Specimens For Electron Microscopy Research Articles

Decay of high-energy electron bound states in crystals

High-energy electrons that are used as a probe of specimens in transmission electron microscopy exhibit a complex and rich behavior due to multiple scattering. Among other things, understanding the dynamical effects is needed for a quantitative analysis of atomic-resolution images and spectroscopic data. In this study, state-correlation functions are computed within the multislice approach that allow to elucidate behaviors of transversely bound states in crystals. These states play an important role as a large fraction of current density can be coupled into them via focused electron probes. We show that bound states are generically unstable and decay monoexponentially with crystal depth. Their attenuation is accompanied by a resonant intensity transfer to Bessel-like wavefunctions that appear as Laue rings in the far-field diffraction patterns. Behaviors of bound states are also quantified when thermal effects are included, as well as point defects. This approach helps to bridge the Bloch wave and multisliced electron propagation pictures of dynamical scattering providing new insights into fundamental solutions of the wave equation, and may assist in developing quantitative STEM/TEM imaging techniques.

Scanning electron microscopic observation on morphology of Carpoglyphus lactis

To observe the morphological characteristics of live Carpoglyphus lactis at different stages of life cycle and its ultrastructure. The live C. lactis specimens were isolated from longan pulp, and firstly rinsed with double distilled water under a light microscope to make slide preparation by the conventional manner, and then were made into scanning electron microscope (SEM) specimens. The specimens were observed under SEM for the ultrastructure characteristics at different developmental stages including egg, larva, nymph and adult (male and female) . The SEM photographs showed that the egg was oval and milky. The larva had three pairs of legs, without genital setae, preanal seta and coxal rod, and there was no trace of genital growth. The nymph had four pairs of legs, genital seta and preanal seta, whereas the genital area was looked still under-developed. The male adult had a conical gnathosoma and a pair of external verticals which were longest setae at the dorsal idiosoma, whereas the other dorsal setae were all short rhabdoid. At the ventral idiosoma, there was an aedoeagus which was like a bent cube, and the top was straight forward. The external sacral setae and post anal were longest setae. The female adult's genital plates were a composite of plastron and cutex inner root, covering the genital tract, and a hole of anus and a pair of anal setae were located at posterior of idiosoma. The morphological characteristics and ultra-structure of C. lactis at different stages of life cycle can be vividly observed under SEM, which provides the morphological bases for further study of the relation between parasitism and disease.

Dimension effect on the in-depth phase transition of MnAs/GaAs(001): From a thin film to a nanorod

We explore the dimension effect on the in-depth phase transition behavior of MnAs thin films on GaAs substrate by investigating the in-depth strain distribution with experiments and simulations. The strain distribution along the film growth direction is verified to be changed when a two-dimensional thin film is thinned into a one-dimensional nanorod as a cross-section transmission electron microscopy (TEM) specimen, thus resulting in a significantly different transition behavior. Our work brings more insights into the transition mechanism of MnAs thin films and illustrates a noteworthy issue in TEM studies of the phase transition at the interface of epitaxial thin films.

Transmission Electron Microscope Specimen Preparation of Si-Based Anode Materials for Li-Ion Battery by Using Focused Ion Beam and Ultramicrotome

Transmission Electron Microscope Specimen Preparation of Si-Based Anode Materials for Li-Ion Battery by Using Focused Ion Beam and Ultramicrotome

Oral Biofilm Formation on Different Materials for Dental Implants.

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Sectioning studies of biomimetic collagen-hydroxyapatite coatings on Ti-6Al-4V substrates using focused ion beam

A biomimetic bone-like collagen-hydroxyapatite (Col-HA) composite coating was formed on a surface-treated Ti-6Al-4V alloy substrate via simultaneous collagen self-assembly and hydroxyapatite nucleation. The coating process has been carried out by immersing sand-blasted, acid-etched and UV irradiated Ti-6Al-4V alloy in type I collagen-containing modified simulated body fluid (m-SBF). The surface morphology and phase composition of the coating were characterized using various techniques. More importantly, dual-beam FIB/SEMs with either gallium ion source (GFIB) or xenon plasma ion source (PFIB) were used to investigate the cross-sectional features of the biomimetic Col-HA composite coating in great details. As a result, the cross-sectional images and thin transmission electron microscopy (TEM) specimens were successfully obtained from the composite coating with no obvious damages or milling ion implantations. Both the cross-sectional SEM and TEM results have confirmed that the Col-HA coating demonstrates a similar microstructure to that of pure HA coating with homogeneously distributed elements across the whole cross section. Both coatings consist of a uniform, crack-free gradient structure with a dense layer adjacent to the interface between the Ti-6Al-4V substrate and the coating facilitating a strong bonding, while a porous structure at the coating surface aiding cell attachment.

The influence of surfaces and interfaces on high spatial resolution vibrational EELS from SiO2.

High-resolution monochromated electron energy-loss spectroscopy has the potential to map vibrational modes at nanometer resolution. Using the SiO2/Si interface as a test case, we observe an initial drop in the SiO2 vibrational signal when the electron probe is 200 nm from the Si due to long-range nature of the Coulomb interaction. However, the distance from the interface at which the SiO2 integrated signal intensity drops to half its maximum value is 5 nm. We show that nanometer resolution is possible when selecting the SiO2/Si interface signal which is at a different energy position than the bulk signal. Calculations also show that, at 60 kV, the signal in the SiO2 can be treated non-relativistically (no retardation) while the signal in the Si, not surprisingly, is dominated by relativistic effects. For typical transmission electron microscope specimen thicknesses, surface coupling effects must also be considered.

Electron Microscopic Study Using Formalin-fixed, Paraffin-embedded Material, with Special Reference to Observation of Microbial Organisms and Endocrine Granules.

In the diagnostic pathology practice, specimens for electron microscopy (EM) are not necessarily handled and fixed under the ideal condition. In the present article, the author describes ultrastructural study using formalin-fixed and/or paraffin-embedded material. The fine morphologic preservation is often acceptable, particularly when small cubes are dug out of paraffin blocks. Particulate structures such as neuroendocrine granules and microbes are consistently observed even using paraffin sections. Paraffin sections signalized with silver particles in Grimelius and Grocott stains or diaminobenzidine products in immunostaining and in situ hybridization are applicable to EM evaluation by using a pre-embedding sequence. The practical merit includes the targeted approach: highly accurate sampling from focal lesions can be achieved for EM analysis, after observing hematoxylin and eosin-stained or specific-signalized paraffin-sections. This allows us pathologists a convenient and practical way for identifying focally infected pathogens, as well as a retrospective ultrastructural analysis of rare lesions long kept as a form of paraffin blocks.

Preparation of Tunable Microchips to Visualize Native Protein Complexes for Single-Particle Electron Microscopy.

Recent advances in technology have enabled single-particle electron microscopy (EM) to rapidly progress as a preferred tool to study protein assemblies. Newly developed materials and methods present viable alternatives to traditional EM specimen preparation. Improved lipid monolayer purification reagents offer considerable flexibility, while ultrathin silicon nitride films provide superior imaging properties to the structural study of protein complexes. Here, we describe the steps for combining monolayer purification with silicon nitride microchips to create a tunable approach for the EM community.

Fine electron biprism on a Si-on-insulator chip for off-axis electron holography

Off-axis electron holography allows both the amplitude and the phase shift of an electron wavefield propagating through a specimen in a transmission electron microscope to be recovered. The technique requires the use of an electron biprism to deflect an object wave and a reference wave to form an interference pattern. Here, we introduce an approach based on semiconductor processing technology to fabricate fine electron biprisms with rectangular cross-sections. By performing electrostatic calculations and preliminary experiments, we demonstrate that such biprisms promise improved performance for electron holography experiments.

Telocytes in the human sinoatrial node.

The sinoatrial node (SAN) is composed mostly of pacemaker, transitional and Purkinje‐like cells. Pacemaker cells, especially in the centre of the SAN, are surrounded by dense fibrous tissue and do not have any contact with transitional cells. We hypothesize that the SAN contains telocytes that have contacts with pacemaker cells and contractile myocardium. Immunohistochemistry using antibodies against HCN4 and antibody combinations against CD34 and HCN4 was carried out on 12 specimens. Confocal laser scanning microscopy (CLSM) with two mixtures of primary antibodies, namely CD34/S100 and vimentin/S100, was performed in three cases. In two cases, CLSM was carried out with CD117 antibody. Specimens for electron microscopy and immunocytochemistry with HCN4 immunogold labelling were taken from another three patients. In our study, we found cells with the immunophenotype of telocytes in the SAN. There were twice as many of these cells in the centre of the SAN as in the periphery (20.3 ± 4.8 versus 10.8 ± 4.4 per high‐power field). They had close contact with pacemaker cells and contractile cardiomyocytes and expressed HCN4. The ultrastructural characteristics of these cells are identical to those of telocytes observed earlier in other organs. Our study provides evidence that telocytes are present in the SAN.

PM-28Three-dimensional Trajectory Simulation of Scattered Electrons in Scanning Electron Microscope Specimen Chamber

PM-28Three-dimensional Trajectory Simulation of Scattered Electrons in Scanning Electron Microscope Specimen Chamber

Maximum usable thickness revisited: Imaging dislocations in Si by modern high-voltage scanning transmission electron microscopy

We have quantitatively evaluated the usable thickness of specimens in scanning transmission electron microscopy (STEM) at 1 MV using a wedge-shaped Si(110) single crystal including artificially introduced high-density dislocations. The width of dislocation images was employed as a criterion for the quantitative evaluation of usable thickness. Superior usable thickness in STEM than in TEM was found; the obtained results were 14.7 µm for STEM and 5.8 µm for TEM. In particular, in STEM, dislocations can be observed as thin lines with 10–15 nm width in the thickness range up to 10 µm. The latest high-voltage STEM is useful for imaging crystal defects in thick semiconductors.

Lanthanoid Staining as a Fast Technology of Preparing Microbiological Specimens for Scanning Electron Microscopy

Lanthanoid Staining as a Fast Technology of Preparing Microbiological Specimens for Scanning Electron Microscopy

Surface relaxation of strained Ga(P,As)/GaP heterostructures investigated by HAADF STEM.

The surfaces of thin transmission electron microscopy (TEM) specimens of strained heterostructures can relax. The resulting bending of the lattice planes significantly influences high-angle annular dark field (HAADF) measurements. We investigate the impact by evaluating the intensities measured at the atomic columns as well as their positions in high-resolution HAADF images. In addition, the consequences in the diffraction plane will be addressed by simulated position averaged convergent beam electron diffraction (PACBED) patterns. The experimental column intensities and positions acquired from a strained Ga(P,As) quantum well (QW) embedded in a in a GaP matrix agree very well with frozen phonon contrast simulations, if the surface relaxation is taken into account by finite element relaxation. Neglecting the surface relaxation the As content of the QW can be significantly underestimated. Taking the effects into account correctly, we find that the lower interface of the investigated Ga(P,As) QW is atomically abrupt whereas the upper one is smeared out.

Numerical modeling of specimen geometry for quantitative energy dispersive X-ray spectroscopy

Transmission electron microscopy specimens typically exhibit local distortion at thin foil edges, which can influence the absorption of X-rays for quantitative energy dispersive X-ray spectroscopy (EDS). Here, we report a numerical, three-dimensional approach to model the geometry of general specimens and its influence on quantification when using single and multiple detector configurations. As a function of specimen tilt, we show that the model correctly predicts the asymmetric nature of X-ray counts and ratios. When using a single detector, we show that complex specimen geometries can introduce significant uncertainty in EDS quantification. Further, we show that this uncertainty can be largely negated by collection with multiple detectors placed symmetrically about the sample such as the FEI Super-X configuration. Based on guidance provided by the model, we propose methods to reduce quantification error introduced by the sample shape. The source code is available at https://github.com/subangstrom/superAngle.

Quantification of Thin Specimens in a Scanning Transmission Electron Microscope at Low Accelerating Voltage using the f-ratio Method

Quantification of Thin Specimens in a Scanning Transmission Electron Microscope at Low Accelerating Voltage using the f-ratio Method

The Crystallographic and Electronic Phase Diagrams of Yttria-Stabilized Zirconia Model Electrolytes

Yttria-stabilized zirconia (YSZ) is known for its high ionic conductivity at elevated temperatures, which makes it ideal for usage as an oxygen ion conducting electrolyte. Thus, it is often utilized in chemical sensors or solid oxide fuel cells. In SOFCs, it is also employed as a component of the anode (a Ni/YSZ cermet). There, besides facilitating the transport of oxygen anions from the electrolyte to the three-phase boundary, it plays a role in the catalytic process involving the activation of various fuels fed to the SOFC. In both cases, the relevant properties – be it the ionic/electronic conductivity or the catalytic activity – are defined by the existence of oxygen vacancies and the electronic structure of the complex oxide. In order to investigate this systematically, thin film model systems with varying compositions, ranging from 3 mol% Y2O3 to 40 mol% Y2O3, were deposited by means of a home-built direct current ion beam sputter source.[1] This source allows for the preparation of thin films (approximately 25 nm) at a controlled growth rate, facilitating the epitaxial growth. As substrates, freshly-cleaved NaCl(001) single crystals were employed, which allowed the thin films to be used as unsupported transmission electron microscopy (TEM) specimens. Using selected area electron diffraction, the crystallography was investigated. However, since the tetragonal and cubic polymorphs contain lattice planes with the same spacings, a distinction is not trivial. Nevertheless, this can be circumvented by calculating the unit cell height, i.e. the lattice parameter c, which is equivalent for the tetragonal and cubic structures, and plotting it as a function of the yttria-content. There, a retention of the unit cell height between 8 and 20 mol% Y2O3 can be observed, which indicates that a phase transition from tetragonal to cubic is present between 8 and 20 mol% yttria as, due to the higher yttria concentration (with Y3+ being the larger ion than Zr4+), the unit cell volume must increase. Hence, the retention of the unit cell height can only be explained by an expansion in the lateral dimensions, i.e. the tetragonal cell becomes cubic.[1] In fact, using magnetron sputtering, the location of the phase transition could be narrowed down to be between 8 and 9.3(4) mol% Y2O3,[2] which is crucial knowledge if, for instance, the cubic structure for the electrolyte is desired. Very similar trends can be found for the direct (by means of valence EELS) and indirect band gaps (UPS), which show a decreasing trend as more Zr is substituted by Y – and, thus, as more oxygen vacancies are generated. However, at the phase transition, there is a discontinuity as the band gaps increase significantly. Furthermore, the electron affinity, i.e. the energy difference between the vacuum level and the conduction band minimum, is shown to follow the same function as well. This parameter exhibits another rare phenomenon: it is negative. Because of this, the vacuum level is within the band gap, which causes electrons that have been excited into the conduction band by, for instance, optical absorption, to be emitted with almost no barrier. This makes these materials also suitable as photocathodes, for example in spin-polarized electron microscopes. With Čerenkov emission spectroscopy, where the light emitted when an electron traverses the sample at a speed larger than the speed of light inside the medium is recorded, another electronic transition at slightly above 3 eV (also displaying the same trend as the lattice parameter as a function of the Y2O3content) was found. By comparison with densities of states from DFT calculations, this can be determined as being the excitation of electrons from the valence band maximum into the unoccupied oxygen vacancy-related gap states, i.e. these values depict the gap state level. With help of these investigations, not only can a crystallographic thin film phase diagram be constructed, but also an electronic structure phase diagram, which displays the relation of various electronic niveaus as a function of the Y2O3content in the YSZ thin films relevant for applications in SOFCs. [1] Götsch, T.; Wallisch, W.; Stöger-Pollach, M.; Klötzer, B.; Penner, S. AIP Adv. 2016, 6 (2),025119 [2] Götsch, T.; Schachinger, T.; Stöger-Pollach, M.; Kaindl, R.; Penner, S. Appl. Surf. Sci. 2017, 402,1–11 Figure 1: The unit cell height (top panel) as well as the band gaps (lower panel) show the same behavior as a function of the yttria content. Figure 1

The biological response chain to pollution: a case study from the "Italian Triangle of Death" assessed with the liverwort Lunularia cruciata.

The liverwort Lunularia cruciata, known for being a species tolerant to pollution able to colonize urban areas, was collected in the town of Acerra (South Italy) to investigate the biological effects of air pollution in one of the three vertices of the so-called Italian Triangle of Death. The ultrastructural damages observed by transmission electron microscopy in specimens collected in Acerra were compared with samples collected in the city center of Naples and in a small rural site far from sources of air pollution (Riccia, Molise, Southern Italy). The biological response chain to air pollution was investigated considering vitality, photosynthetic efficiency, heat shock protein 70 (Hsp70) induction and gene expression levels, and chlorophyll degradation and related ultrastructural alterations. Particularly, a significant increment in Hsp70 expression and occurrence, and modifications in the chloroplasts' ultrastructure can be strictly related to the environmental pollution conditions in the three sites. The results could be interpreted in relation to the use of these parameters as biomarkers for environmental pollution.

Influence of surface relaxation of strained layers on atomic resolution ADF imaging

Surface relaxation of thin transmission electron microscopy (TEM) specimens of strained layers results in a severe bending of lattice planes. This bending significantly displaces atoms from their ideal channeling positions which has a strong impact on the measured annular dark field (ADF) intensity.With the example of GaAs quantum wells (QW) embedded in a GaP barrier, we model the resulting displacements by elastic theory using the finite element (FE) formalism. Relaxed and unrelaxed super cells served as input for state of the art frozen phonon simulation of atomic resolution ADF images.We systematically investigate the dependencies on the sample´s geometric parameters, i.e. QW width and TEM sample thickness, by evaluating the simulated intensities at the atomic column´s positions as well as at the background positions in between. Depending on the geometry the ADF intensity can be affected in a range several nm from the actual interface.Moreover, we investigate the influence of the surface relaxation on the angular distribution of the scattered intensity. At high scattering angles we observe an intensity reduction at the interface as well as in the GaP barrier due to de-channeling. The amount of intensity reduction at an atomic column is directly proportional to its mean square displacement. On the contrary we find a clearly increased intensity at low angles caused by additional diffuse scattering. We discuss the implications for quantitative evaluations as well as strategies to compensate for the reduced intensities.