Real-space visualization of intrinsic magnetic fields of an antiferromagnet.

Characterizing magnetic structures down to atomic dimensions is central to the design and control of nanoscale magnetism in materials and devices. However, real-space visualization of magnetic fields at such dimensions has been extremely challenging. In recent years, atomic-resolution differential phase contrast scanning transmission electron microscopy (DPC STEM)1 has enabled direct imaging of electric field distribution even inside single atoms2. Here we show real-space visualization of magnetic field distribution inside antiferromagnetic haematite (α-Fe2O3) using atomic-resolution DPC STEM in a magnetic-field-free environment3. After removing the phase-shift component due to atomic electric fields and improving the signal-to-noise ratio by unit-cell averaging, real-space visualization of the intrinsic magnetic fields in α-Fe2O3 is realized. These results open a new possibility for real-space characterization of many magnetic structures.

Regulatory features aid interpretation of 3'UTR variants.

Our ability to determine the clinical impact of variants in 3' untranslated regions (UTRs) of genes remains poor. We provide a thorough analysis of 3' UTR variants from several datasets. Variants in putative regulatory elements, including RNA-binding protein motifs, eCLIP peaks, and microRNA sites, are up to 16 times more likely than variants not in these elements to have gene expression and phenotype associations. Variants in regulatory motifs result in allele-specific protein binding in cell lines and allele-specific gene expression differences in population studies. In addition, variants in shared regions of alternatively polyadenylated isoforms and those proximal to polyA sites are more likely to affect gene expression and phenotype. Finally, pathogenic 3' UTR variants in ClinVar are up to 20 times more likely than benign variants to fall in a regulatory site. We incorporated these findings into RegVar, a software tool that interprets regulatory elements and annotations for any 3' UTR variant and predicts whether the variant is likely to affect gene expression or phenotype. This tool will help prioritize variants for experimental studies and identify pathogenic variants in individuals.

Determining the Projected Crystal Structure from Four-dimensional Scanning Transmission Electron Microscopy via the Scattering Matrix.

We present a gradient-descent-based approach to determining the projected electrostatic potential from four-dimensional scanning transmission electron microscopy measurements of a periodic, crystalline material even when dynamical scattering occurs. The method solves for the scattering matrix as an intermediate step, but overcomes the so-called truncation problem that limited previous scattering-matrix-based projected structure determination methods. Gradient descent is made efficient by using analytic expressions for the gradients. Through simulated case studies, we show that iteratively improving the scattering matrix determination can significantly improve the accuracy of the projected structure determination.

Scattering Matrix Determination in Crystalline Materials from 4D Scanning Transmission Electron Microscopy at a Single Defocus Value.

Recent work has revived interest in the scattering matrix formulation of electron scattering in transmission electron microscopy as a stepping stone toward atomic-resolution structure determination in the presence of multiple scattering. We discuss ways of visualizing the scattering matrix that make its properties clear. Through a simulation-based case study incorporating shot noise, we shown how regularizing on this continuity enables the scattering matrix to be reconstructed from 4D scanning transmission electron microscopy (STEM) measurements from a single defocus value. Intriguingly, for crystalline samples, this process also yields the sample thickness to nanometer accuracy with no a priori knowledge about the sample structure. The reconstruction quality is gauged by using the reconstructed scattering matrix to simulate STEM images at defocus values different from that of the data from which it was reconstructed.

Optimizing Experimental Conditions for Accurate Quantitative Energy-Dispersive X-ray Analysis of Interfaces at the Atomic Scale.

The invention of silicon drift detectors has resulted in an unprecedented improvement in detection efficiency for energy-dispersive X-ray (EDX) spectroscopy in the scanning transmission electron microscope. The result is numerous beautiful atomic-scale maps, which provide insights into the internal structure of a variety of materials. However, the task still remains to understand exactly where the X-ray signal comes from and how accurately it can be quantified. Unfortunately, when crystals are aligned with a low-order zone axis parallel to the incident beam direction, as is necessary for atomic-resolution imaging, the electron beam channels. When the beam becomes localized in this way, the relationship between the concentration of a particular element and its spectroscopic X-ray signal is generally nonlinear. Here, we discuss the combined effect of both spatial integration and sample tilt for ameliorating the effects of channeling and improving the accuracy of EDX quantification. Both simulations and experimental results will be presented for a perovskite-based oxide interface. We examine how the scattering and spreading of the electron beam can lead to erroneous interpretation of interface compositions, and what approaches can be made to improve our understanding of the underlying atomic structure.

Inelastic Scattering in Electron Backscatter Diffraction and Electron Channeling Contrast Imaging.

Electron backscatter diffraction (EBSD) and electron channeling contrast imaging (ECCI) are used to extract crystallographic information from bulk samples, such as their crystal structure and orientation as well as the presence of any dislocation and grain boundary defects. These techniques rely on the backscattered electron signal, which has a large distribution in electron energy. Here, the influence of plasmon excitations on EBSD patterns and ECCI dislocation images is uncovered by multislice simulations including inelastic scattering. It is shown that the Kikuchi band contrast in an EBSD pattern for silicon is maximum at small energy loss (i.e., few plasmon scattering events following backscattering), consistent with previous energy-filtered EBSD measurements. On the other hand, plasmon excitation has very little effect on the ECCI image of a dislocation. These results are explained by examining the role of the characteristic plasmon scattering angle on the intrinsic contrast mechanisms in EBSD and ECCI.

Direct Visualization of Local Electromagnetic Field Structures by Scanning Transmission Electron Microscopy.

The functional properties of materials and devices are critically determined by the electromagnetic field structures formed inside them, especially at nanointerface and surface regions, because such structures are strongly associated with the dynamics of electrons, holes and ions. To understand the fundamental origin of many exotic properties in modern materials and devices, it is essential to directly characterize local electromagnetic field structures at such defect regions, even down to atomic dimensions. In recent years, rapid progress in the development of high-speed area detectors for aberration-corrected scanning transmission electron microscopy (STEM) with sub-angstrom spatial resolution has opened new possibilities to directly image such electromagnetic field structures at very high-resolution. In this Account, we give an overview of our recent development of differential phase contrast (DPC) microscopy for aberration-corrected STEM and its application to many materials problems. In recent years, we have developed segmented-type STEM detectors which divide the detector plane into 16 segments and enable simultaneous imaging of 16 STEM images which are sensitive to the positions and angles of transmitted/scattered electrons on the detector plane. These detectors also have atomic-resolution imaging capability. Using these segmented-type STEM detectors, we show DPC STEM imaging to be a very powerful tool for directly imaging local electromagnetic field structures in materials and devices in real space. For example, DPC STEM can clearly visualize the local electric field variation due to the abrupt potential change across a p-n junction in a GaAs semiconductor, which cannot be observed by normal in-focus bright-field or annular type dark-field STEM imaging modes. DPC STEM is also very effective for imaging magnetic field structures in magnetic materials, such as magnetic domains and skyrmions. Moreover, real-time imaging of electromagnetic field structures can now be realized through very fast data acquisition, processing, and reconstruction algorithms. If we use DPC STEM for atomic-resolution imaging using a sub-angstrom size electron probe, it has been shown that we can directly observe the atomic electric field inside atoms within crystals and even inside single atoms, the field between the atomic nucleus and the surrounding electron cloud, which possesses information about the atomic species, local chemical bonding and charge redistribution between bonded atoms. This possibility may open an alternative way for directly visualizing atoms and nanostructures, that is, seeing atoms as an entity of electromagnetic fields that reflect the intra- and interatomic electronic structures. In this Account, the current status of aberration-corrected DPC STEM is highlighted, along with some applications in real material and device studies.

Brief Report: Common Genetic Variation in Chromosome 10 q22.1 Shows a Strong Sex Bias in Human Embryonic Stem Cell Lines and Directly Controls the Novel Alternative Splicing of Human NODAL which is Associated with XIST Expression in Female Cell Lines.

The potential use of pluripotent stem cells for personalized regenerative medicine necessitates an improved understanding of how germ-line genetic variation may affect pluripotency. Given previous reports of a female bias in established human embryonic stem cell (hESC) lines, sex-specific differences must also be considered. Herein we describe, for the first time, how genetic polymorphisms may affect the establishment of widely used hESC lines. We demonstrate that the minor allele of the human single nucleotide polymorphism (SNP) rs2231947 found within the NODAL gene locus is under-represented in male but not female hESC lines. We also show that this SNP is highly functional in hESC lines. The SNP rs2231947 directly controls the alternative splicing of a novel NODAL transcript isoform. Thus we demonstrate that genetic variation drastically affects the expression of a gene that plays a major role in the regulation of pluripotency and cell fate. Our work helps detail how genetic heterogeneity is manifested in hESC biology and highlights the need to identify how specific genetic variants can explain important differences between pluripotent cell line models both within and between species.

Assessing breast cancer cell lines as tumour models by comparison of mRNA expression profiles.

INTRODUCTION: Breast cancer researchers use cell lines to model myriad phenomena ranging from DNA repair to cancer stem cell phenotypes. Though appropriate, and even requisite, for many studies, the suitability of cell lines as tumour models has come into question owing to possibilities of tissue culture artefacts and clonal selection. These issues are compounded by the inability of cancer cells grown in isolation to fully model the in situ tumour environment, which also contains a plethora of non-tumour cell types. It is thus important to understand similarities and differences between cancer cell lines and the tumours that they represent so that the optimal tumour models can be chosen to answer specific research questions. METHODS: In the present study, we compared the RNA-sequencing transcriptomes of a collection of breast cancer cell lines to transcriptomes obtained from hundreds of tumours using The Cancer Genome Atlas. Tumour purity was accounted for by analysis of stromal and immune scores using the ESTIMATE algorithm so that differences likely resulting from non-tumour cells could be accounted for. RESULTS: We found the transcriptional characteristics of breast cancer cell lines to mirror those of the tumours. We identified basal and luminal cell lines that are most transcriptionally similar to their respective breast tumours. Our comparison of expression profiles revealed pronounced differences between breast cancer cell lines and tumours, which could largely be attributed to the absence of stromal and immune components in cell culture. A focus on the Wnt pathway revealed the transcriptional downregulation or absence of several secreted Wnt antagonists in culture. Gene set enrichment analysis suggests that cancer cell lines have enhanced proliferation and glycolysis independent of stromal and immune contributions compared with breast cancer cells in situ. CONCLUSIONS: This study demonstrates that many of the differences between breast cancer cell lines and tumours are due to the absence of stromal and immune components in vitro. Hence, extra precautions should be taken when modelling extracellular proteins in vitro. The specific differences discovered emphasize the importance of choosing an appropriate model for each research question.

Accurate Nanoscale Crystallography in Real-Space Using Scanning Transmission Electron Microscopy.

Here, we report reproducible and accurate measurement of crystallographic parameters using scanning transmission electron microscopy. This is made possible by removing drift and residual scan distortion. We demonstrate real-space lattice parameter measurements with <0.1% error for complex-layered chalcogenides Bi2Te3, Bi2Se3, and a Bi2Te2.7Se0.3 nanostructured alloy. Pairing the technique with atomic resolution spectroscopy, we connect local structure with chemistry and bonding. Combining these results with density functional theory, we show that the incorporation of Se into Bi2Te3 causes charge redistribution that anomalously increases the van der Waals gap between building blocks of the layered structure. The results show that atomic resolution imaging with electrons can accurately and robustly quantify crystallography at the nanoscale.

Three-dimensional location of a single dopant with atomic precision by aberration-corrected scanning transmission electron microscopy.

Materials properties, such as optical and electronic response, can be greatly enhanced by isolated single dopants. Determining the full three-dimensional single-dopant defect structure and spatial distribution is therefore critical to understanding and adequately tuning functional properties. Combining quantitative Z-contrast scanning transmission electron microscopy images with image simulations, we show the direct determination of the atomic-scale depth location of an optically active, single atom Ce dopant embedded within wurtzite-type AlN. The method represents a powerful new tool for reconstructing three-dimensional information from a single, two-dimensional image.

Room-temperature polar ferromagnet ScFeO3 transformed from a high-pressure orthorhombic perovskite phase.

Multiferroic materials have been the subject of intense study, but it remains a great challenge to synthesize those presenting both magnetic and ferroelectric polarizations at room temperature. In this work, we have successfully obtained LiNbO3-type ScFeO3, a metastable phase converted from the orthorhombic perovskite formed under 15 GPa at elevated temperatures. A combined structure analysis by synchrotron X-ray and neutron powder diffraction and high-angle annular dark-field scanning transmission electron microscopy imaging reveals that this compound adopts the polar R3c symmetry with a fully ordered arrangement of trivalent Sc and Fe ions, forming highly distorted ScO6 and FeO6 octahedra. The calculated spontaneous polarization along the hexagonal c-axis is as large as 100 µC/cm(2). The magnetic studies show that LiNbO3-type ScFeO3 is a weak ferromagnet with TN = 545 K due to a canted G-type antiferromagnetic ordering of Fe(3+) spins, representing the first example of LiNbO3-type oxides with magnetic ordering far above room temperature. A comparison of the present compound and rare-earth orthorhombic perovskites RFeO3 (R = La-Lu and Y), all of which possess the corner-shared FeO6 octahedral network, allows us to find a correlation between TN and the Fe-O-Fe bond angle, indicating that the A-site cation-size-dependent octahedral tilting dominates the magnetic transition through the Fe-O-Fe superexchange interaction. This work provides a general and versatile strategy to create materials in which ferroelectricity and ferromagnetism coexist at high temperatures.

Direct observation of dopant atom diffusion in a bulk semiconductor crystal enhanced by a large size mismatch.

Diffusion is one of the fundamental processes that govern the structure, processing, and properties of materials and it plays a crucial role in determining device lifetimes. However, direct observations of diffusion processes have been elusive and limited only to the surfaces of materials. Here we use an aberration-corrected electron microscope to locally excite and directly image the diffusion of single Ce and Mn dopants inside bulk wurtzite-type AlN single crystals, identifying correlated vacancy-dopant and interstitial-dopant kick-out mechanisms. Using a 200 kV electron beam to supply energy, we observe a higher frequency of dopant jumps for the larger and heavier Ce atoms than the smaller Mn atoms. These observations confirm density-functional-theory-based predictions of a decrease in diffusion barrier for large substitutional atoms. The results show that combining depth sensitive microscopy with theoretical calculations represents a new methodology to investigate diffusion mechanisms, not restricted to surface phenomena, but within bulk materials.

Quantitative annular dark field electron microscopy using single electron signals.

One of the difficulties in analyzing atomic resolution electron microscope images is that the sample thickness is usually unknown or has to be fitted from parameters that are not precisely known. An accurate measure of thickness, ideally on a column-by-column basis, parameter free, and with single atom accuracy, would be of great value for many applications, such as matching to simulations. Here we propose such a quantification method for annular dark field scanning transmission electron microscopy by using the single electron intensity level of the detector. This method has the advantage that we can routinely quantify annular dark field images operating at both low and high beam currents, and under high dynamic range conditions, which is useful for the quantification of ultra-thin or light-element materials. To facilitate atom counting at the atomic scale we use the mean intensity in an annular dark field image averaged over a primitive cell, with no free parameters to be fitted. To illustrate the potential of our method, we demonstrate counting the number of Al (or N) atoms in a wurtzite-type aluminum nitride single crystal at each primitive cell over the range of 3-99 atoms.

Practical aspects of removing the effects of elastic and thermal diffuse scattering from spectroscopic data for single crystals.

A method to remove the effects of elastic and thermal diffuse scattering (TDS) of the incident electron probe from electron energy-loss and energy-dispersive X-ray spectroscopy data for atomically resolved spectrum images of single crystals of known thickness is presented. By calculating the distribution of the probe within a specimen of known structure, it is possible to deconvolve the channeling of the probe and TDS from experimental data by reformulating the inelastic cross-section as an inverse problem. In electron energy-loss spectroscopy this allows valid comparisons with first principles fine-structure calculations to be made. In energy-dispersive X-ray spectroscopy, direct compositional analyses such as ζ-factor and Cliff-Lorimer k-factor analysis can be performed without the complications of channeling and TDS. We explore in detail how this method can be incorporated into existing multislice programs, and demonstrate practical considerations in implementing this method using a simulated test specimen. We show the importance of taking into account the scattering of the probe in k-factor analysis in a zone axis orientation. The applicability and limitations of the method are discussed.

Quantifying negative selection in human 3' UTRs uncovers constrained targets of RNA-binding proteins.

Many non-coding variants associated with phenotypes occur in 3' untranslated regions (3' UTRs), and may affect interactions with RNA-binding proteins (RBPs) to regulate gene expression post-transcriptionally. However, identifying functional 3' UTR variants has proven difficult. We use allele frequencies from the Genome Aggregation Database (gnomAD) to identify classes of 3' UTR variants under strong negative selection in humans. We develop intergenic mutability-adjusted proportion singleton (iMAPS), a generalized measure related to MAPS, to quantify negative selection in non-coding regions. This approach, in conjunction with in vitro and in vivo binding data, identifies precise RBP binding sites, miRNA target sites, and polyadenylation signals (PASs) under strong selection. For each class of sites, we identify thousands of gnomAD variants under selection comparable to missense coding variants, and find that sites in core 3' UTR regions upstream of the most-used PAS are under strongest selection. Together, this work improves our understanding of selection on human genes and validates approaches for interpreting genetic variants in human 3' UTRs.

Functional complex point-defect structure in a huge-size-mismatch system.

Cubic boron nitride is a promising system for photonics and optoelectronics. Determining the inclusion mechanisms for dopants with a large size mismatch, such as luminous rare-earth elements, is prerequisite to understanding their functional properties and to effective doping control. Combining evidence from subangstrom resolution scanning transmission electron microscopy, imaging simulations, and first-principles calculations, we show that cationic Ce(3+) single dopants are not located at cationic B sites but rather at anionic N sites surrounded by B-site vacancies.

Regulatory features aid interpretation of 3'UTR Variants.

Our ability to determine the clinical impact of variants in 3' untranslated regions (UTRs) of genes remains poor. We provide a thorough analysis of 3'UTR variants from several datasets. Variants in putative regulatory elements including RNA-binding protein motifs, eCLIP peaks, and microRNA sites are up to 16 times more likely than other variants to have gene expression and phenotype associations. Heterozygous variants in regulatory motifs result in allele-specific protein binding in cell lines and allele-specific gene expression differences in population studies. In addition, variants in shared regions of alternatively polyadenylated isoforms and those proximal to polyA sites are more likely to affect gene expression and phenotype. Finally, pathogenic 3'UTR variants in ClinVar are 20 times more likely than benign variants to fall in a regulatory site. We incorporated these findings into RegVar, a software tool that interprets regulatory elements and annotations for any 3'UTR variant, and predicts whether the variant is likely to affect gene expression or phenotype. This tool will help prioritize variants for experimental studies and identify pathogenic variants in patients.

RNA cytosine methyltransferase NSUN5 promotes protein synthesis and tumorigenic phenotypes in glioblastoma.

Glioblastoma (GBM) is the most common and aggressive malignant primary brain tumor in adults. The standard treatment achieves a median overall survival for GBM patients of only 15 months. Hence, novel therapies based on an increased understanding of the mechanistic underpinnings of GBM are desperately needed. In this study, we show that elevated expression of 28S rRNA (cytosine-C(5))-methyltransferase NSUN5, which methylates cytosine 3782 of 28S rRNA in GBM cells, is strongly associated with the poor survival of GBM patients. Moreover, we demonstrate that overexpression of NSUN5 increases protein synthesis in GBM cells. NSUN5 knockdown decreased protein synthesis, cell proliferation, sphere formation, migration, and resistance to temozolomide in GBM cell lines. NSUN5 knockdown also decreased the number and size of GBM neurospheres in vitro. As a corollary, mice harboring U251 tumors wherein NSUN5 was knocked down survived longer than mice harboring control tumors. Taken together, our results suggest that NSUN5 plays a protumorigenic role in GBM by enabling the enhanced protein synthesis requisite for tumor progression. Accordingly, NSUN5 may be a hitherto unappreciated target for the treatment of GBM.

Systematic identification of disease-causing promoter and untranslated region variants in 8,040 undiagnosed individuals with rare disease.

Background: Both promoters and untranslated regions (UTRs) have critical regulatory roles, yet variants in these regions are largely excluded from clinical genetic testing due to difficulty in interpreting pathogenicity. The extent to which these regions may harbour diagnoses for individuals with rare disease is currently unknown. Methods: We present a framework for the identification and annotation of potentially deleterious proximal promoter and UTR variants in known dominant disease genes. We use this framework to annotate de novo variants (DNVs) in 8,040 undiagnosed individuals in the Genomics England 100,000 genomes project, which were subject to strict region-based filtering, clinical review, and validation studies where possible. In addition, we performed region and variant annotation-based burden testing in 7,862 unrelated probands against matched unaffected controls. Results: We prioritised eleven DNVs and identified an additional variant overlapping one of the eleven. Ten of these twelve variants (82%) are in genes that are a strong match to the individual's phenotype and six had not previously been identified. Through burden testing, we did not observe a significant enrichment of potentially deleterious promoter and/or UTR variants in individuals with rare disease collectively across any of our region or variant annotations. Conclusions: Overall, we demonstrate the value of screening promoters and UTRs to uncover additional diagnoses for previously undiagnosed individuals with rare disease and provide a framework for doing so without dramatically increasing interpretation burden.