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Visualizing the Structural Basis of Genome SilencingFussner, Eden Margaret 19 June 2014 (has links)
Eukaryotic genomes must be folded and compacted to fit within the restricted volume of the nucleus. This folding, and the subsequent organization of the genome, reflects both the transcription profile of the cell and of the specific cell type. A dispersed, mesh-like chromatin configuration, for example, is characteristic of a pluripotent stem cell. Here we show that the acquisition of the pluripotent state during somatic cell reprogramming is coincident with the disruption of compact heterochromatin domains. Using Electron Spectroscopic Imaging (ESI), I made the surprising observation that the heterochromatin domains of the induced pluripotent and of the parental somatic cell contained 10 nm chromatin fibres. Since ESI generates projection images, the precise three-dimensional organization of all chromatin fibres within these domains could not be elucidated. To circumvent this limitation, I developed an electron microscopy technique that combines ESI with tomography. Using this approach, I found that both heterochromatin domains and the surrounding euchromatin of murine pluripotent cells, fibroblasts, and somatic tissues are in fact organized entirely as 10 nm chromatin fibres. This challenges the current paradigm that most, if not all, of the genome exists as 30 nm and higher-order chromatin fibre assemblies. Rather than transitions between 10 nm and 30 nm fibres, I propose that the organization and thus the regulation of the genome is achieved by the bending and folding of 10 nm chromatin fibres into discrete domains in a cell type-specific manner.
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Novel scanning probe microscope instrumentation with applications in nanotechnologyHumphry, Martin James January 2000 (has links)
A versatile scanning probe microscope controller has been constructed. Its suitability for the control of a range of different scanning probe microscope heads has been demonstrated. These include an ultra high vacuum scanning tunnelling microscope, with which atomic resolution images of Si surfaces was obtained, a custom-built atomic force microscope, and a custom-built photon emission scanning tunnelling microscope. The controller has been designed specifically to facilitate data acquisition during molecular manipulation experiments. Using the controller, the fullerene molecule C60 has been successfully manipulated on Si(100)-2x1 surfaces and detailed data has been acquired during the manipulation process. Evidence for two distinct modes of manipulation have been observed. A repulsive mode with success rates up to 90% was found to occur with tunnel gap impedances below 2GΩ, while between 2GΩ and 8GΩ attractive manipulation events were observed, with a maximum success rate of ~8%. It was also found that the step size between feedback updates had a significant effect on tip stability, and that dwell time of the STM tip at each data point had a critical effect on manipulation probability. A multi-function scanning probe microscope head has been developed capable of operation as a scanning tunnelling microscope and an atomic force microscope in vacuum and a magnetic field of 7T. The custom-built controller also presented here was used to control the head. A three-axis inertial sliding motor was developed for the head, capable of reproducible step sizes of <1000Å. In addition, an optical fibre interferometer was constructed with a sensitivity of 0.2Å/ √Hz. Preliminary development of a magnetic resonance force microscope mode has also been performed, with initial results showing such a system to be feasible.
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Hydrodynamics of the atomic force microscopeClarke, Richard John January 2005 (has links)
With a proven ability to uncover fundamental biological processes, the atomic force microscope (AFM) represents one of the most valuable and versatile tools available to the biophysical sciences. We study the unsteady small-scale flows generated within the AFM by its sensing probe (a long thin cantilever), which have received relatively little attention to date, yet which are increasingly relevant in an age of microdevices. The early parts of this thesis investigate some canonical two-dimensional flows driven by oscillations of an infinite-length rigid cantilever. These prove amenable to analysis and enable us to investigate many of the important physical phenomena and compile a comprehensive collection of asymptotic expressions for the drag. The corresponding results lay out the influence of a nearby wall, geometry and oscillation frequency. The limitations of a two-dimensional approach are then explored through the development of a novel unsteady slender-body theory (USBT) for finite-length cylinders, an asymptotic treatment of which offers corrections to traditional resistive-force-theory (RFT) methods by accounting for geometric factors and flow inertia. These ideas are then extended to the study of thin rectangular plates. Two key parameters are identified which promote two-dimensionality in the flow, namely the frequency of oscillation and the proximity of a nearby boundary. We then examine flexible cylinders and plates by coupling the hydrodynamics to linearized elastic beam and plate equations, which simulate the hydrodynamically-damped high-speed deformable motion of the AFM's cantilever, when driven either externally or by Brownian motion. In the latter case, we adopt an approach which offers notable improvements over the most advanced method currently available to the AFM community.
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Non-scanning fluorescence confocal microscopy using laser speckle illuminationJiang, Shihong January 2005 (has links)
Confocal scanning microscopy (CSM) is a much used and advantageous form of microscopy. Although CSM is superior to conventional microscopy in many respects, a major disadvantage is the complexity of the scanning process and the sometimes long time to perform the scan. In this thesis a novel non-scanning fluorescence confocal microscopy is investigated. The method uses a random time-varying speckle pattern to illuminate the specimen, recording a large number of independent full-field frames without the need for a scanning system. The recorded frames are then processed in a suitable way to give a confocal image. The goal of this research project is to confirm the effectiveness and practicality of speckle-illumination microscopy and to develop this proposal into a functioning microscope system. The issues to be addressed include modelling of the system performance, setting up experiments, computer control and image processing. This work makes the following contributions to knowledge: * The development of criteria for system performance evaluation * The development of methods for speckle processing, whereby the number of frames required for an image of acceptable quality can be reduced * The implementation of non-scanning fluorescence confocal microscopy based upon separate recording of the speckle patterns and the fluorescence frames, demonstrating the practicality and effectiveness of this method * The realisation of real-time image processing by optically addressed spatial light modulator, showing how this new form of optical arrangement may be used in practice The thesis is organised into three main segments. Chapters 1-2 review related work and introduce the concepts of fluorescence confocal microscopy. Chapters 3-5 discuss system modelling and present results of performance evaluation. Chapters 6-8 present experimental results based upon the separate recording scheme and the spatial light modulation scheme, draw conclusions and offer some speculative suggestions for future research.
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Microscopy and surface chemical investigations of dyed cellulose textilesChettra, Satinderjeet Kaur January 2006 (has links)
Cotton is a vital material for the textile industry, providing the fundamental raw component for the manufacture of numerous and varied garments. It has been thoroughly characterised both in terms of its constitution; behaviour under a variety of environmental and manufacturing conditions; and several mechanisms by which it takes up dyestuffs. Recently the availability of a range of high-performance surface analysis tools has allowed researchers to begin to assess the contribution of the surface interface to the overall properties of cellulose in cotton. In particular, these approaches offer considerable potential to address the current lack of fundamental experimental data in support of the proposed dye-uptake mechanisms in cotton fibres. The absence of a detailed molecular model for the process makes it difficult to predict dye performance and to identify the key characteristics of the cotton which influence dyeing. For example, the existence and location of dye binding sites is still unclear. It has been postulated that the occurrence of crystalline or amorphous regions in cotton may play a role in such binding and in dye uptake. A deeper understanding of the dyeing mechanisms therefore requires knowledge of the interplay between the physical chemistry of the dye, its adsorption/diffusion onto the surface of and within cotton fibres, and the related physical and chemical characteristics of the cotton itself. Here we begin to address these broad questions through the application of atomic force microscopy (AFM) and other complimentary surface analytical techniques, to analyse a range of dyed and undyed cellulose based textiles. We provide high-resolution surface morphological image data, which show nanometre scale detail of the surface of dyed and undyed cotton fibres. It is believed that the dyeing process induces an increase in crystallinity, due to breakages in hydrogen bonds between cellulose chain molecules during swelling of fibres within the dyebath, thus allowing dye molecules to enter and become entrapped within the fibre matrix. We provide evidence in the form of image data that suggest a difference in the crystal structure between undyed and dyed cellulose fibres to support this theory. We also reveal possible crystalline and amorphous regions within the substrate through AFM phase imaging using modified tips and successfully fingerprint regions of dyed and undyed cellulose fibres. Complimentary surface chemical analysis of dyed and undyed fibres, provide qualitative and quantitative data to show the presence of dye molecules on the surface of dyed cellulose textiles. Novel investigations of dyed fibres through X-ray photoelectron spectroscopy (XPS), determine the amount of dye present at the surface of cotton fibres. Using the N 1s atomic orbital region as a diagnostic peak, the level of dye loading could be directly attributed to the concentration levels of dye within the dyebath. The XPS data also provided strong evidence for possible dye-uptake mechanisms. We observed that certain stages of the dyeing process directly influenced the amount of dye entering cotton fibres. XPS showed that an increase in salt (NaCl) content within the dyebath produced dyed fibres with increased presence of dye compound at the surface. The knowledge obtained from these studies will help to improve the dyes and dyeing mechanisms for cotton and other textiles, thus improving the quality of dyed garments offered to the consumer.
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Atomic force microscopy investigations of peptide self-assemblySedman, Victoria L. January 2006 (has links)
The ability of short peptide fragments to self-assemble in isolation as amyloid and amyloid-like structures has prompted their use as model systems for the study of amyloid formation and recently also for their utilisation as novel nanofibrillar material. The atomic force microscope (AFM) is used here to investigate the self-assembly of two peptide systems and the development of strategies to directly manipulate and control the structures they form. The studies presented in Chapter 2 address the self-assembly of a peptide fragment of the human amylin polypeptide; amylin (20-29). In the opening study we use ex situ AFM imaging to characterise the early stages of amylin (20-29) fibril formation. High-resolution images reveal that following an initial lag phase, fibrils displaying a globular appearance are formed, which over time are replaced by flat ribbon-like fibrils with no periodicity displaying a range of polymorphic structures and assemblies. Following on from these findings, we investigate the influence of solution conditions on amylin (20-29) fibril formation utilising in situ AFM imaging. Altering the pH and electrolyte composition affords a range of morphologies including, truncated and long branched or unbranched flexible fibrils and globular aggregates. Following on from this characterisation chapter, in Chapter 3 a strategy to assemble specifically functionalised fibrillar material from chemically modified amylin (20-29) peptides was investigated. Azide and alkyne moieties were successfully coupled to the amylin (20-29) peptides. Ex situ AFM imaging and Congo red binding confirmed that the additional steric bulk had no detrimental effects on the fibril forming capacity of the peptides. Finally, in Chapter 4 the focus turns to the self-assembly of a dipeptide of phenylalanine which corresponds to the core recognition motif of the beta-amyloid polypeptide. Here, the AFM is used to study the physical properties of the well-ordered, discrete, hollow nanotubes which are formed. Their chemical stability in organic solvents and considerable thermal stability under both dry and wet heating conditions is revealed. Finally, the use of strong magnetic fields to directly control and orientate the diphenylalanine nanotubes was examined by AFM. The results presented throughout this thesis demonstrate the versatility of self-assembling peptides for the generation of fibrillar nanostructures that can be directly modified and controlled to generate novel architectures and functionalised well ordered nanomaterials.
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Scanning optical microscopy of semiconductor devicesMcCabe, Eithne January 1987 (has links)
A new method to display low contrast OBIC images has been used to highlight defects in semiconductor devices. In addition an exciting novel method to obtain spatial information on the distribution of defects at the silicon/silicon-dioxide interface in metal oxide semiconductor devices has been found. This method can examine many defects which cause serious problems for device manufacturers including the effect of radiation damage on device performance. Other non-destructive techniques which can complement OBIC imaging are explored including photoluminescence and infrared transmission imaging. Additional research is proposed for the future. This research in conjunction with the research in this thesis would allow a comprehensive and powerful examination approach of both static and dynamic conditions of semiconductor devices.
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Colloidal oxide and sulphide interactions in aqueous electrolyte studied by atomic force microscopy /Toikka, Gary. Unknown Date (has links)
Thesis (PhD)-- University of South Australia, 1997
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Wavelength effects on in vivo confocal scanning laser microscopy/Luedtke, Michael A. Papazoglou, Elisabeth S. January 2007 (has links)
Thesis (M.S.)--Drexel University, 2007. / Includes abstract. Includes bibliographical references (leaves 50-51).
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Colloidal suspension flow and transport behavior in small channels by magnetic resonance microscopyBrown, Jennifer Ruth. January 2007 (has links) (PDF)
Thesis (Ph. D.)--Montana State University--Bozeman, 2007. / Typescript. Chairperson, Graduate Committee: Joseph D. Seymour. Includes bibliographical references (leaves 189-201).
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