Effective Impedance Method for In situ Ellipsometry Analysis of Magnetic Films

The method of effective surface impedance is proposed and applied for in situ characterisation of magnetic structures. For any ellipsometry investigations a proper choice of a physical model is important for solving the inverse problem. Reasonable approximations used for in situ ellipsometry monitoring are assumptions of a constant rate of layer growth and stable optical parameters. Standard ellipsometry analysis requires the model response to be calculated from every layer in the structure. Errors from underlying layers propagate through the entire structure and accumulate. In this case a method of a pseudosubstrate is used which approximates the underlying structure as a single interface (so called virtual interface), rather than tracking the entire sample history. The virtual interface is placed at some level and growth is modelled on this interface with no knowledge retained for the underlying structure. There are various methods for describing the virtual interface. In this paper, the concept of the effective impedance is used which requires only three measurement data points and is convenient for combined investigation of optical and magneto-optical properties. The algorithm is based on the calculation of the characteristic matrix of the layer (Abeles matrix) and surface impedance of the virtual interface using two ellipsometric experimental data points. The method is successfully used to analyse Co / SiCo films. When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/3358

Structural studies of influenza A virus by cryo-electron tomography

Influenza A virus (IAV) is a pleomorphic, enveloped virus known for its yearly epidemics and occasional, but fatal pandemics. The outer surface glycoprotein hemagglutinin (HA) together with the matrix protein 1 (M1) are the most abundant protein components of assembled virions. HA, located at the outside of virions, is involved in cell receptor recognition, membrane fusion and is the most relevant protein for antibody binding. Therefore the structure of isolated HA has been extensively characterised by X-ray crystallography. However, it remains unclear to which extent the structure of isolated HA corresponds to the in situ HA structure on the surface of IAV. M1 determines the morphology of the virus by forming a matrix layer underneath the viral membrane. A high resolution structure of full length M1 is missing and the lack of information about the in situ arrangement of the M1 matrix layer currently limits our understanding of how M1 functions. Here, I set out to determine the structures of HA and M1 directly from IAV particles using high resolution cryo-electron tomography (cryoET) and subtomogram averaging. I found that virus purification can affect the integrity of the virus HA glycoprotein layer and the morphology of virus particles. I therefore adapted a workflow which allows studying the structure of viral proteins directly from viruses in the vicinity of virus-producing cells. Biosafety regulations required inactivation of IAV samples by chemical fixation prior to cryoEM imaging. To assess effects of fixation, I complemented structural studies of HA from pathogenic, fixed IAV particles with studies of HA from non-infectious, unfixed virus-like particles (VLPs). These studies revealed that fixation captures HA in an open conformation while HA structures determined from unfixed samples perfectly match the closed conformation observed in the trimeric crystal structure. In concordance with recent work by others, this observation suggests that fixation captures HA in a an open, otherwise transient conformation, which is part of a constant opening and closing motion known as breathing motion. To characterise the in situ structure and arrangement of M1, I established a subtomogram averaging workflow to cope with the challenges presented by the small size of M1. I successfully obtained two independent structures of M1 directly from viruses and VLPs. Comparisons of my structures to existing high resolution models of the N-terminal domain (NTD) of M1 revealed that M1 monomers arrange as parallel strands, with a helical propensity and directly underneath the membrane. For the first time, my data allow to describe the M1-membrane interface as well as relevant M1-M1 interfaces within the matrix layer. Finally, I have gained first structural insights into the M1 C-terminal domain (CTD). I further combined the obtained structural information for M1 with a theoretical model of the mechanics of M1 polymerization and membrane deformation during virus assembly. The obtained results suggest that linear polymerization of M1 into multiple parallel strands efficiently provides energy to drive assembly of new virus particles. The results presented in this thesis improve our understanding of the arrangement and structure of the two influenza proteins HA and M1 in situ which has implications for current models of HA-mediated membrane fusion, virus architecture and virus assembly

Preparation of nuclear matrices from cultured cells: subfractionation of nuclei in situ

Analyses of the different structural systems of the nucleus and the proteins associated with them pose many problems. Because these systems are largely overlapping, in situ localization studies that preserve the in vivo location of proteins and cellular structures often are not satisfactory. In contrast, biochemical cell fractionation may provide artifactual results due to cross-contamination of extracts and structures. To overcome these problems, we have developed a method that combines biochemical cell fractionation and in situ localization and leads to the preparation of a residual cellular skeleton (nuclear matrix and cytoskeletal elements) from cultured cells. This method's main feature is that cell fractionation is performed in situ. Therefore, structures not solubilized in a particular extraction step remain attached to the substrate and retain their morphology. Before and after each extraction step they can be analyzed for the presence and location of the protein under study by using immunological or cytochemical techniques. Thereby the in vivo origin of a protein solubilized in a particular extraction step is determined. The solubilized protein then may be further characterized biochemically. In addition, to allow analyses of proteins associated with the residual cellular skeleton, we have developed conditions for its solubilization that do not interfere with enzymatic and immunological studies

Pseudo-binary phase diagram for Zr-based in situ ß phase composites

The pseudo-binary (quasi-equilibrium) phase diagram for Zr-based bulk metallic glasses with crystalline in situ precipitates (ß phase) has been constructed from high-temperature phase information and chemical composition analysis. The phase evolution was detected in situ by high-energy synchrotron x-ray diffraction followed by Rietveld analysis of the data for volume fraction estimation. The phase diagram delineates phase fields and allows the control of phase fractions. Combined with related previous work by the authors, this diagram offers a unique opportunity to control both the morphology and volume of the dendritic ß phase precipitates to enhance the properties of the composites

X-Ray Diffraction Detects D-Periodic Location of Native Collagen Crosslinks In Situ and Those Resulting from Non- Enzymatic Glycation

Synchrotron based X-ray diffraction experiments can be highly effective in the study of mammalian connective tissues and related disease. It has been employed here to observe changes in the structure of Extra-Cellular Matrix (ECM), induced in an ex vivo tissue based model of the disease process underlying diabetes. Pathological changes to the structure and organization of the fibrillar collagens within the ECM, such as the formation of non-enzymatic crosslinks in diabetes and normal aging, have been shown to play an important role in the progression of such maladies. However, without direct, quantified and specific knowledge of where in the molecular packing these changes occur, development of therapeutic interventions has been impeded. In vivo, the result of non-enzymatic glycosylation i.e. glycation, is the formation of sugar-mediated crosslinks, aka advanced glycation end-products (AGEs), within the native D-periodic structure of type I collagen. The locations for the formation of these crosslinks have, until now, been inferred from indirect or comparatively low resolution data under conditions likely to induce experimental artifacts. We present here X-ray diffraction derived data, collected from whole hydrated and intact isomorphously derivatized tendons, that indicate the location of both native (existing) and AGE crosslinks in situ of D-periodic fibrillar collagen

Kinetics and crystallization path of a Fe-based metallic glass alloy

The thermal stability and the quantification of the different transformation processes involved in the overall crystallization of the Fe50Cr15Mo14C15B6 amorphous alloy were investigated by several characterization techniques. Formation of various metastable and stable phases during the devitrification process in the sequence a-Fe, ¿-Cr6Fe18Mo5, M23(C,B)6, M7C3, ¿-Fe3Mo3C and FeMo2B2 (with M = Fe, Cr, Mo), was observed by in-situ synchrotron high energy X-ray diffraction and in-situ transmission electron microscopy. By combining these techniques with differential scanning calorimetry data, the crystallization states and their temperature range of stability under continuous heating were related with the evolution of the crystallized fraction and the phase sequence as a function of temperature, revealing structural and chemical details of the different transformation mechanisms.Postprint (published version