Over the last few years, TEM has become a powerful technique to study cracks and specially crack tips. However, the number of publications including TEM results has not grown as it was expected. The main reason for this might be difficulties in the sample preparation. In this work we present a novel FIB sample preparation technique which has proved to be an ideal tool for preparing cross sectional samples containing crack tips. The morphology of intergranular stress corrosion cracking (IGSCC) has been investigated in Alloy 600 subjected to constant load and slow strain rate tests in simulated primary circuit pressurized water reactor conditions. Cracks were observed to nucleate at high-angle grain boundaries and propagate to depths of a few tens of micrometer along such boundaries, still in the initiation stage. Electron diffraction, energy dispersive x-ray (EDX) and electron energy loss spectroscopy (EELS) have been used to identify the different corrosion products and precipitates. Elemental mapping was employed to reveal changes in composition in the crack tip area. Major observations at cracks and grain boundaries include: the presence of different oxides in different locations, differences in grain boundary oxides and open crack/free surface oxides. These observations suggest that IGSCC involves oxygen diffusion through a porous oxide region along grain boundaries to the bare metal. This is a novel concept that offers an alternative to previous mechanisms proposed in the literature e.g. H embrittlement, slip-dissolution, etc., for which no supporting evidence has been found.

Most living eukaryotes are currently classified into one of five or six ‘supergroups’, which are in turn often divided between two assemblages: ‘unikonts’ and ‘bikonts’. This thesis explores the cytoskeletal morphology and phylogeny of three lineages that do not belong to any supergroup: ancyromonads, apusomonads, and breviates, likely relatives of supergroups Opisthokonta and Amoebozoa. It also investigates the phylogeny of malawimonads (basal members of supergroup Excavata) and collodictyonids (another unaffiliated lineage). Serial-section transmission electron microscopy was used to model the flagellar apparatus cytoskeletons of the ancyromonad Ancyromonas sigmoides, the breviate Breviata anathema, and the apusomonad Thecamonas trahens. Each has two main posterior microtubular roots and at least one anterior root (two in Ancyromonas). All three possess splitting posterior right microtubular roots and supernumerary singlets, features also characteristic of basal members of the supergroup Excavata (‘typical excavates’). One peripheral microtubule system in Ancyromonas, and the ‘right ribbon’ in Thecamonas, are likely homologous to dorsal fans in Breviata and ‘typical excavates’, and to the ‘r2’ root of myxogastrid Amoebozoa. One of the branches of the splitting root in Breviata and Thecamonas joins the right and intermediate roots, similarly to some myxogastrids. This implies that myxogastrids, and not the simpler pelobionts, represent the ancestral state for Amoebozoa. A phylogenomic analysis was performed focussing on apusomonads breviates, ancyromonads, and the problematic ‘typical excavate’ malawimonads, based on new transcriptomic data from Ancyromonas and an undescribed malawimonad. Rapid-site- removal analyses recover the ‘unikont’/‘bikont’ partition, and do not support the previously demonstrated affiliation between breviates and the ‘unikont’ supergroup Amoebozoa. Specifically, they group apusomonads with the ‘unikont’ supergroup Opisthokonta, and ancyromonads with breviates. Taxon-removal analyses group ancyromonads, breviates, and apusomonads together. Most analyses group malawimonads (perhaps with collodictyonids, another problematic group) between ‘unikonts’ and (other) ‘bikonts’, while other excavates are in a basal position amongst other ‘bikonts’. Combining these morphological and phylogenetic results suggests that splitting right roots, supernumerary intermediate singlets, and dorsal fans are found in multiple ‘basal’ lineages in both ‘unikont’ and ‘bikont’ portions of the eukaryotic tree, are likely characters of the last common ancestor of most or all living eukaryotes.

Leukocytes play a central role in protecting the body against infectious organisms and their research is essential for understanding the mechanisms of immunity. By studying leukocytes across a range of species, insights are provided into differing strategies employed to ensure resistance to disease. Surprisingly, the structure and function of marsupial leukocytes has received very limited study. Marsupials represent a major evolutionary pathway with distinct differences in reproduction and development from placental mammals. These differences in the life history of marsupials place unique challenges on the immune system, and differences in leukocyte structure and function could be reasonably expected. In this thesis, studies were undertaken to examine the cytochemical, ultrastructural and functional features of leukocytes from species of marsupials, belonging to the family Macropodidae (kangaroos and wallabies). The aim of these studies was to elucidate the characteristics of macropodid leukocytes and to compare and contrast these features with the known characteristics of other mammalian leukocytes. Leukocytes from two species of macropodid, the tammar wallaby (Macropus eugenii) and the western grey kangaroo (Macropus fuliginosis), formed the basis of this study with additional material provided from quokka (Setonix brachyurus), woylie (Bettongia pencillata) and red kangaroo (Macropus rufus). Staining characteristics of cells were examined following reaction with Sudan black B, peroxidase, chloroacetate esterase, naphthyl butyrate esterase, alkaline phosphatase and periodic acid-Schiff. Peroxidase and Sudan Black B reactions were similar to domestic animal species but chloroacetate esterase and naphthyl butyrate esterase were unreliable as markers for macropodid neutrophils and monocytes, respectively. Significant variation in staining for alkaline phosphatase was seen between species of macropodid. Tammar wallabies and quokka demonstrated strong neutrophil alkaline phosphatase activity whereas western grey kangaroos, red kangaroos and woylies contained no activity within their leukocytes. Peroxidase and alkaline phosphatase cytochemistry were also assessed at the ultrastructural level with transmission electron microscopy. This allowed the identification of distinct granule populations within macropodid neutrophils. Two subcellular compartments containing alkaline phosphatase activity were identified within tammar wallaby neutrophils. These were considered equivalent to secretory vesicles and a subpopulation of specific granules. Tubular vesicles containing alkaline phosphatase were also identified within the eosinophils of tammar wallabies. These structures were a novel finding having not been reported previously in the eosinophils of other animal species. In addition to cytochemistry, the general ultrastructure of leukocytes from tammar wallabies and western grey kangaroos were reported. Results were similar to previous reports for other marsupial species. The current body of knowledge was extended by the first detailed description of the ultrastructure of basophils in a marsupial. To assess functional aspects of macropdid neutrophils, flow cytometric assays were performed examining oxidative burst responses and phagocytosis. Reactive oxygen species were generated by neutrophils from tammar wallabies and western grey kangaroos in response to phorbol 12-myristate 13-acetate but not N-formyl-Met-Leu-Phe or opsonised bacteria. Phagocytosis of opsonised bacteria was also measured in neutrophils from tammar wallabies, which was poor in contrast to ovine neutrophils. However, flow cytometric studies were limited by sample preparation. Further optimisation of isolation methods for tammar wallaby leukocytes should be undertaken before dogmatic conclusions are drawn. Overall, the results of this thesis demonstrate that, in the areas examined, the general characteristics of leukocyte structure and function of mammals are present in macropodids. However differences were identified both within and outside of the macropodid group. These differences have important ramifications for the use of ‘model’ species in the study of leukocyte biology in marsupials. The results also provide potentially useful tools for the clinical diagnosis of haematological disease in macropodids and may be of interest to those studying comparative and evolutionary aspects of leukocyte structure and function.

Thesis (M.S.)--Ball State University, 2009. / Title from PDF t.p. (viewed on Apr. 16, 2010). Includes bibliographical references (p. 36-37).

Chronic kidney disease (CKD) and end stage renal disease (ESRD) are significant causes of adult morbidity and mortality worldwide. Though these conditions are common, the mechanisms of pathogenesis in kidney disease are poorly understood. Genetic predisposition has been established in the African American population; however this does not explain the ubiquity of CKD in the United States and abroad. Diabetes and hypertension are the two most frequently occurring co-morbidities in kidney disease and both have been identified as putative sources of injury to the delicate filtering structures of the kidney. Furthermore, the intrinsic functional relationship between the cardiovascular and renal organ systems adds to the plausibility of a hemodynamic cause. In light of this knowledge, we aim to explore the roles of genetic predisposition and hypertension in the pathogenesis and progression of CKD. The filtering apparatus of the kidney, the glomerulus, is a looping tuft of capillaries specialized to allow the passage of water and certain substances from the blood while restricting others. Glomeruli at the corticomedullary boundary of the kidney experience blood pressures closer to those in systemic arterioles and are subject to similar hemodynamic stresses. To evaluate the role of hypertension in CKD, we employed a well-known model of hypertensive kidney disease in mice involving uninephrectomy (UNX), subcutaneous implantation of a timed-release pellet containing the active aldosterone precursor deoxycorticosterone acetate (DOCA), and a high-salt diet. Given the role of heritability in human CKD pathogenesis, we applied the DOCA-UNX model in two strains of mice with differing susceptibility to kidney damage, the 129S6 and C57BL/6 strains, to evaluate the effects of genetic predisposition. Mice were subjected to varying lengths of hypertension exposure and their kidneys were subsequently examined by transmission electron microscopy (TEM). Ultrastructural lesions of glomeruli were evaluated by a renal pathologist and assigned subjective pathology scores based on the extent and severity of involvement. We hypothesized that certain glomerular lesions, particularly those involving the podocytes of the visceral epithelium, would increase in severity in mice with heritable susceptibility (129S6) as well as those with longer exposure to glomerular hypertension. Our observations demonstrate these hypotheses are partially correct. By TEM histopathology, mouse strain was found to have a significant effect on the severity of certain epithelial lesions while duration of hypertension had a significant effect on the overall morphological pathology of the podocytes, glomerular basement membrane, and glomerulus as a whole. These results provide a promising foundation for further investigation of the pathogenesis of CKD in mice.

Developments in scanning transmission electron microscopy (STEM) have opened up new possibilities for time-resolved imaging at the atomic scale. However, rapid imaging of single atom dynamics brings with it a new set of challenges, particularly regarding noise and the interaction between the electron beam and the specimen. This thesis develops a set of analytical tools for capturing atomic motion and analyzing the dynamic behaviour of materials at the atomic scale. Machine learning is increasingly playing an important role in the analysis of electron microscopy data. In this light, new unsupervised learning tools are developed here for noise removal under low-dose imaging conditions and for identifying the motion of surface atoms. The scope for real-time processing and analysis is also explored, which is of rising importance as electron microscopy datasets grow in size and complexity. These advances in image processing and analysis are combined with computational modelling to uncover new chemical and physical insights into the motion of atoms adsorbed onto surfaces. Of particular interest are systems for heterogeneous catalysis, where the catalytic activity can depend intimately on the atomic environment. The study of Cu atoms on a graphene oxide support reveals that the atoms undergo anomalous diffusion as a result of spatial and energetic disorder present in the substrate. The investigation is extended to examine the structure and stability of small Cu clusters on graphene oxide, with atomistic modelling used to understand the significant role played by the substrate. Finally, the analytical methods are used to study the surface reconstruction of silicon alongside the electron beam-induced motion of adatoms on the surface. Taken together, these studies demonstrate the materials insights that can be obtained with time-resolved STEM imaging, and highlight the importance of combining state-ofthe- art imaging with computational analysis and atomistic modelling to quantitatively characterize the behaviour of materials with atomic resolution.

Metallophthalocyanine-single walled carbon nanotube conjugates were successfully synthesized and applied in the electrochemical characterizations of pesticides (amitrole and diuron) and 2-mercaptoethanol (2-ME). The formation of conjugates was confirmed through the use of the following analytical techniques: UV-vis, FTIR, Raman and XRD spectroscopies, atomic force and transmission electron microscopies and voltammetry. Chemically linking SWCNT to MPcs created platforms that offered efficient transfer of electrons and this was confirmed through electrochemical impedance studies (EIS) and voltammetry as shown by lower ΔEp values observed in conjugates. Carboxy carrying MPcs have very poor electron transfer kinetics (both tetrasubstituted and low symmetry), but the presence of SWCNTs activates their catalysis. All electrochemical studies were done at pH 4. Cyclic voltammetry, rotating disk linear sweep voltammetry, chronoamperometry and EIS were used in the electrochemical characterization of 2-ME and the pesticides on poly-Ni(OH)TAPc and MPc-SWCNT modified glassy carbon electrodes (GCEs). High Tafel slopes were observed for the pesticides relative to 2-ME, an indication of the passivating nature of their oxidation products. However, conjugates showed very high resistances to passivation and were easily regenerated by shaking in methanol. Improved catalysis of the conjugates is also indicated by the high catalytic rate constants for the analytes, observed on these electrodes. Conjugates of low symmetry MPcs with SWCNTs gave the highest catalytic rate constants, confirming better catalysis on these electrode surfaces. The nature of SWCNT functionalization also affected catalysis, with amine functionalized SWCNTs inducing better catalytic properties into the MPcs than carboxylic acid terminated CNTs. The presence of amine functionalized SWCNTs activates the catalysis of non-catalytic carboxy-carrying MPcs and this is more pronounced in conjugates of tetrasubstituted MPcs relative to those of low symmetry Pcs. Ethylene amine (EA) functionalized SWCNTs reduced redox overpotentials of the MPcs more than the phenyl-amine (PA) functionalized counterparts. Poly-NiTAPc was successfully converted to poly-Ni(OH)TAPc through cyclisation in pH 4 buffer and showed very good catalytic properties towards diuron, relative to the former.

abstract: Because of their favorable ionic and/or electronic conductivity, non-stoichiometric oxides are utilized for energy storage, energy conversion, sensing, catalysis, gas separation, and information technologies, both potential and commercialized. Charge transport in these materials is influenced strongly by grain boundaries, which exhibit fluctuations in composition, chemistry and atomic structure within Ångstroms or nanometers. Here, studies are presented that elucidate the interplay between macroscopic electrical conductivity, microscopic character, and local composition and electronic structure of grain boundaries in polycrystalline ceria-based (CeO2) solid solutions. AC impedance spectroscopy is employed to measure macroscopic electrical conductivity of grain boundaries, and electron energy-loss spectroscopy (EELS) in the aberration-correction scanning transmission electron microscope (AC-STEM) is used to quantify local composition and electronic structure. Electron diffraction orientation imaging microscopy is employed to assess microscopic grain boundary character, and links these macro- and nanoscopic techniques across length scales. A model system, CaxCe1-xO2-x-δ, is used to systematically investigate relationships between nominal Ca2+ concentration, grain boundary ionic conductivity, microscale character, and local solute concentration. Grain boundary conductivity varied by several orders of magnitude over the composition range, and assessment of grain boundary character highlighted the critical influence of local composition on conductivity. Ceria containing Gd3+ and Pr3+/4+ was also investigated following previous theoretical work predicting superior ionic conductivity relative to state-of-the-art GdxCe1-xO2-x/2-δ. The grain boundary conductivity was nearly 100 times greater than expected and a factor four enrichment of Pr concentration was observed at the grain boundary, which suggested electronic conduction that was cited as the origin of the enhanced conductivity. This finding inspired the development of two EELS-based experimental approaches to elucidate the effect of Pr enrichment on grain boundary conductivity. One employed ultra-high energy resolution (~10 meV) monochromated EELS to characterize Pr inter-bandgap electronic states. Alternatively, STEM nanodiffraction orientation imaging coupled with AC-STEM EELS was employed to estimate the composition of the entire grain boundary population in a polycrystalline material. These compositional data were the input to a thermodynamic model used to predict electrical properties of the grain boundary population. These results suggest improved DC ionic conduction and enhanced electronic conduction under AC conditions. / Dissertation/Thesis / Doctoral Dissertation Materials Science and Engineering 2016

abstract: Nanocrystalline (nc) thin films exhibit a wide range of enhanced mechanical properties compared to their coarse-grained counterparts. Furthermore, the mechanical behavior and microstructure of nc films is intimately related. Thus, precise control of the size, aspect ratio and spatial distribution of grains can enable the synthesis of thin films with exceptional mechanical properties. However, conventional bottom-up techniques for synthesizing thin films are incapable of achieving the microstructural control required to explicitly tune their properties. This dissertation focuses on developing a novel technique to synthesize metallic alloy thin films with precisely controlled microstructures and subsequently characterizing their mechanical properties using in situ transmission electron microscopy (TEM). Control over the grain size and distribution was achieved by controlling the recrystallization process of amorphous films by the use of thin crystalline seed layers. The novel technique was used to manipulate the microstructure of structural (TiAl) and functional (NiTi) thin films thereby exhibiting its capability and versatility. Following the synthesis of thin films with tailored microstructures, in situ TEM techniques were employed to probe their mechanical properties. Firstly, a novel technique was developed to measure local atomic level elastic strains in metallic glass thin films during in situ TEM straining. This technique was used to detect structural changes and anelastic deformation in metallic glass thin films. Finally, as the electron beam (e-beam) in TEMs is known to cause radiation damage to specimen, systematic experiments were carried out to quantify the effect of the e-beam on the stress-strain response of nc metals. Experiments conducted on Al and Au films revealed that the e-beam enhances dislocation activity leading to stress relaxation. / Dissertation/Thesis / Supplementary Video S1 / Supplementary Video S2 / Supplementary Video S3 / Doctoral Dissertation Materials Science and Engineering 2017

Tomography of crystal defects in the electron microscope was first attempted in 2005 by the author and colleagues. This thesis further develops the technique, using a variety of samples and methods. Use of a more optimised, commercial tomographic reconstruction program on the original GaN weak beam dark-field (WBDF) tilt series gave a finer reconstruction with lower background, line width 10-20 nm. Four WBDF tilt series were obtained of a microcrack surrounded by dislocations in a sample of indented silicon, tilt axes parallel to g = 220, 220, 400 and 040. Moiré fringes in the defect impaired alignment and reconstruction. The effect on reconstruction of moiré fringe motion with tilt was simulated, resulting in an array of rods, not a flat plane. Dislocations in a TiAl alloy were reconstructed from WBDF images with no thickness contours, giving an exceptionally clear reconstruction. The effect of misalignment of the tilt axis with systematic row g(ng) was assessed by simulating tilt series with diffraction condition variation across the tilt range of Δn = 0, 1 and 2. Misalignment changed the inclination of the reconstructed dislocation with the foil surfaces, and elongated the reconstruction in the foil normal direction; this may explain elongation additional to the missing wedge effect in experiments. Tomography from annular dark-field (ADF) STEM dislocation images was also attempted. A tilt series was obtained from the GaN sample; the reconstructed dislocations had a core of bright intensity of comparable width to WBDF reconstructions, with a surrounding region of low intensity to 60 nm width. An ADF STEM reconstruction was obtained from the Si sample at the same microcrack as for WBDF; here automatic specimen drift correction in tomography acquisition software succeeded, a significant improvement. The microcrack surfaces in Si reconstructed as faint planes and dislocations were recovered as less fragmented lines than from the WBDF reconstruction. ADF STEM tomography was also carried out on the TiAl sample, using a detector inner angle (βin) that included the first order Bragg spots (in other series βin had been 4-6θ B). Extinctions occurred which were dependent on tilt; this produced only weak lines in the reconstruction. Bragg scattering in the ADF STEM image was estimated by summing simulated dark-field dislocation images from all Bragg beams at a zone axis; a double line was produced. It was hypothised that choosing the inner detector angle to omit these first Bragg peaks may preclude most dynamical image features. Additional thermal diffuse scattering (TDS) intensity due to dilatation around an edge dislocation was estimated and found to be insignificant. The Huang scattering cross section was estimated and found to be 9Å, ten times thinner than experimental ADF STEM dislocation images. The remaining intensity may be from changes to TDS from Bloch wave transitions at the dislocation; assessing this as a function of tilt is for further work. On simple assessment, only three possible axial channeling orientations were found over the tilt range for GaN; if this is typical, dechanneling contrast probably does not apply to defect tomography.