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Segmented and pixelated detectors on scanning transmission electron microscopes enable the complex specimen transmission function to be reconstructed. Imaging the transmission function is key to interpreting the electric and magnetic properties of the specimen, and as such four-dimensional scanning transmission electron microscopy (4D-STEM) imaging techniques are crucial for our understanding of functional materials. Many of the algorithms used in the reconstruction of the transmission function rely on the multiplicative approximation and the (weak) phase object approximation, which are not valid for many materials, particularly at high resolution. Herein, we study the breakdown of simple phase imaging in thicker samples. We demonstrate the behavior of integrated center of mass imaging, single-side band ptychography, and Wigner distribution deconvolution over a thickness series of simulated GaN 4D-STEM datasets. We further give guidance as to the optimal focal conditions for obtaining a more interpretable dataset using these algorithms.

Increasing interest in three-dimensional nanostructures adds impetus to electron microscopy techniques capable of imaging at or below the nanoscale in three dimensions. We present a reconstruction algorithm that takes as input a focal series of four-dimensional scanning transmission electron microscopy (4D-STEM) data. We apply the approach to a lead iridate, PbIrO, and yttrium-stabilized zirconia, YZrO, heterostructure from data acquired with the specimen in a single plan-view orientation, with the epitaxial layers stacked along the beam direction. We demonstrate that Pb–Ir atomic columns are visible in the uppermost layers of the reconstructed volume. We compare this approach to the alternative techniques of depth sectioning using differential phase contrast scanning transmission electron microscopy (DPC-STEM) and multislice ptychographic reconstruction.

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.

PDF file - 1.2MB, Nodal antibody used for IHC labels same cells as R&D antibody used in previous studies, but with less non-specific background staining

PDF file - 1MB, Nodal co-localizes with CD31 in human breast cancer

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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)

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 post-transcriptional gene expression. However, identifying functional 3’ UTR variants has proven difficult. We used allele frequencies from the Genome Aggregation Database (gnomAD) to identify classes of 3’ UTR variants under strong negative selection in humans. We developed intergenic mutability-adjusted proportion singleton (iMAPS), a generalized measure related to MAPS, to quantify negative selection in non-coding regions. This approach, in conjunction within vitroandin vivobinding data, identifies precise RBP binding sites, miRNA target sites, and polyadenylation signals (PASs) under strong selection. For each class of sites, we identified thousands of gnomAD variants under selection comparable to missense coding variants, and found 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.

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.

CellTrace™ Violet (CTV) is a powerful tool for tracking cell proliferation by permanently binding cellular proteins and rendering the cell fluorescent. After cell division, each daughter cell contains half of the parent cell's fluorescence, enabling quantification of proliferation via flow cytometry. This method enables monitoring of several generations of cell division and tracking of different cell populations in co-culture. Here we describe the use of CellTrace™ Violet in different cell types, and we share important observations we made during protocol optimization.

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.

Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5’ Untranslated Regions (5’UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. The accumulation of the corresponding proteins induces plasticity and “fate-switching” toward stem cell-like phenotypes. Mechanistically, we observe that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem-cell-like phenotypes. These effects are overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB), suggesting that the Integrated Stress Response drives breast cancer plasticity. Collectively, our findings reveal a mechanism of induction of plasticity of breast cancer cells and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis., Protein synthesis suppression protects breast cancer cells from clinically relevant stresses like hypoxia. Here, the authors show that unique mRNA isoforms that govern stem cell-like phenotypes escape translational repression to drive tumor progression and chemoresistance.

We propose a linear imaging theory for differential phase contrast under the weak-phase-weak-amplitude object approximation. Contrast transfer functions are defined for thin and thick weak objects, and they successfully describe several imaging characteristics of differential phase contrast. We discuss the defocus dependence of the contrast for several examples: atomic resolution, a p-n junction, a heterointerface, and grain boundaries. Understanding the imaging characteristics helps in adjusting aberrations in DPC STEM.

Atomic-resolution electron microscopes utilize high-power magnetic lenses to produce magnified images of the atomic details of matter. Doing so involves placing samples inside the magnetic objective lens, where magnetic fields of up to a few tesla are always exerted. This can largely alter, or even destroy, the magnetic and physical structures of interest. Here, we describe a newly developed magnetic objective lens system that realizes a magnetic field free environment at the sample position. Combined with a higher-order aberration corrector, we achieve direct, atom-resolved imaging with sub-Å spatial resolution with a residual magnetic field of less than 0.2 mT at the sample position. This capability enables direct atom-resolved imaging of magnetic materials such as silicon steels. Removing the need to subject samples to high magnetic field environments enables a new stage in atomic resolution electron microscopy that realizes direct, atomic-level observation of samples without unwanted high magnetic field effects., Electron microscopy typically requires strong magnetic lenses in order to reach atomic resolution, prohibiting the possibility to measure magnetic materials. The authors here present a lens design that enables atomic-resolution electron microscopy of magnetic materials by providing a field-free sample region.

Author(s): Pelz, PM; Brown, HG; Stonemeyer, S; Findlay, SD; Zettl, A; Ercius, P; Zhang, Y; Ciston, J; Scott, MC; Ophus, C | Abstract: Three-dimensional phase-contrast imaging of multiply-scattering samples in x-ray and electron microscopy is challenging due to small numerical apertures, the unavailability of wave front shaping optics, and the highly nonlinear inversion required from intensity-only measurements. In this work, we present an algorithm using the scattering matrix formalism to solve the scattering from a noncrystalline medium from scanning diffraction measurements and simultaneously recover the illumination aberrations. We demonstrate our method experimentally in a scanning transmission electron microscope, recovering the scattering matrix of a heterogeneous sample with two layers of multiwall carbon nanotubes filled with TaTe2 core-shell structures, spaced 10nm apart in the axial direction. Our work enables phase contrast imaging and materials characterization in multiply-scattering samples at high resolution for a wide range of materials.

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.

Angular resolved scanning transmission electron microscopy is an important tool for investigating the properties of materials. However, several recent studies have observed appreciable discrepancies in the angular scattering distribution between experiment and theory. In this paper we discuss a general approach to low-loss inelastic scattering which, when incorporated in the simulations, resolves this problem and also closely reproduces experimental data taken over an extended angular range. We also explore the role of ionic bonding, temperature factors, amorphous layers on the surfaces of the specimen, and static displacements of atoms on the angular scattering distribution. The incorporation of low-loss inelastic scattering in simulations will improve the quantitative usefulness of techniques such as low-angle annular dark-field imaging and position-averaged convergent beam electron diffraction, especially for thicker specimens.

Material properties are sensitive to atomistic structure defects such as vacancies or impurities, and it is therefore important to determine not only the local atomic configuration but also their chemical bonding state. Annular dark-field scanning transmission electron microscopy (STEM) combined with electron energy-loss spectroscopy has been utilized to investigate the local electronic structures of such defects down to the level of single atoms. However, it is still challenging to two-dimensionally map the local bonding states, because the electronic fine-structure signal from a single atom is extremely weak. Here, we show that atomic-resolution differential phase-contrast STEM imaging can directly visualize the anisotropy of single Si atomic electric fields in monolayer graphene. We also visualize the atomic electric fields of Stone–Wales defects and nanopores in graphene. Our results open the way to directly examine the local chemistry of the defective structures in materials at atomistic dimensions.

Probing the charge density distributions in materials at atomic scale remains an extremely demanding task, particularly in real space. However, recent advances in differential phase contrast-scanning transmission electron microscopy (DPC-STEM) bring this possibility closer by directly visualizing the atomic electric field. DPC-STEM at atomic resolutions measures how a sub-angstrom electron probe passing through a material is affected by the atomic electric field, the field between the nucleus and the surrounding electrons. Here, we perform a fully quantitative analysis which allows us to probe the charge density distributions inside atoms, including both the positive nuclear and the screening electronic charges, with subatomic resolution and in real space. By combining state-of-the-art DPC-STEM experiments with advanced electron scattering simulations we are able to map the spatial distribution of the electron cloud within individual atomic columns. This work constitutes a crucial step toward the direct atomic scale determination of the local charge redistributions and modulations taking place in materials systems.

// Abdulraheem Alshareef 1, 2 , Meredith S. Irwin 3 , Nidhi Gupta 2 , Hai-Feng Zhang 2, 4 , Moinul Haque 2 , Scott D. Findlay 5, 6 , Bo Kyung Alex Seong 7 , Justine Lai 2 , Mohammed Rayis 8 , Sadeq Al-Dandan 9 and Raymond Lai 2, 5, 10 1 Department of Applied Medical Sciences, Taibah University, Medina, Saudi Arabia 2 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Canada 3 Division of Haematology-Oncology, Department of Pediatrics, The Hospital for Sick Children, University of Toronto, Ontario, Canada 4 Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, Canada 5 Department of Oncology, University of Alberta, Edmonton, Canada 6 Department of Anatomy and Cell Biology, Faculty of Medicine and Dentistry, University of Western Ontario, London, Canada 7 Department of Medical Biophysics, University of Toronto, Toronto, Canada 8 Department of Pediatric Oncology, King Fahad Medical City, Riyadh, Saudi Arabia 9 Department of Anatomical Pathology, King Fahad Medical City, King Saud bin Abdulaziz University, Riyadh, Saudi Arabia 10 DynaLIFE Medical Laboratories, Edmonton, Canada Correspondence to: Raymond Lai, email: rlai@ualberta.ca Keywords: neuroblastoma; anaplastic lymphoma kinase; alk-expressing human cancers; intron-retention transcript; prognostic markers Received: January 04, 2017 Accepted: December 28, 2017 Published: January 12, 2018 ABSTRACT ALK missense mutations are detected in 8% of neuroblastoma (NB) tumors at diagnosis and confer gain-of-function oncogenic effects. The mechanisms by which the expression of wild-type or mutant ALK , which is detectable in the majority of cases, is regulated are not well understood. We have identified a novel ALK transcript characterized by the retention of intron 19 ( ALK-I19 ). ALK-I19 was detected in 4/4 NB cell lines, but not other non-NB cells with ALK aberrations. The functional significance of ALK-I19 was determined by specific siRNA knockdown of this transcript, which resulted in substantially decreased expression of the fully-spliced ALK transcripts (FS- ALK ) and a significant reduction in cell growth. We also demonstrate that ALK-I19 is a precursor of FS- ALK . ALK-I19 was detected in 14/37 (38%) tumors from patients with newly diagnosed NB. ALK-I19 expression correlated with undifferentiated histology and strong ALK protein expression detectable by immunohistochemistry. Importantly, patients with tumors that did not express ALK-I19 and lacked MYCN amplification had an excellent clinical outcome, with 19/19 patients survived at 5-years. In conclusion, ALK-I19 is a novel ALK transcript that likely represents a marker of undifferentiated NB cells. The absence of ALK-I19 and MYCN amplification is a useful prognostic marker for NB patients.

We assess a selection of electron probes in terms of the spatial resolution with which information can be derived about the structure of a specimen, as opposed to the nominal image resolution. Using Ge [001] as a study case, we investigate the scattering dynamics of these probes and determine their relative merits in terms of two qualitative criteria: interaction volume and interpretability. This analysis provides a 'menu of probes' from which an optimum probe for tackling a given materials science question can be selected. Hollow cone, vortex and spherical wave fronts are considered, from unit cell to Ångstrom size, and for different defocus and specimen orientations.

Phonon energy-loss spectroscopy using electrons has both high resolution and low resolution components, associated with short- and long-range interactions, respectively. In this paper, we discuss how these two contributions arise from a fundamental quantum mechanical perspective. Starting from a correlated model for the atomic motion we show how short range 'impact' scattering and long range 'dipole' scattering arises. The latter dominates in aloof beam imaging, an imaging geometry in which radiation damage can be avoided.

Electric field mapping using segmented detectors in the scanning transmission electron microscope has recently been achieved at the nanometre scale. However, converting these results to quantitative field measurements involves assumptions whose validity is unclear for thick specimens. We consider three approaches to quantitative reconstruction of the projected electric potential using segmented detectors: a segmented detector approximation to differential phase contrast and two variants on ptychographical reconstruction. Limitations to these approaches are also studied, particularly errors arising from detector segment size, inelastic scattering, and non-periodic boundary conditions. A simple calibration experiment is described which corrects the differential phase contrast reconstruction to give reliable quantitative results despite the finite detector segment size and the effects of plasmon scattering in thick specimens. A plasmon scattering correction to the segmented detector ptychography approaches is also given. Avoiding the imposition of periodic boundary conditions on the reconstructed projected electric potential leads to more realistic reconstructions.

Differential phase contrast in scanning transmission electron microscopy can visualize local electromagnetic fields inside specimens. The contrast derived from first moments, the so-called center of mass, of the diffraction patterns for each probe position can be quantitatively related to the local electromagnetic field under the phase object approximation. While only approximate first moments can be obtained with a segmented detector, in weak phase objects the fields can be accurately quantified on the basis of a phase contrast transfer function. Through systematic image simulations we further show that the quantification based on the approximated first moment is a good approximation also for strong phase objects.

The increasing use of energy dispersive X-ray spectroscopy in atomic resolution scanning transmission electron microscopy invites the question of whether its success in precision composition determination at lower magnifications can be replicated in the atomic resolution regime. In this paper, we explore, through simulation, the prospects for composition measurement via the model system of AlxGa1-xAs, discussing the approximations used in the modelling, the variability in the signal due to changes in configuration at constant composition, and the ability to distinguish between different compositions. Results are presented in such a way that the number of X-ray counts, and thus the expected variation due to counting statistics, can be gauged for a range of operating conditions.

Linking the atomic level kinetic precipitation pathways induced by elemental additions to the resulting microstructure is fundamentally desirable for the design of new classes of light alloys. Aberration-corrected scanning transmission electron microscopy (AC-STEM) and first principles calculations were used to investigate the influence of trace Au (200 ppm) additions on precipitation in an Al-Cu-Au alloy. These Au additions resulted in a significant enhancement of the low-temperature age hardening, which was demonstrated to be associated with accelerated precipitate nucleation and growth. Atomic-resolution annular dark field (ADF) imaging showed the clearly reduced critical length and thickness of θ′ precipitates with Au additions, therefore accelerating the nucleation of θ′. Agglomerated Au clusters were observed in θ′ precipitates, which were demonstrated to correspond to a localised energetically favourable state. These observations have been explained through first-principles calculations and relevant thermodynamic modelling. This work provides a potential way to refine the alloy microstructure for improving the mechanical behaviour of light alloys.

Here we report the influence of key experimental parameters on atomically resolved energy dispersive X-ray spectroscopy (EDX). In particular, we examine the role of the probe forming convergence semi-angle, sample thickness, lattice spacing, and dwell/collection time. We show that an optimum specimen-dependent probe forming convergence angle exists to maximize the signal-to-noise ratio of the atomically resolved signal in EDX mapping. Furthermore, we highlight that it can be important to select an appropriate dwell time to efficiently process the X-ray signal. These practical considerations provide insight for experimental parameters in atomic resolution energy dispersive X-ray analysis.

Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5’ Untranslated Regions (5’UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. This leads to accumulation of corresponding proteins which induce plasticity and “fate-switching” toward stem-cell like phenotypes. Surprisingly, we observed that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem cell-like phenotypes. Strikingly, these effects can be overcome with drugs that antagonize translational reprogramming caused by eIF2α phosphorylation (e.g. ISRIB). Collectively, our findings unravel a hitherto unappreciated mechanism of induction of plasticity of breast cancer cells, and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.

The projected electrostatic potential of a thick crystal is reconstructed at atomic-resolution from experimental scanning transmission electron microscopy data recorded using a new generation fast- readout electron camera. This practical and deterministic inversion of the equations encapsulating multiple scattering that were written down by Bethe in 1928 removes the restriction of established methods to ultrathin ($\lesssim 50$ {\AA}) samples. Instruments already coming on-line can overcome the remaining resolution-limiting effects in this method due to finite probe-forming aperture size, spatial incoherence and residual lens aberrations., Comment: 6 pages, 3 figures

It is well known that dynamical diffraction varies with changes in sample thickness and local crystal orientation (due to sample bending). In differential phase contrast scanning transmission electron microscopy (DPC-STEM), this can produce contrast comparable to that arising from the long-range electromagnetic fields probed by this technique. Through simulation we explore the scale of these dynamical diffraction artefacts and introduce a metric for the magnitude of their contribution to the contrast. We show that precession over an angular range of a few milliradian can suppress this contribution to the contrast by one-to-two orders of magnitude. Our exploration centres around a case study of GaAs near the [011] zone-axis orientation using a probe-forming aperture semiangle on the order of 0.1 mrad at 300 keV, but the trends found and methodology used are expected to apply more generally.

To correlate atomistic structure with longer range electric field distribution within materials, it is necessary to use atomically fine electron probes and specimens in on-axis orientation. However, electric field mapping via low magnification differential phase contrast imaging under these conditions raises challenges: electron scattering tends to reduce the beam deflection due to the electric field strength from what simple models predict, and other effects, most notably crystal mistilt, can lead to asymmetric intensity redistribution in the diffraction pattern which is difficult to distinguish from that produced by long range electric fields. Using electron scattering simulations, we explore the effects of such factors on the reliable interpretation and measurement of electric field distributions. In addition to these limitations of principle, some limitations of practice when seeking to perform such measurements using segmented detector systems are also discussed.

Quantitative agreement on an absolute scale is demonstrated between experiment and simulation for two-dimensional, atomic-resolution elemental mapping via energy dispersive X-ray spectroscopy. This requires all experimental parameters to be carefully characterized. The agreement is good, but some discrepancies remain. The most likely contributing factors are identified and discussed. Previous predictions that increasing the probe forming aperture helps to suppress the channelling enhancement in the average signal are confirmed experimentally. It is emphasized that simple column-by-column analysis requires a choice of sample thickness that compromises between being thick enough to yield a good signal-to-noise ratio while being thin enough that the overwhelming majority of the EDX signal derives from the column on which the probe is placed, despite strong electron scattering effects.

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.