Beyond simple imaging with energetic beams – nanopatterning, correlative microscopy and statistical analysis of inclusions

Electron microscopy (EM) has advanced in an exponential way since the first transmission electron microscope (TEM) was built in the 1930’s. The urge to ‘see’ things is an essential part of human nature (talk of ‘seeing is believing’) and apart from scanning tunnel microscopes which give information about the surface, EM is the only imaging technology capable of really visualising atomic structures in depth down to single atoms. With the development of nanotechnology the demand to image and analyse small things has become even greater and electron microscopes have found their way from highly delicate and sophisticated research grade instruments to key-turn and even bench-top instruments for everyday use in every materials research lab on the planet. The semiconductor industry is as dependent on the use of EM as life sciences and pharmaceutical industry. With this generalisation of use for imaging, the need to deploy advanced uses of EM has become more and more apparent. The combination of several coinciding beams (electron, ion and even light) to create DualBeam or TripleBeam instruments for instance enhances the usefulness from pure imaging to manipulating on the nanoscale. And when it comes to the analytic power of EM with the many ways the highly energetic electrons and ions interact with the matter in the specimen there is a plethora of niches which evolved during the last two decades, specialising in every kind of analysis that can be thought of and combined with EM. In the course of this study the emphasis was placed on the application of these advanced analytical EM techniques in the context of multiscale and multimodal microscopy – multiscale meaning across length scales from micrometres or larger to nanometres, multimodal meaning numerous techniques applied to the same sample volume in a correlative manner. In order to demonstrate the breadth and potential of the multiscale and multimodal concept an integration of it was attempted in two areas: I) Biocompatible materials using polycrystalline stainless steel and II) Semiconductors using thin multiferroic films. I) The motivation to use stainless steel (316L medical grade) comes from the potential modulation of endothelial cell growth which can have a big impact on the improvement of cardio-vascular stents – which are mainly made of 316L – through nano-texturing of the stent surface by focused ion beam (FIB) lithography. Patterning with FIB has never been reported before in connection with stents and cell growth and in order to gain a better understanding of the beam-substrate interaction during patterning a correlative microscopy approach was used to illuminate the patterning process from many possible angles. Electron backscattering diffraction (EBSD) was used to analyse the crystallographic structure, FIB was used for the patterning and simultaneously visualising the crystal structure as part of the monitoring process, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were employed to analyse the topography and the final step being 3D visualisation through serial FIB/SEM sectioning. II) The motivation for the use of thin multiferroic films stems from the ever-growing demand for increased data storage at lesser and lesser energy consumption. The Aurivillius phase material used in this study has a high potential in this area. Yet it is necessary to show clearly that the film is really multiferroic and no second phase inclusions are present even at very low concentrations – ~0.1vol% could already be problematic. Thus, in this study a technique was developed to analyse ultra-low density inclusions in thin multiferroic films down to concentrations of 0.01%. The goal achieved was a complete structural and compositional analysis of the films which required identification of second phase inclusions (through elemental analysis EDX(Energy Dispersive X-ray)), localise them (employing 72 hour EDX mapping in the SEM), isolate them for the TEM (using FIB) and give an upper confidence limit of 99.5% to the influence of the inclusions on the magnetic behaviour of the main phase (statistical analysis).

The five-parameter grain boundary character and energy distributions of a fully austenitic high-manganese steel using three dimensional data

The three-dimensional interfacial grain boundary network in a fully austenitic high-manganese steel was studied as a function of all five macroscopic crystallographic parameters (i.e. lattice misorientation and grain boundary plane normal) using electron backscattering diffraction mapping in conjunction with focused ion beam serial sectioning. The relative grain boundary area and energy distributions were strongly influenced by both the grain boundary plane orientation and the lattice misorientation. Grain boundaries terminated by (1 1 1) plane orientations revealed relatively higher populations and lower energies compared with other boundaries. The most frequently observed grain boundaries were {1 1 1} symmetric twist boundaries with the Σ3 misorientation, which also had the lowest energy. On average, the relative areas of different grain boundary types were inversely correlated to their energies. A comparison between the current result and previously reported observations (e.g. high-purity Ni) revealed that polycrystals with the same atomic structure (e.g. face-centered cubic) have very similar grain boundary character and energy distributions. © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

The Developing Left Superior Cervical Ganglion of Pacas (Agouti Paca)

In this study the main question investigated was the number and size of both binucleate and mononucleate superior cervical ganglion (SCG) neurons and, whether post-natal development would affect these parameters. Twenty left SCGs from 20 male pacas were used. Four different ages were investigated, that is newborn (4 days), young (45 days), adult (2 years), and aged animals (7 years). By using design-based stereo-logical methods, that is the Cavalieri principle and a physical disector combined with serial sectioning, the total volume of ganglion and total number of mononucleate and binucleate neurons were estimated. Furthermore, the mean perikaryal (somal) volume of mononucleate and binucleate neurons was estimated using the vertical nucleator. The main findings of this study were a 154% increase in the SCG volume, a 95% increase in the total number of mononucleate SCG neurons and a 50% increase in the total volume of SCG neurons. In conclusion, apart from neuron number, different adaptive mechanisms may coexist in the autonomic nervous system to guarantee a functional homeostasis during ageing, which is not always associated with neuron losses. Anat Rec, 292:966-975, 2009. (C) 2009 Wiley-Liss, Inc.

Three-Dimensional Characterization of Pores in Ti-6Al-4V Alloy

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Female reproductive system of the decapitating fly Pseudacteon wasmanni Schmitz (Diptera : Phoridae)

Pseudacteon wasmanni is a South American decapitating fly that parasitizes workers of Solenopsis fire ants. We used light microscopy (historesin serial-sectioning stained with Haematoxylin/Eosin) and scanning electron microscopy to show and analyze internal and whole external views of the female reproductive system. All specimens analyzed (n = 9) by light microscopy showed post-vitellogenic oocytes inside the ovaries. The lack of typical follicles (oocyte-nurse cell complexes) in all specimens suggests that oogenesis occurs during the pupal stage. The total number of eggs found ranged from 31 to 280 (X = 142 +/- 73, SD). The egg has a slugform or torpedo shape (about 130 by 20 mum) with a pointed apex at the posterior pole as defined by the fly; the micropyle appears to be in a depression or invagination at the anterior pole. An acute hypodermic-like ovipositor is evaginated from the hard sclerotized external genitalia during egg laying. The existence of a muscular bulb associated with the end of the common oviduct suggests that the egg is injected into the ant's body by a strong contraction of the bulb which probably is stimulated by bending of several ventral sensilla. During contraction, the abdomen extends out along a large fold between the sixth and seventh tergites in such a way that the sclerotized genitalia is rotated ventrally into a slightly anterior orientation in preparation for oviposition. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Metástases de carcinoma de mama em cadelas: avaliação da técnica de linfonodo sentinela

Pós-graduação em Medicina Veterinária - FMVZ

Three dimensional (3D) microstructure-based modeling of interfacial decohesion in particle reinforced metal matrix composites

Modeling and prediction of the overall elastic–plastic response and local damage mechanisms in heterogeneous materials, in particular particle reinforced composites, is a very complex problem. Microstructural complexities such as the inhomogeneous spatial distribution of particles, irregular morphology of the particles, and anisotropy in particle orientation after secondary processing, such as extrusion, significantly affect deformation behavior. We have studied the effect of particle/matrix interface debonding in SiC particle reinforced Al alloy matrix composites with (a) actual microstructure consisting of angular SiC particles and (b) idealized ellipsoidal SiC particles. Tensile deformation in SiC particle reinforced Al matrix composites was modeled using actual microstructures reconstructed from serial sectioning approach. Interfacial debonding was modeled using user-defined cohesive zone elements. Modeling with the actual microstructure (versus idealized ellipsoids) has a significant influence on: (a) localized stresses and strains in particle and matrix, and (b) far-field strain at which localized debonding takes place. The angular particles exhibited higher degree of load transfer and are more sensitive to interfacial debonding. Larger decreases in stress are observed in the angular particles, because of the flat surfaces, normal to the loading axis, which bear load. Furthermore, simplification of particle morphology may lead to erroneous results.

Nonmuscle myosin II-B is required for normal development of the mouse heart

We used targeted gene disruption in mice to ablate nonmuscle myosin heavy chain B (NMHC-B), one of the two isoforms of nonmuscle myosin II present in all vertebrate cells. Approximately 65% of the NMHC-B−/− embryos died prior to birth, and those that were born suffered from congestive heart failure and died during the first day. No abnormalities were detected in NMHC-B+/− mice. The absence of NMHC-B resulted in a significant increase in the transverse diameters of the cardiac myocytes from 7.8 ± 1.8 μm (right ventricle) and 7.8 ± 1.3 μm (left ventricle) in NMHC-B+/+ and B+/− mice to 14.7 ± 1.1 μm and 13.8 ± 2.3 μm, respectively, in NMHC-B−/− mice (in both cases, P < 0.001). The increase in size of the cardiac myocytes was seen as early as embryonic day 12.5 (4.5 ± 0.2 μm for NMHC-B+/+ and B+/− vs. 7.2 ± 0.6 μm for NMHC-B−/− mice (P < 0.01)). Six of seven NMHC-B−/− newborn mice analyzed by serial sectioning also showed structural cardiac defects, including a ventricular septal defect, an aortic root that either straddled the defect or originated from the right ventricle, and muscular obstruction to right ventricular outflow. Some of the hearts of NMHC-B−/− mice showed evidence for up-regulation of NMHC-A protein. These studies suggest that nonmuscle myosin II-B is required for normal cardiac myocyte development and that its absence results in structural defects resembling, in part, two common human congenital heart diseases, tetralogy of Fallot and double outlet right ventricle.

cut11+: A Gene Required for Cell Cycle-dependent Spindle Pole Body Anchoring in the Nuclear Envelope and Bipolar Spindle Formation in Schizosaccharomyces pombe

The “cut” mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11+ suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11+ encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.

As-cast morphology of iron-intermetallics in Al-Si foundry alloys

The as-cast three-dimensional morphologies of alpha-Al-15(Fe,Mn)(3)Si-2 and beta-Al5FeSi intermetallics were investigated by serial sectioning. Large beta-Al5FeSi intermetallics were observed to grow around pre-existing dendrite arms. The alpha-Al-15(Fe,Mn)(3)Si-2 intermetallic particle was observed to have a central polyhedral particle and an external highly convoluted three-dimensional structure. (c) 2005 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Three-dimensional analysis of eutectic grains in hypoeutectic Al-Si alloys

An approach to the qualitative analysis of quenched microstructures in three dimensions is presented and demonstrated on unmodified and Sr-modified Al-10% Si samples. The samples were repeatedly polished to obtain a series of digital images through the depth of the microstructure. A three-dimensional reconstruction of the microstructure was obtained by assembling the images of the serial sections. Reconstructions were made of unmodified and Sr-modified Al-Si eutectic grains that were quenched during eutectic solidification. The three-dimensional reconstructions show that strontium modification changes the size and morphology of the Al-Si eutectic grains. Sr-modified eutectic grains are large approximately spherical grains and grow with a high interface velocity. In the unmodified alloy, many small eutectic grains grow from the dendrite arm tips. The unmodified eutectic grains appear to grow from the dendrite tips into the undercooled liquid, rather than back-filling the dendrite envelope, possibly continuing to grow in the same manner as the equiaxed dendrites. (C) 2004 Elsevier B.V. All rights reserved.

Penetration of chlorhexidine into human skin

This study evaluated a model of skin permeation to determine the depth of delivery of chlorhexidine into full-thickness excised human skin following topical application of 2% (wt/vol) aqueous chlorhexidine digluconate. Skin permeation studies were performed on full-thickness human skin using Franz diffusion cells with exposure to chlorhexidine for 2 min, 30 min, and 24 h. The concentration of chlorhexidine extracted from skin sections was determined to a depth of 1,500 µm following serial sectioning of the skin using a microtome and analysis by high-performance liquid chromatography. Poor penetration of chlorhexidine into skin following 2-min and 30-min exposures to chlorhexidine was observed (0.157 ± 0.047 and 0.077 ± 0.015 µg/mg tissue within the top 100 µm), and levels of chlorhexidine were minimal at deeper skin depths (less than 0.002 µg/mg tissue below 300 µm). After 24 h of exposure, there was more chlorhexidine within the upper 100-µm sections (7.88 ± 1.37 µg/mg tissue); however, the levels remained low (less than 1 µg/mg tissue) at depths below 300 µm. There was no detectable penetration through the full-thickness skin. The model presented in this study can be used to assess the permeation of antiseptic agents through various layers of skin in vitro. Aqueous chlorhexidine demonstrated poor permeation into the deeper layers of the skin, which may restrict the efficacy of skin antisepsis with this agent. This study lays the foundation for further research in adopting alternative strategies for enhanced skin antisepsis in clinical practice.

Studies of skin antisepsis and enhanced penetration of chlorhexidine

Current evidence-based guidelines recommend that 2% (w/v) chlorhexidine digluconate (CHG), preferentially in 70% (v/v) isopropyl alcohol (IIPA), is used for skin antisepsis prior to incision of the skin. In this current study, the antimicrobial efficacy of CHG, six essential oils [tea tree oil (TTO), thymol, eucalyptus oil (EO), juniper oil, lavender oil and citronella] and novel benzylidenecarboxamidrazone and thiosemicarbazone compounds were determined against a panel of microorganisms commonly associated with skin infection (Staphylococcus epidermidis, S. aureus, meticillin-resistant S. aureus, Propionibacterium acnes, Acinetobacter spp., Pseudomonas aeruginosa and Candida albicans) The results demonstrated synergistic activity of CHG in combination with EO against biofilm cultures of S. epidermidis, with significantly reduced concentrations of CHG and EO required to inhibit biofilm growth compared to CHG or EO alone. Skin permeation of CHG was subsequently investigated using an in vitro human skin model (Franz cell) and the penetration profile was determined by serial sectioning of the full thickness human skin. Two percent (w/v) CHG in aqueous solution and in 70% (v/v) IPA demonstrated poor skin permeation; however, the skin permeation was significantly enhanced in combination with 5% - 50% (v/v) EO. Detectable levels of CHG did not permeate through full thickness skin in 24 h. Skin permeation of 2% (w/v) CHG in 70% (v/v) IPA in the presence of 10% (v/v) EO was subsequently studied. The results demonstrated a significantly enhanced skin penetration of CHG after a 2 min application, with CHG detected at significant levels to a depth of 600 m with CHG in combination with EO and IPA compared to 100 m with IPA alone. Combination antisepsis comprising CHG and EO may be beneficial for skin antisepsis prior to invasive procedures to reduce the number of microorganisms on and within the skin due to enhanced skin penetration of CHG and improved efficacy against S. epidermidis in a biofilm mode of growth.

Microscopic anatomy of the temporal bone; a photographic survey of serial sections of the temporal bone cut in the three routine planes of sectioning human specimens,

1971 ed. published under title: Surgical and microscopic anatomy of the temporal bone.