the presence of multiple high angle boundaries between adjacent crystallites with rotations about the c-axis. These and other observations enable a more rigorous understanding of the relationship between HAp structures and the physical properties of dental enamel.

Correlating Crystallographic Orientation and Ferroic Properties of Twin Domains in Metal Halide Perovskites.

Metal halide perovskite (MHP) solar cells have attracted worldwide research interest. Although it has been well established that grain, grain boundary, and grain facet affect MHPs optoelectronic properties, less is known about subgrain structures. Recently, MHP twin stripes, a subgrain feature, have stimulated extensive discussion due to the potential for both beneficial and detrimental effects of ferroelectricity on optoelectronic properties. Connecting the ferroic behavior of twin stripes in MHPs with crystal orientation will be a vital step to understand the ferroic nature and the effects of twin stripes. In this work, we studied the crystallographic orientation and ferroic properties of CH3NH3PbI3 twin stripes, using electron backscatter diffraction (EBSD) and advanced piezoresponse force microscopy (PFM), respectively. Using EBSD, we discovered that the orientation relationship across the twin walls in CH3NH3PbI3 is a 90° rotation about ⟨1̅1̅0⟩, with the ⟨030⟩ and ⟨111⟩ directions parallel to the direction normal to the surface. By careful inspection of CH3NH3PbI3 PFM results including in-plane and out-of-plane PFM measurements, we demonstrate some nonferroelectric contributions to the PFM responses of this CH3NH3PbI3 sample, suggesting that the PFM signal in this CH3NH3PbI3 sample is affected by nonferroelectric and nonpiezoelectric forces. If there is piezoelectric response, it is below the detection sensitivity of our interferometric displacement sensor PFM (<0.615 pm/V). Overall, this work offers an integrated picture describing the crystallographic orientations and the origin of PFM signal of MHPs twin stripes, which is critical to understanding the ferroicity in MHPs.

Quaternion-based disorientation coloring of orientation maps.

The authors of the present work propose a color coding technique using quaternions for the display of crystallographic orientation data such as EBSD maps. The main difference to existing color coding techniques in this field is that it creates a color space, within which the perceived color differences are approximately proportional to Euclidean distances in the corresponding 3D quaternion vector space, and thus approximately proportional to mutual disorientation angles. Since all disorientation parameters (axis and angle pairs, but represented as quaternions) are taken into account, color ambiguities appearing in maps created by other techniques are successfully avoided, and the sub-grain orientation differences within grains can be unambiguously visualized.

Insights into the Silver Reflection Layer of a Vertical LED for Light Emission Optimization.

In this work, Ag as a highly reflective mirror layer of gallium nitride (GaN)-based blue vertical light-emitting diodes (VLEDs) has been systematically investigated by correlating scanning electron microscopy/energy dispersive X-ray spectroscopy/transmission Kikuchi diffraction/electron backscatter diffraction, aberration-corrected scanning transmission electron microscopy, and atomic force microscopy techniques. In the context of high-efficiency lighting, three critical aspects have been scrutinized on the nanoscale: (1) chemical diffusion, (2) grain morphology, and (3) surface topography of the Ag layer. We found that nanoscale inhomogeneous distribution of In in InGaN/GaN quantum wells (QWs), interfacial diffusion (In/Ga out-diffusion into the Ag layer and diffusion of Ag into p-GaN and QWs), and Ag agglomeration deteriorate the light reflectivity, which account for the decreased luminous efficiency in VLEDs. Meanwhile, the surface morphology and topographical analyses revealed the nanomorphology of the Ag layer, where a nanograin size of ∼300 nm with special nanotwinned boundaries and an extremely smooth surface of ∼3-4 nm are strongly desired for better reflectivity. Further, on the basis of these microscopy results, suggestions on light extraction optimization are given to improve the performance of GaN-based blue VLEDs. Our findings enable fresh and deep understanding of performance-microstructure correlation of LEDs on the nanoscale, providing guidance to the design and manufacture of high-performance LED devices.

Relocation is the key to successful correlative fluorescence and scanning electron microscopy.

In this chapter the authors report on an automated hardware and software solution enabling swift correlative sample array mapping of fluorescently stained molecules within cells and tissues across length scales. Samples are first observed utilizing wide-field optical and fluorescence microscopy, followed by scanning electron microscopy, using calibration points on a dedicated sample-relocation holder. We investigated HeLa cells in vitro, fluorescently labeled for monosialoganglioside one (GM-1), across both imaging platforms within tens of minutes of initial sample preparation. This resulted in a high-throughput and high spatially resolved correlative fluorescence and electron microscopy analysis and allowed us to collect complementary nanoscopic information on the molecular and structural composition of two differently distinct HeLa cell populations expressing different levels of GM-1. Furthermore, using the small zebrafish animal model Danio rerio, we showed the versatility and relocation accuracy of the sample-relocation holder to locate fluo-tagged macromolecular complexes within large volumes using long ribbons of serial tissue sections. The subsequent electron microscopy imaging of the tissue arrays of interest enabled the generation of correlated information on the fine distribution of albumin within hepatic and kidney tissue. Our approach underpins the merits that an automated sample-relocation holder solution brings in support of results-driven research, where relevant biological questions can be answered, and high-throughput data can be generated in a rigorous statistical manner.

Characterization of Ultra-fine Grained and Nanocrystalline Materials Using Transmission Kikuchi Diffraction.

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and nanocrystalline materials. The traditional techniques associated with scanning electron microscopy (SEM), such as electron backscatter diffraction (EBSD), do not possess the required spatial resolution due to the large interaction volume between the electrons from the beam and the atoms of the material. Transmission electron microscopy (TEM) has the required spatial resolution. However, due to a lack of automation in the analysis system, the rate of data acquisition is slow which limits the area of the specimen that can be characterized. This paper presents a new characterization technique, Transmission Kikuchi Diffraction (TKD), which enables the analysis of the microstructure of UFG and nanocrystalline materials using an SEM equipped with a standard EBSD system. The spatial resolution of this technique can reach 2 nm. This technique can be applied to a large range of materials that would be difficult to analyze using traditional EBSD. After presenting the experimental set up and describing the different steps necessary to realize a TKD analysis, examples of its use on metal alloys and minerals are shown to illustrate the resolution of the technique and its flexibility in term of material to be characterized.

Correlating Atom Probe Crystallographic Measurements with Transmission Kikuchi Diffraction Data.

Correlative microscopy approaches offer synergistic solutions to many research problems. One such combination, that has been studied in limited detail, is the use of atom probe tomography (APT) and transmission Kikuchi diffraction (TKD) on the same tip specimen. By combining these two powerful microscopy techniques, the microstructure of important engineering alloys can be studied in greater detail. For the first time, the accuracy of crystallographic measurements made using APT will be independently verified using TKD. Experimental data from two atom probe tips, one a nanocrystalline Al-0.5Ag alloy specimen collected on a straight flight-path atom probe and the other a high purity Mo specimen collected on a reflectron-fitted instrument, will be compared. We find that the average minimum misorientation angle, calculated from calibrated atom probe reconstructions with two different pole combinations, deviate 0.7° and 1.4°, respectively, from the TKD results. The type of atom probe and experimental conditions appear to have some impact on this accuracy and the reconstruction and measurement procedures are likely to contribute further to degradation in angular resolution. The challenges and implications of this correlative approach will also be discussed.

Laser-Assisted Atom Probe Tomography of Deformed Minerals: A Zircon Case Study.

The application of atom probe tomography to the study of minerals is a rapidly growing area. Picosecond-pulsed, ultraviolet laser (UV-355 nm) assisted atom probe tomography has been used to analyze trace element mobility within dislocations and low-angle boundaries in plastically deformed specimens of the nonconductive mineral zircon (ZrSiO4), a key material to date the earth's geological events. Here we discuss important experimental aspects inherent in the atom probe tomography investigation of this important mineral, providing insights into the challenges in atom probe tomography characterization of minerals as a whole. We studied the influence of atom probe tomography analysis parameters on features of the mass spectra, such as the thermal tail, as well as the overall data quality. Three zircon samples with different uranium and lead content were analyzed, and particular attention was paid to ion identification in the mass spectra and detection limits of the key trace elements, lead and uranium. We also discuss the correlative use of electron backscattered diffraction in a scanning electron microscope to map the deformation in the zircon grains, and the combined use of transmission Kikuchi diffraction and focused ion beam sample preparation to assist preparation of the final atom probe tip.

Visualization of the structural changes in plywood and gypsum board during the growth of Chaetomium globosum and Stachybotrys chartarum.

Fungal growth in indoor environments is associated with many negative health effects. Many studies focus on brown- and white-rot fungi and their effect on wood, but there is none that reveals the influence of soft-rot fungi, such as Stachybotrys spp. and Chaetomium spp., on the structure of building materials such as plywood and gypsum wallboard. This study focuses on using micro-computed tomography (microCT) to investigate changes of the structure of plywood and gypsum wallboard during fungal degradation by S. chartarum and C. globosum. Changes in the materials as a result of dampness and fungal growth were determined by measuring porosity and pore shape via microCT. The results show that the composition of the building material influenced the level of penetration by fungi as shown by scanning electron microscopy (SEM). Plywood appeared to be the most affected, with the penetration of moisture and fungi throughout the whole thickness of the sample. Conversely, fungi grew only on the top cardboard in the gypsum wallboard and they did not have significant influence on the gypsum wallboard structure. The majority of the observed changes in gypsum wallboard occurred due to moisture. This paper suggests that the mycelium distribution within building materials and the structural changes, caused by dampness and fungal growth, depend on the type of the material.

Redox-freezing and nucleation of diamond via magnetite formation in the Earth's mantle.

Diamonds and their inclusions are unique probes into the deep Earth, tracking the deep carbon cycle to >800 km. Understanding the mechanisms of carbon mobilization and freezing is a prerequisite for quantifying the fluxes of carbon in the deep Earth. Here we show direct evidence for the formation of diamond by redox reactions involving FeNi sulfides. Transmission Kikuchi Diffraction identifies an arrested redox reaction from pyrrhotite to magnetite included in diamond. The magnetite corona shows coherent epitaxy with relict pyrrhotite and diamond, indicating that diamond nucleated on magnetite. Furthermore, structures inherited from h-Fe3O4 define a phase transformation at depths of 320-330 km, the base of the Kaapvaal lithosphere. The oxidation of pyrrhotite to magnetite is an important trigger of diamond precipitation in the upper mantle, explaining the presence of these phases in diamonds.

Deformation-induced trace element redistribution in zircon revealed using atom probe tomography.

Trace elements diffuse negligible distances through the pristine crystal lattice in minerals: this is a fundamental assumption when using them to decipher geological processes. For example, the reliable use of the mineral zircon (ZrSiO4) as a U-Th-Pb geochronometer and trace element monitor requires minimal radiogenic isotope and trace element mobility. Here, using atom probe tomography, we document the effects of crystal-plastic deformation on atomic-scale elemental distributions in zircon revealing sub-micrometre-scale mechanisms of trace element mobility. Dislocations that move through the lattice accumulate U and other trace elements. Pipe diffusion along dislocation arrays connected to a chemical or structural sink results in continuous removal of selected elements (for example, Pb), even after deformation has ceased. However, in disconnected dislocations, trace elements remain locked. Our findings have important implications for the use of zircon as a geochronometer, and highlight the importance of deformation on trace element redistribution in minerals and engineering materials.

Effect of triple junctions on deformation twinning in a nanostructured Cu-Zn alloy: A statistical study using transmission Kikuchi diffraction.

Scanning electron microscopy transmission Kikuchi diffraction is able to identify twins in nanocrystalline material, regardless of their crystallographic orientation. In this study, it was employed to characterize deformation twins in Cu/10 wt % Zn processed by high-pressure torsion. It was found that in 83% of grains containing twins, at least one twin intersects with a triple junction. This suggests that triple junctions could have promoted the nucleation of deformation twins. It should be cautioned that this technique might be unable to detect extremely small nanoscale twins thinner than its step size.

An automated method of quantifying ferrite microstructures using electron backscatter diffraction (EBSD) data.

The identification and quantification of the different ferrite microconstituents in steels has long been a major challenge for metallurgists. Manual point counting from images obtained by optical and scanning electron microscopy (SEM) is commonly used for this purpose. While classification systems exist, the complexity of steel microstructures means that identifying and quantifying these phases is still a great challenge. Moreover, point counting is extremely tedious, time consuming, and subject to operator bias. This paper presents a new automated identification and quantification technique for the characterisation of complex ferrite microstructures by electron backscatter diffraction (EBSD). This technique takes advantage of the fact that different classes of ferrite exhibit preferential grain boundary misorientations, aspect ratios and mean misorientation, all of which can be detected using current EBSD software. These characteristics are set as criteria for identification and linked to grain size to determine the area fractions. The results of this method were evaluated by comparing the new automated technique with point counting results. The technique could easily be applied to a range of other steel microstructures.

Structural analysis of reactionary dentin formed in response to polymicrobial invasion.

In response to microbial invasion of dentin odontoblasts secrete an altered calcified matrix termed reactionary dentin (Rd). 3D reconstruction of focused-ion-beam scanning electron microscopy (FIB-SEM) image slices revealed helical tubular structures in Rd that contrasted with regular cylindrical tubules characteristic of dentin from healthy teeth and affected so-called physiological dentin (Pd) lying exterior to Rd. This helical structure in Rd provided effective constriction of tubule lumen diameter that formed a barrier to bacterial advance towards the dental pulp. SEM of resin cast preparations revealed altered extension of odontoblast processes through Rd. The distribution of key mineral elements was studied by combination of 3D reconstruction of focused-ion-beam based X-ray microanalysis (FIB-EDS), laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). There was a marked redistribution of calcium and phosphorous in Rd together with an increase of diffusely deposited magnesium compatible with the mineral deposition phase of synthesis of this altered matrix. Changes in tubule structure and mineral content characteristic of Rd are consistent with reduced hardness and lower elastic modulus reported for this matrix. Findings provide insight into the unique structure of Rd synthesised as a primary response to infection.

Vulnerability of the paper Nautilus (Argonauta nodosa) shell to a climate-change ocean: potential for extinction by dissolution.

Shell calcification in argonauts is unique. Only females of these cephalopods construct the paper nautilus shell, which is used as a brood chamber for developing embryos in the pelagic realm. As one of the thinnest (225 µm) known adult mollusc shells, and lacking an outer protective periostracum-like cover, this shell may be susceptible to dissolution as the ocean warms and decreases in pH. Vulnerability of the A. nodosa shell was investigated through immersion of shell fragments in multifactorial experiments of control (19 °C/pH 8.1; pCO(2) 419; Ω(Ca) = 4.23) and near-future conditions (24 °C/pH 7.8-7.6; pCO(2) 932-1525; Ω(Ca) = 2.72-1.55) for 14 days. More extreme pH treatments (pH 7.4-7.2; pCO(2) 2454-3882; Ω(Ca) = 1.20-0.67) were used to assess tipping points in shell dissolution. X-ray diffractometry revealed no change in mineralogy between untreated and treated shells. Reduced shell weight due to dissolution was evident in shells incubated at pH 7.8 (projected for 2070) after 14 days at control temperature, with increased dissolution in warmer and lower pH treatments. The greatest dissolution was recorded at 24 °C (projected for local waters by 2100) compared to control temperature across all low-pH treatments. Scanning electron microscopy revealed dissolution and etching of shell mineral in experimental treatments. In the absence of compensatory mineralization, the uncovered female brood chamber will be susceptible to dissolution as ocean pH decreases. Since the shell was a crucial adaptation for the evolution of the argonauts' holopelagic existence, persistence of A. nodosa may be compromised by shell dissolution in an ocean-change world.

The first occurrence in the fossil record of an aquatic avian twig-nest with Phoenicopteriformes eggs: evolutionary implications.

BACKGROUND: We describe the first occurrence in the fossil record of an aquatic avian twig-nest with five eggs in situ (Early Miocene Tudela Formation, Ebro Basin, Spain). Extensive outcrops of this formation reveal autochthonous avian osteological and oological fossils that represent a single taxon identified as a basal phoenicopterid. Although the eggshell structure is definitively phoenicopterid, the characteristics of both the nest and the eggs are similar to those of modern grebes. These observations allow us to address the origin of the disparities between the sister taxa Podicipedidae and Phoenicopteridae crown clades, and traces the evolution of the nesting and reproductive environments for phoenicopteriforms. METHODOLOGY/PRINCIPAL FINDINGS: Multi-disciplinary analyses performed on fossilized vegetation and eggshells from the eggs in the nest and its embedding sediments indicate that this new phoenicopterid thrived under a semi-arid climate in an oligohaline (seasonally mesohaline) shallow endorheic lacustine environment. High-end microcharacterizations including SEM, TEM, and EBSD techniques were pivotal to identifying these phoenicopterid eggshells. Anatomical comparisons of the fossil bones with those of Phoenicopteriformes and Podicipediformes crown clades and extinct palaelodids confirm that this avian fossil assemblage belongs to a new and basal phoenicopterid. CONCLUSIONS/SIGNIFICANCE: Although the Podicipediformes-Phoenicopteriformes sister group relationship is now well supported, flamingos and grebes exhibit feeding, reproductive, and nesting strategies that diverge significantly. Our multi-disciplinary study is the first to reveal that the phoenicopteriform reproductive behaviour, nesting ecology and nest characteristics derived from grebe-like type strategies to reach the extremely specialized conditions observed in modern flamingo crown groups. Furthermore, our study enables us to map ecological and reproductive characters on the Phoenicopteriformes evolutionary lineage. Our results demonstrate that the nesting paleoenvironments of flamingos were closely linked to the unique ecology of this locality, which is a direct result of special climatic (high evaporitic regime) and geological (fault system) conditions.

Orientation mapping of nanostructured materials using transmission Kikuchi diffraction in the scanning electron microscope.

In this study, the new technique of transmission Kikuchi diffraction (TKD) in the scanning electron microscope (SEM) has been applied for the first time to enable orientation mapping of bulk, nanostructured metals. The results show how the improved spatial resolution of SEM-TKD, compared to conventional EBSD, enables reliable mapping of truly nanostructured metals and alloys, with mean grain sizes in the 40-200 nm range. The spatial resolution of the technique is significantly below 10nm, and contrasting examples are shown from both dense (Ni) and lighter (Al-alloy) materials. Despite the burden of preparing thin, electron-transparent samples, orientation mapping using SEM-TKD is likely to become invaluable for routine characterisation of nanocrystalline and, potentially, highly deformed microstructures.