Fluorochromes such as tetracycline have been used to label bone for histomorphometric analysis, measuring bone formation, growth, maintenance, and pathology. More recently, similar fluorescence has been observed in ancient human bone. Attributed to tetracycline (TC) exposure, this phenomenon could affect various aspects of health during life and/or preservation of remains postmortem. Standard epifluorescence microscopy is the most common tool employed in the analysis of these labels. Though valuable, this technique is limited by its inability to penetrate bone three-dimensionally and its inclusion of out-of-focus light, possibly disrupting accurate analysis. Confocal Laser Scanning Microscopy (CLSM) has been demonstrated as a valuable tool for three-dimensional histology. Its application to the study of compact bone fluorescence has been lacking, especially in archaeological and forensic sciences. In the following two papers, modern TC-controlled bone is compared to well preserved archaeological bone recovered from the Dakhleh Oasis, Egypt, using both standard wide-field and more modern confocal techniques for imaging and analysis. Spectral analysis via CLSM shows that both modern and ancient fluorescent labels in bone share the exact same fluorescence emission peak at 525 nm. Differences in the shape of the spectral curve and photobleaching characteristics are discussed. In addition, CLSM's high-resolution two- and three-dimensional imaging capabilities (in polarized light, scattered light, and fluorescence light) are found to increase the flexibility and creativity of investigations into the occurrence of tetracycline labels in archaeological bone and could have added benefits for modern medical and anatomical experimentation.

The goal of current research is probing the relationship between catalyst features and the fuel cell performance with a range of in-depth structural analysis. The study investigated different catalyst systems including core-shell structured catalyst, catalysts with unique carbon-transition metal oxide supports. PtRu catalysts nanoparticles with unique core-shell structure, one of the most practical catalysts in PEMFC technology, have been successfully obtained with the evidence from the characterization results. It is found that the enhanced CO oxidation may be achieved through the interactions between the Pt shell and Ru core atoms, which can modify the electronic structure of the Pt surface by the presence of subsurface Ru atoms or by disrupting the Pt surface arrangement. Furthermore, the possibility of presence of the compressive strain within the Pt rich shell is proved by the lattice measurements, which could significantly affect the catalytic activity. Pt catalysts supported on complex oxide and carbon support were studied to investigate the relationship between the catalyst and its support. Observations from STEM images and HAADF and energy dispersive X-ray spectrometry demonstrate the preferential distribution of Pt nanoparticles on the hybrid supports, which include Nb2O3 / C, Ta2O5 / C, (Nb2O3+TiOx) / C, (Ta2O5+TiOx) / C, and (WO3+TiOx)/C). Such evidence indicates the interaction between the catalyst and support is based on the presence of an interconnected oxide network over the carbon support and the presence of Pt strongly connected to the oxide network. In addition, using electron energy loss spectroscopy (EELS), the electronic structure of the catalyst support under various conditions was also studied to provide further evidence of the strong metal support interaction effect. / Thesis / Doctor of Philosophy (PhD)

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The conventional atomic force microscope (AFM) is equipped with a single optical detection system. Probe-sample separation is determined in an independent deflection with respect to AFM z-translation experiment. This method of determining probe-surface separation is relative, susceptible to drift and does not provide real time separation information. The evanescent wave atomic force microscope (EW-AFM) utilizes a second, independent detection system to determine absolute probe-surface separation in real time. The EW-AFM can simultaneously acquire real-time force and probe-sample separation information using the optical lever and evanescent scattering detection systems, respectively. The EW-AFM may be configured with feedback on the optical-lever system for constant force applications or with feedback on evanescent wave scattering intensity for constant height applications. Scattering of the evanescent wave exponential decay profile is used to determine probe-surface separation. Sub-micron sized dielectric and metallic probes show exponential scattering profiles, micron sized polystyrene and borosilicate microspheres show non-exponential profiles when they are affixed beneath the cantilever tip. By affixing the microspheres to the end of the AFM cantilever exponential and non-exponential profiles were observed. The EW-AFM can be used to conduct force-distance and imaging experiments. The EW-AFM was used to measure the thickness of surfactant bilayers formed at the silica-solution interface using silicon nitride AFM tips. The presence of a refractive index difference between the surfactant bilayer and the solution does not influence the accuracy of the surfactant bilayer thickness measurement. The EW-AFM was used to scan a 2 x 2 micron area in constant height mode. The probe was brought to within 6 nanometers of a planar dielectric surface using the evanescent wave intensity as a height reference with accuracy of ± 1 nm. This capability may be utilized to observe charge heterogeneity at the solid-liquid interface with nanometer lateral resolution or to map chemical functional group heterogeneity based on perturbations to the electrical double layer. The EW-AFM evanescent scattering system has an absolute separation resolution of 0.3 nm compared to 1.0 nm relative separation resolution for the optical lever system. In constant scattering (constant height) mode the real time separation precision is about 2 nm. / Ph. D.

The intensity of light produced from synchrotrons enables X-ray imaging down to the micron and submicron scale. This high degree of resolution is necessary to study metals in hydrated biological samples, where trace (metal) elements are found in the lowest concentration. Water within these aqueous samples will undergo radiolysis and produce various reactive oxygen species, which degrades the quality of information gathered from the sample during X-ray imaging. Studies have shown that the best way to counter the effects of radiolysis and preserve samples in their metabolic state during X-ray imaging is to keep them cryogenically frozen. We have developed affordable cryostats and novel protocol to not only improve cryo-imaging at current third-generation synchrotrons, but also enable cryo-imaging at existing synchrotrons that have limited accessibility. This dissertation will provide a detailed description of the tasks that were accomplished to contribute to the development cryo-imaging. The first task was the fabrication of a portable cryostage. The cryostage's discreet profile and unique design successfully enabled it to be effortlessly adapted into three beamlines across two different DOE facilities and facilitate multiple imaging modalities, i.e., correlative imaging. With the next task, we explored adding an ice frame about the stage to help reduce the accumulation of frost on the surface of a frozen sample that was explored. The addition of the ice frame significantly improved the imaging of frozen samples, nearly doubling the overall image clarity in comparison to when it was absent. The final task saw the application of a cryostream, in place of a cryostage, to provide a cooled convective flux across the sample for 2D and 3D visualization for cryo X-ray imaging. / Doctor of Philosophy / Synchrotrons are light producing particle accelerators that enable X-ray imaging down to the micron and submicron scale. This high degree of resolution is necessary to study metals in hydrated biological samples, where trace elements are found in low concentrations. The X-ray beam from the synchrotron will force any water within these aqueous samples to undergo radiation induced water decomposition, i.e., radiolysis, and produce hydroxyl radicals that will degrade the quality of information gathered from the sample during X-ray imaging. Early studies have shown that the best counter to the effects of radiolysis, while also preserving samples in their metabolic state during X-ray imaging, is to keep them cryogenically frozen. We have developed affordable cryostats and novel protocols to not only improve cryo-imaging at current third-generation synchrotrons, but also enable cryo-imaging at existing synchrotrons that have limited accessibility. This dissertation will describe, in detail, three tasks that were accomplished. The first task was to the fabrication of a portable cryostage. The cryostage unique design successfully enabled it to be used within different beamlines and for multiple imaging perspectives. With the next task, an ice frame to help reduce the accumulation of frost on the surface of a frozen sample being explored. The ice frame was shown to significantly improve the imaging of frozen samples. The final task saw the application of a cryostream, a jet stream of cold nitrogen gas, to enable an alternative approach for 2D and 3D visualization for cryo X-ray imaging.

The ability of cells to probe their mechanical environment and react to external stimuli is critical for maintenance of their normal structure and function. Through connections to the extracellular matrix, cells can sense mechanical cues such as substrate rigidity and stretch, and through force transmission across their contractile cytoskeleton can react accordingly to those signals by applying contractile traction forces to their surrounding environment. Healthy cells can react to these mechanical cues to maintain their cytoskeletal prestress (tension) at a set or homeostatic level over time, a phenomenon known as tensional homeostasis. Progression of certain diseases such as asthma, atherosclerosis, and cancer have been linked to a loss of tensional homeostasis. As such, tools for quantifying the traction forces that adhered cells apply to their substrate are crucial for gaining a better understanding of not just how healthy cells interact mechanically with their environment, but also how changes to the extracellular matrix or mutations within the cell can impact their ability to maintain tensional homeostasis and therefore remain both functional and viable. Our group has previously developed a method of quantifying cellular traction forces using indirectly pattered, soft hydrogel substrates known as micropattern traction microscopy. This method was initially developed to create discrete grid micropatterns, which while useful for measuring cellular traction forces does not offer any ability for the user to control cell growth area shape or size. This technique was further improved on through the creation of a protocol for changing discrete grid patterns into isolated island micropatterns, but this two-step process was challenging and generated islands of inconsistent shape and size. Here, we propose a new method for generating isolated island micropatterns of essentially any desired shape and size in a single step, as well as a corresponding image analysis algorithm for calculating cellular traction forces from these island micropatterns. Additionally, this dissertation also includes an investigation into the impact of distinct Epithelial-cadherin mutations on the ability of gastric adenocarcinoma (AGS) cells to achieve tensional homeostasis. Disruption of tensional homeostasis in the epithelium is a hallmark of certain cancers, and mutations in E-cadherin proteins have been identified in malignant epithelial cells. Here, through analysis of AGS cell traction force data collected previously by Dr. Han Xu during her dissertation work, we have found that two distinct mutations in the intracellular domain of E-cadherins have an impact on the ability of AGS cells to achieve tensional homeostasis.

Current magnetic data storage technology is encountering certain fundamental limitations that present roadblocks to its scalability to areal densities of 1 Tbit/in^2 and beyond. Next generation magnetic storage technology is expected to use optical near field techniques to heat the magnetic film locally to write data bits. This requires experimental measurement of thermal conductivity of materials with sub--100 nm resolution. This is essential for the tailoring of the thin film stack to optimize the heat transfer of the process. This can be accomplished with a simple modification to a traditional atomic force microscopy (AFM) system. The modification requires the deposition of a thin metal film on the AFM cantilever thus creating a bimetallic cantilever. The curvature of a bimetallic cantilever is sensitive to temperature. Another modification is the use of a heating laser to raise the temperature of the cantilever so that when it scans across a sample with areas of varying thermal conductivity the bimetallic deformation of the heated cantilever is altered. The resulting system is sensitive to local variations in thermal conductivity with nanoscale resolution. Nanoscale thermal conductivity measurements can then be used to optimize the heat transfer properties of the materials used in a heat assisted magnetic recording system. AFM technology can also play a key role in the development of next generation solid-state chemical sensors. An AFM can be used to measure the workfunction of a material with near atomic resolution thus enabling the study of chemical reactions with high spatial resolution. Since chemical sensors typically use a chemical reaction at their front end to monitor the prescience of a gas, an AFM system can thus be used to understand and optimize the properties of the chemical reaction by monitoring the local workfunction. In this thesis, I explain the use of atomic force microscopy in measuring thermal and chemical properties of materials with applications towards the magnetic storage industry and chemical sensing.

Semiconductor nanostructures have attracted great interest owing to their unique physical properties and potential applications in nanoscale functional devices. The enhancement of the physical properties of semiconductor nanostructures and their performance in devices requires a deeper understanding of their fundamental microstructural properties. Thus this thesis is focused on the experimental and theoretical studies of the microstructural properties of two important semiconductor nanostructures: axial heterostructured silicon nanowires with varying doping and indium nitride colloidal nanoparticles. In this thesis, axial heterostructured silicon nanowires with varying doping were synthesized on an oxide-removed Si{111} substrate using a vapour-liquid-solid approach. Their fundamental microstructural properties, including the crystalline structure, wire growth direction and morphologies, were studied using various characterization techniques. It is found that a very small fraction of the silicon nanowires crystallize in a hexagonal (wurtzite) phase, which is thermodynamically unstable in bulk silicon under ambient conditions, while a large majority of the synthesized silicon nanowires exhibit the expected diamond cubic crystalline structure. About 75% of the diamond cubic silicon nanowires synthesized grow in a single <111> direction, while the rest contain growth-related kinks, where the nanowire switches to another direction during the growth. The ~109° silicon nanowire kinks are the most commonly observed, and the growth direction before and after such ~109° kink are both <111>. The sidewalls of silicon nanowires do not change abruptly at the ~109° kink, but exhibit an elbow-shaped structure. It is also found that the nanowire sidewalls exhibit periodic nanofaceting, which is strongly doping-dependent. The nanofaceting is found to occur during the enhanced sidewall growth that arises when the diborane dopant gas is introduced. A thermodynamic model predicting the dependence of nanofacet period on the wire diameter is developed. Another semiconductor nanostructure studied in this thesis is indium nitride colloidal nanoparticles, which were grown using a solution-phase chemical method. The formation of such indium nitride colloidal nanoparticles is confirmed by studying their compositions, crystalline structures and shape using various electron microscopy techniques. The size of the indium nitride colloidal nanoparticles was controlled by varying the time of solution-phase reactions. The most probable size of the colloidal nanoparticles increases and the size distribution broadens with the increase of reaction time. The crystalline structures of the indium nitride colloidal nanoparticles are found to be particle size dependent. The observed dependence of the band gap blueshift of the indium nitride colloidal nanoparticles on the reaction time (hence the particle size) is explained by the quantum-size effect.

In recent years, the confocal and two photon microscopes have become ubiquitous tools in life science laboratories. The reason for this is that both these systems can acquire three dimensional image data from biological specimens. Specifically, this is done by acquiring a series of two-dimensional images from a set of equally spaced planes within the specimen. The resulting image stack can be manipulated and displayed on a computer to reveal a wealth of information. These systems can also be used in time lapse studies to monitor the dynamical behaviour of specimens by recording a number of image stacks at a sequence of time points. The time resolution in this situation is, however, limited by the maximum speed at which each constituent image stack can be acquired. Various techniques have emerged to speed up image acquisition and in most practical implementations a single, in-focus, image can be acquired very quickly. However, the real bottleneck in three dimensional imaging is the process of refocusing the system to image different planes. This is commonly done by physically changing the distance between the specimen and imaging lens, which is a relatively slow process. It is clear with the ever-increasing need to image biologically relevant specimens quickly that the speed limitation imposed by the refocusing process must be overcome. This thesis concerns the acquisition of data from a range of specimen depths without requiring the specimen to be moved. A new technique is demonstrated for two photon microscopy that enables data from a whole range of specimen depths to be acquired simultaneously so that a single two dimensional scan records extended depth of field image data directly. This circumvents the need to acquire a full three dimensional image stack and hence leads to a significant improvement in the temporal resolution for acquiring such data by more than an order of magnitude. In the remainder of this thesis, a new microscope architecture is presented that enables scanning to be carried out in three dimensions at high speed without moving the objective lens or specimen. Aberrations introduced by the objective lens are compensated by the introduction of an equal and opposite aberration with a second lens within the system enabling diffraction limited performance over a large range of specimen depths. Focusing is achieved by moving a very small mirror, allowing axial scan rates of several kHz; an improvement of some two orders of magnitude. This approach is extremely general and can be applied to any form of optical microscope with the very great advantage that the specimen is not disturbed. This technique is developed theoretically and experimental results are shown that demonstrate its potential application to a broad range of sectioning methods in microscopy.

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