Resolution of Virtual Depth Sectioning from Four-Dimensional Scanning Transmission Electron Microscopy.

One approach to three-dimensional structure determination using the wealth of scattering data in four-dimensional (4D) scanning transmission electron microscopy (STEM) is the parallax method proposed by Ophus et al. (2019. Advanced phase reconstruction methods enabled by 4D scanning transmission electron microscopy, Microsc Microanal25, 10-11), which determines the scattering matrix and uses it to synthesize a virtual depth-sectioning reconstruction of the sample structure. Drawing on an equivalence with a hypothetical confocal imaging mode, we derive contrast transfer and point spread functions for this parallax method applied to weakly scattering objects, showing them identical to earlier depth-sectioning STEM modes when only bright field signal is used, but that improved depth resolution is possible if dark field signal can be used. Through a simulation-based study of doped Si, we show that this depth resolution is preserved for thicker samples, explore the impact of shot noise on the parallax reconstructions, discuss challenges to making use of dark field signal, and identify cases where the interpretation of the parallax reconstruction breaks down.

Relative roles of multiple scattering and Fresnel diffraction in the imaging of small molecules using electrons, Part II: Differential Holographic Tomography.

It has been argued that in atomic-resolution transmission electron microscopy (TEM) of sparse weakly scattering structures, such as small biological molecules, multiple electron scattering usually has only a small effect, while the in-molecule Fresnel diffraction can be significant due to the intrinsically shallow depth of focus. These facts suggest that the three-dimensional reconstruction of such structures from defocus image series collected at multiple rotational orientations of a molecule can be effectively performed for each atom separately, using the incoherent first Born approximation. The corresponding reconstruction method, termed here Differential Holographic Tomography, is developed theoretically and demonstrated computationally on several numerical models of biological molecules. It is shown that the method is capable of accurate reconstruction of the locations of atoms in a molecule from TEM data collected at a small number of random orientations of the molecule, with one or more defocus images per orientation. Possible applications to cryogenic electron microscopy and other areas are briefly discussed.

Factors limiting quantitative phase retrieval in atomic-resolution differential phase contrast scanning transmission electron microscopy using a segmented detector.

Quantitative differential phase contrast imaging of materials in atomic-resolution scanning transmission electron microscopy using segmented detectors is limited by various factors, including coherent and incoherent aberrations, detector positioning and uniformity, and scan-distortion. By comparing experimental case studies of monolayer and few-layer graphene with image simulations, we explore which parameters require the most precise characterisation for reliable and quantitative interpretation of the reconstructed phases. Coherent and incoherent lens aberrations are found to have the most significant impact. For images over a large field of view, the impact of noise and non-periodic boundary conditions are appreciable, but in this case study have less of an impact than artefacts introduced by beam deflections coupling to beam scanning (imperfect tilt-shift purity).

Large angle illumination enabling accurate structure reconstruction from thick samples in scanning transmission electron microscopy.

Most reconstructions of the electrostatic potential of a specimen at atomic resolution assume a thin and weakly scattering sample, restricting accurate quantification to specimens only tens of Ångströms thick. We demonstrate that using large-angle-illumination scanning transmission electron microscopy (STEM)-a probe forming aperture with convergence angle larger than about 50 mrad-allows us to better meet the weak phase object approximation and thereby accurately reconstruct the electrostatic potential in samples thicker than the order of 100 Å.

A quantum mechanical exploration of phonon energy-loss spectroscopy using electrons in the aloof beam geometry.

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.

Structure Retrieval at Atomic Resolution in the Presence of Multiple Scattering of the Electron Probe.

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 (≲50 Å) 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.

Measuring nanometre-scale electric fields in scanning transmission electron microscopy using segmented detectors.

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.

Atomic resolution elemental mapping using energy-filtered imaging scanning transmission electron microscopy with chromatic aberration correction.

This paper addresses a novel approach to atomic resolution elemental mapping, demonstrating a method that produces elemental maps with a similar resolution to the established method of electron energy-loss spectroscopy in scanning transmission electron microscopy. Dubbed energy-filtered imaging scanning transmission electron microscopy (EFISTEM) this mode of imaging is, by the quantum mechanical principle of reciprocity, equivalent to tilting the probe in energy-filtered transmission electron microscopy (EFTEM) through a cone and incoherently averaging the results. In this paper we present a proof-of-principle EFISTEM experimental study on strontium titanate. The present approach, made possible by chromatic aberration correction, has the advantage that it provides elemental maps which are immune to spatial incoherence in the electron source, coherent aberrations in the probe-forming lens and probe jitter. The veracity of the experiment is supported by quantum mechanical image simulations, which provide an insight into the image-forming process. Elemental maps obtained in EFTEM suffer from the effect known as preservation of elastic contrast, which, for example, can lead to a given atomic species appearing to be in atomic columns where it is not to be found. EFISTEM very substantially reduces the preservation of elastic contrast and yields images which show stability of contrast with changing thickness. The experimental application is demonstrated in a proof-of-principle study on strontium titanate.

A new method to detect and correct sample tilt in scanning transmission electron microscopy bright-field imaging.

Important properties of functional materials, such as ferroelectric shifts and octahedral distortions, are associated with displacements of the positions of lighter atoms in the unit cell. Annular bright-field scanning transmission electron microscopy is a good experimental method for investigating such phenomena due to its ability to image light and heavy atoms simultaneously. To map atomic positions at the required accuracy precise angular alignment of the sample with the microscope optical axis is necessary, since misalignment (tilt) of the specimen contributes to errors in position measurements of lighter elements in annular bright-field imaging. In this paper it is shown that it is possible to detect tilt with the aid of images recorded using a central bright-field detector placed within the inner radius of the annular bright-field detector. For a probe focus near the middle of the specimen the central bright-field image becomes especially sensitive to tilt and we demonstrate experimentally that misalignment can be detected with a precision of less than a milliradian, as we also confirm in simulation. Coma in the probe, an aberration that can be misidentified as tilt of the specimen, is also investigated and it is shown how the effects of coma and tilt can be differentiated. The effects of tilt may be offset to a large extent by shifting the diffraction plane detector an amount equivalent to the specimen tilt and we provide an experimental proof of principle of this using a segmented detector system.

Addressing preservation of elastic contrast in energy-filtered transmission electron microscopy.

Energy-filtered transmission electron microscopy (EFTEM) images with resolutions of the order of an Ångström can be obtained using modern microscopes corrected for chromatic aberration. However, the delocalized nature of the transition potentials for atomic ionization often confounds direct interpretation of EFTEM images, leading to what is known as "preservation of elastic contrast". In this paper we demonstrate how more interpretable images might be obtained by scanning with a focused coherent probe and incoherently averaging the energy-filtered images over probe position. We dub this new imaging technique energy-filtered imaging scanning transmission electron microscopy (EFISTEM). We develop a theoretical framework for EFISTEM and show that it is in fact equivalent to precession EFTEM, where the plane wave illumination is precessed through a range of tilts spanning the same range of angles as the probe forming aperture in EFISTEM. It is demonstrated that EFISTEM delivers similar results to scanning transmission electron microscopy with an electron energy-loss spectrometer but has the advantage that it is immune to coherent aberrations and spatial incoherence of the probe and is also more resilient to scan distortions.

Surface determination through atomically resolved secondary-electron imaging.

Unique determination of the atomic structure of technologically relevant surfaces is often limited by both a need for homogeneous crystals and ambiguity of registration between the surface and bulk. Atomically resolved secondary-electron imaging is extremely sensitive to this registration and is compatible with faceted nanomaterials, but has not been previously utilized for surface structure determination. Here we report a detailed experimental atomic-resolution secondary-electron microscopy analysis of the c(6 × 2) reconstruction on strontium titanate (001) coupled with careful simulation of secondary-electron images, density functional theory calculations and surface monolayer-sensitive aberration-corrected plan-view high-resolution transmission electron microscopy. Our work reveals several unexpected findings, including an amended registry of the surface on the bulk and strontium atoms with unusual seven-fold coordination within a typically high surface coverage of square pyramidal TiO5 units. Dielectric screening is found to play a critical role in attenuating secondary-electron generation processes from valence orbitals.

Rapidly fatal neuropathies and an ALS clinical presentation.

OBJECTIVES - The objective of this report is to describe patients with an unusually severe, rapidly fatal acquired polyneuropathy. METHODS - The clinical, electrodiagnostic (EDX), laboratory, and pathological findings in three patients with a distinctive form of neuropathic illness are discussed. RESULTS - Three patients, ages 67, 54, and 50, had clinical findings that met accepted clinical criteria for amyotrophic lateral sclerosis (ALS) - definite in two and probable in one. The EDX studies in these patients had abnormalities that would be highly atypical for ALS. There were features consistent with an asymmetrical, non-length-dependent process as well findings consistent with demyelination - features consistent with a chronic acquired polyneuropathy. All patients had a rapidly progressive course with death because of respiratory failure in 4-30 months. The patients did not respond to immunomodulating therapies. Extensive evaluations in these patients did not reveal a cause for the patients' neuropathies. Postmortem examination in two of the patients did not reveal evidence for ALS. In one of these patients, there were pathological findings thought possibly consistent with an immunologically mediated process. CONCLUSION - This report emphasizes the importance of considering other diagnoses in patients who clinically appear to have ALS and raise the specter of an as of yet poorly understood, severe neuropathic illness.

Cutaneous wound analysis using hyperspectral imaging.

A correlative bright-field and hyperspectral analysis of full-thickness, cutaneous wounds in a porcine model was undertaken to investigate the efficacy of hyperspectral imaging as an alternate method for wound identification. Analysis of a randomly selected specimen yielded distinct spectral signatures for cutaneous regions of interest including the epidermis, injured dermis, and normal dermis. The scanning of the entire specimen group using these hyperspectral signatures revealed an exclusionary, pseudo-color pattern whereby a central wound region was consistently defined by a unique spectral signature. An algorithm was derived as an objective tool for the comparison of the wound regions defined by the hyperspectral classification versus the pathologists' manual tracings. The dimensions of the wound identified in the hyperspectral assay did not differ significantly from the wound region identified by the pathologists using standard bright-field microscopy. These data indicate that hyperspectral analysis may provide a high-throughput alternative for wound estimation that approximates standard bright-field imaging and pathologist evaluation.

Interobserver variability associated with the MIB-1 labeling index: high levels suggest limited prognostic usefulness for patients with primary brain tumors.

BACKGROUND: The use of the MIB-1 labeling index (LI) as a potential prognostic marker for patients with primary brain tumors is controversial. Many studies advocating its prognostic usefulness have suggested discrete MIB-1 LI cut-off values, above which patients have significantly worse outcomes. However, interobserver variability associated previously with MIB-1 LI calculation has not been reported despite the fact that the degree of interobserver variability impacts the clinical usefulness of such cut-off values. METHODS: MIB-1 LIs were calculated independently using a standardized protocol by six pathologist observers for 50 astrocytic gliomas of varying grades. The level of interobserver agreement was determined by calculating kappa statistics for pairwise pathologist comparisons using MIB-1 LI cut-off values of 2.5%, 5.0%, 8.0%, 11.0%, and 15.0%. Spearman rank correlation coefficients were used to assess the pairwise associations between observer MIB-1 LIs. RESULTS: Although there was general agreement among pathologists regarding whether an MIB-1 LI for a given astroglial tumor was low, moderate, or high based on the analysis of correlation, a high level of interobserver variability was associated with the determination of specific MIB-1 LIs. The highest level of agreement occurred using a cut-off value of 5.0%, with pairwise kappa statistics for this value ranging from 0.52 to 0.80. CONCLUSIONS: The high level of interobserver variability suggests that proposed discrete MIB-1 LI prognostic cut-off values most likely are not useful clinically for predicting outcome for individual patients with primary brain tumors. Further prospective studies are needed investigating the prognostic usefulness of MIB-1 LI ranges that optimize interobserver agreement.

January 2001: A 37 year old man with a history of Hodgkin's disease.

The January Cases of the Month (COM): A case of intracranial metastatic nodular sclerosing Hodgkin's disease without dural attachment in a 37-year-old previously stage III male is presented with a brief review of the literature. Both the primary tumor in the lymph node biopsy and the metastatic brain tumor showed similar histopathology and a immunohistochemical profile typical for Hodgkin's Disease. After chemotherapy, there are no signs of recurrence or systemic disease on follow-up for five months.

Management of neurocytomas: case report and review of the literature.

The purpose of this report is to review the available literature on the presentation, pathology, and treatment of central nervous system (CNS) neurocytomas. A case report of an extraventricular neurocytoma is presented along with a comprehensive literature search of patients with a diagnosis of CNS neurocytoma. CNS neurocytomas are rare neoplasms, with fewer than 240 cases reported in the literature. The majority of neurocytomas are found in the ventricular system of the brain. Immunohistochemistry is frequently used to help distinguish this tumor from other CNS neoplasms. MIB-1 proliferation index is commonly used in an attempt to predict biologic behavior. Little is known about the management of patients with this tumor, because most reports are from the pathologic literature and contain sparse information regarding clinical management. Neurocytomas are rare CNS tumors with varied biologic behavior. MIB-1 index may help direct adjuvant therapy. An excellent prognosis can be expected if a gross total resection is achieved. Postoperative radiation therapy (RT) may be considered after subtotal resection. Otherwise, RT is an option for medically inoperable or recurrent disease.

Thermal lethality of Salmonella Senftenberg and Listeria innocua in fully cooked and packaged chicken breast strips via steam pasteurization.

Fully cooked chicken breast strips were surface inoculated to contain 9 log10 (CFU/g) Salmonella Senftenberg or Listeria innocua. The inoculated products were vacuum packaged in 0.2-mm-thick barrier bags (241 by 114 mm), then steam pasteurized at 88 degrees C in a continuous process for 26 to 40 min or in a batch process for 33 to 41 min. After the treatments, the products were analyzed for the survivors of Salmonella or Listeria. The models were developed to correlate the surviving rate of Salmonella and Listeria with cooking time for both continuous and batch processes. A cooking time of 34 min was needed to achieve 7 logs of the reduction in a batch process. To achieve the same log reduction, a longer (6 min) cooking time was needed in a batch process than in a continuous process. The results from this study will be useful for processors to evaluate postcooking treatment procedures for ready-to-eat meat products.

"Large cell/anaplastic" medulloblastomas: a Pediatric Oncology Group Study.

495 medulloblastomas (MBs) from 6 Pediatric Oncology Group (POG) protocols were reviewed to assess the incidence and prognostic significance of "large cell" and "anaplastic" variants. "Large cell" medulloblastomas (LC MBs) were those with focal or diffuse, large, round neoplastic cells with prominent nucleoli. "Anaplastic" MBs (A MBs) were those with nuclei that were also large but markedly atypical with coarse chromatin and irregular shapes. Twenty-one cases were identified in the combined LC/A MB group, comprising about 4% of all MBs. Survival curves and Kaplan-Meier estimates of survival probabilities were examined separately for the LC/A MB and control groups. The logrank test for detecting poorer survival in the 21 cases was significant (p < 0.0001). Fluorescence in situ hybridization for c-myc showed amplification in 4 of 11 cases of the LC/A phenotype and 1 additional case of high level gain at 8q24 was disclosed by comparative genomic hybridization. Comparative genomic hybridization confirmed c-myc amplification and found evidence for isochromosome 17q in 3 of 4 LC/A cases studied successfully. One additional tumor showed high level gain restricted to 2p13 consistent with n-myc amplification. Monosomy 22, common in atypical teratoid/rhabdoid tumors, was not found. These results suggest that LC/A MB phenotype could be, at least in part, a correlate of c-myc, and possibly n-myc, amplification. The study thus confirms original observations about the LC MB in regard to histological features, immunohistochemical findings, c-myc amplification, cytogenetic findings, and poor prognosis.

Intracranial leiomyosarcoma in a patient with AIDS.

We report an intracranial leiomyosarcoma in the pontine cistern of a 34-year-old woman infected with the human immunodeficiency virus (HIV). The clinical, radiological and pathological data are reviewed. The tumor was Epstein-Barr virus (EBV) positive by in situ hybridization. This case emphasizes that smooth muscle neoplasms arising in the setting of immunocompromise can occur intracranially, and corroborates a hypothesis that EBV coinfection may have a role in development of these tumors.

Neurofilament-L homopolymers are less mechanically stable than native neurofilaments.

Neurofilaments are cytoskeletal components of neurones that are thought to play an important structural role in the axon. Specific functions of neurofilaments are not yet well defined; however, other intermediate filaments are known to have structural and mechanical functions in different cell types. The atomic force microscope (AFM) can be used to visualize and manipulate biological structures through direct physical contact. This allows the AFM to be used to probe the mechanical properties of these structures. In this paper we present AFM images of native neurofilaments isolated from bovine spinal cord, composed of NF-L, NF-M and NF-H, and filaments polymerized in vitro from purified NF-L. Morphologically these structures, in solution and under ambient conditions, are in agreement with previous data from electron microscopy. However, repeated scanning of NF-L homopolymers (in solution) produced significant disruptions of segments of filaments, both within and at the ends of the filaments. This disruption resulted in complete loss of portions of the filaments and in breaks in the continuity of the filaments. Repeated scanning of isolated native neurofilaments under similar conditions produced no detectable structural changes. Under extremely high applied forces the native neurofilaments were bent and distorted by the action of the AFM tip, but were never broken. These data suggest that purified NF-L is not sufficient to confer complete mechanical stability to neurofilaments.