The work discussed in this dissertation is aimed at creating novel approaches to chemical imaging that ultimately allow for submicron resolution. This goal has been approached from two direction using laser based desorption and coupling it with an AFM using apertureless tip-enhanced laser ablation/ionization. The second direction was through the development a new approach to thermal desorption based mass spectrometry experiments by using a proximal probe to spatially desorb the surface and ionizing the plume of neutrals using a secondary ionization source at atmospheric pressure. The thermal desorption approach allows for the easy scaling of the technique all the way from the millimeter to the nanometer regime. In the nanometer regime an AFM platform with silicon based heating AFM probes is used to locally desorb material from nanometer sized craters. The final work in this thesis focused on trying to improve laser based desorption through a secondary ionization of the neutrals plume by capturing the laser desorption plume into a liquid and then electrospaying the solution into a MS. The added benefit of being able to capture the desorption plume into a liquid is the ability to carry out post sampling processing of the captured analyte via high performance liquid chromatography. The ability to clean up a sample via HPLC also allow for the detection of isobaric compounds as well as trace level materials which otherwise would be obscured by matrix effects in complicated sample matrixes like tissues. This application of laser desorption with a secondary ionization by capture into a liquid could be envisioned to be applied to AFM based laser desorption techniques where boosting the ionization efficiency is crucial for signal detection.

<p>The work in this thesis deals with pre-clinical development of rapid in vivo ³¹P mag- netic resonance spectroscopy (MRS) techniques. Current MRI literature of ³¹P spec- troscopy presents evidence of increased concentrations of phosphomonoesters (PME), and phosphodiester (PDE) as well as inorganic phosphate concentrations in tumor tissue. Human breast cancer studies have demonstrated correlation between disease progression and both PME and PDE peaks. Furthermore, ³¹P MRS can be used to detect, grade tumours and monitor response to chemo and radiation therapy.<br />Tumor measurements are typically static (i.e. single measurement per scan). In other experiments, on muscle for example, dynamic measures are required the purpose of which is to assess temporal function and recovery. In all ³¹P acquisitions there are problems surrounding RF coil design, pulse sequence speed, localization and system calibration. The work presented here focused on improving all these aspects and provide easy and reliable work flow to use ³¹P MRS in a clinical setting.<br />One of the aspects of this thesis lies in designing and construction of an RF coil that is well suited for integration with a clinical MRI breast imaging and biopsy system. The designed coil was tuned for simultaneous operation at ³¹P (51.73 MHz) and ¹H (127.88MHz) Larmor frequencies. This design has advantages in the fact that complex pulse sequences with heteronuclear decoupling could be performed easily. The additional features of the coil design is that it is possible to swap it into the breast imaging system without moving the patient. Along with the designed coil, custom software was written to assist with transmit gain calibration of ³¹P RF pulses, to ensure maximum MR signal. The automated prescan ensures easy work flow and minimizes the operator variability and patient time inside the MR scanner.<br />Another aspect of this thesis deals with rapid pulse sequence development, to further speed up the ³¹P MRS data acquisition. Echo planar spectroscopic imaging (EPSI) with a fly–back gradient trajectory is currently one of the most reliable and robust techniques for speeding up chemical shift imaging (CSI) acquisitions. A ³¹P EPSI sequence was written to acquire spectroscopic imaging data at 1, 2 and 2.6 cm spatial resolution and spectral bandwidth of 3125 Hz. The sequence showed an ability to speed up data acquisition up to 16 times, where SNR permits.<br />Phantom studies were used to verify the double tuned coil and EPSI sequence en- suring proper and safe operation. In vivo measurements of an exercising muscle demonstrated the ability of ³¹P EPSI to play an important role in rapidly acquiring spatially localized ³¹P spectroscopic data.<br />With these preclinical developments in place a clinical trial is possible using ³¹P MRS rapidly and efficiently. Furthermore the increased usability of ³¹P MRS provided by the tools developed in this thesis can prove to be beneficial by integrating ³¹P MRS into existing clinical protocols.</p> / Doctor of Science (PhD)

<p>Soft condensed matter systems are a very diverse and challenging subject to study. To understand the complex macro-properties of such systems one approach is to characterize the microscopic structure and dynamics. A powerful technique for determining micro and nanoscale properties is scattering of radiation sources. Light, electron and neutron scattering techniques provide insight into the complicated molecular structures and the processes happening on these small scales.</p> <p>We have used neutron and x-ray scattering techniques to determine structural and dynamical information from two different types of soft condensed matter systems. The microscopic nature of a cobalt magnetic fluid was studied using neutron scattering, and the structure and dynamics of molecules within lipid bilayers was studied with the use of both neutron and x-ray scattering.</p> <p>Under strong magnetic fields, our cobalt fluid's small magnetic particles formed short chains, which we observed using neutron scattering.</p> <p>In the lipid bilayer systems which were studied we determined the positional orientation of cholesterol, Aspirin, and ethanol molecules, observed the effect of temperature on some of these systems, characterized domains and dynamics, and recreated the molecular structures of Alzheimer's protein in a brain-like membrane.</p> / Master of Science (MSc)

<p>A key problem in the origin of life is to understand how an autocatalytic, self-replicating biopolymer system may have originated from a non-living chemical system. This thesis presents mathematical and computational models that address this issue. We consider a reaction system in which monomers (nucleotides) and polymers (RNAs) can be formed by chemical reactions at a slow spontaneous rate, and can also be formed at a high rate by catalysis, if polymer catalysts (ribozymes) are present. The system has two steady states: a ‘dead’ state with a low concentration of ribozymes and a ‘living’ state with a high concentration of ribozymes. Using stochastic simulations, we show that if a small number of ribozymes is formed spontaneously, this can drive the system from the dead to the living state. In the well mixed limit, this transition occurs most easily in volumes of intermediate size. In a spatially-extended two-dimensional system with finite diffusion rate, there is an optimal diffusion rate at which the transition to life is very much faster than in the well-mixed case. We therefore argue that the origin of life is a spatially localized stochastic transition. Once life has arisen in one place by a rare stochastic event, the living state spreads deterministically through the rest of the system. We show that similar autocatalytic states can be controlled by nucleotide synthases as well as by polymerase ribozymes, and that the same mechanism can also work with recombinases, if the recombination reaction is not perfectly reversible. Chirality is introduced into the polymerization model by considering simultaneous synthesis and polymerization of left- and right-handed monomers. We show that there is a racemic non-living state and two chiral living states. In this model, the origin of life and the origin of homochirality may occur simultaneously due to the same stochastic transition.</p> / Doctor of Philosophy (PhD)

<p>Biological membranes play host to a number of processes essential for cellular function and are the most important biological interface. The structurally complex and highly dynamic nature of the membrane poses significant measurement challenges, requiring an experimental technique capable of accessing very short, nanometer length scales, and fast, micro-pico second time scales.</p> <p>The experimental work presented in this thesis uses a variety of neutron scattering techniques to study the structure and dynamics of biologically relevant model membrane systems. The main body of this work can be sub-divided into two distinct topics: (1) lateral diffusion of lipid molecules in a bilayer; and (2) the measurement of domains in the membrane.</p> <p>Diffusion is the fundamental mechanism for lipids and proteins to move throughout the lipid matrix of a biological membrane. Despite a strong effort to model lipid diffusion, there is still no coherent model which describes the motion of lipid molecules from less than a lipid-lipid distance to macroscopic length scales. The experiments presented on this topic attempt to extend the range over which diffusion is typically measured by neutron scattering, to initiate the development of a more complete lipid diffusion model.</p> <p>Lipid domains and rafts are thought be platforms for many cellular functions; however, their small size and transient nature makes them notoriously difficult to observe. The penultimate chapter of this thesis provides evidence supporting the existence of domains in a model lipid/cholesterol system by probing of the dynamics of the system. The challenge of observing these structures directly was addressed by modifying the traditional neutron triple-axis spectrometry setup to increase its sensitivity to systems with short-range order. This technique was employed to examine the coexistence of fluid and gel domains in a single-component lipid bilayer system, as well as the presence of highly ordered lipid domains in a model membrane containing cholesterol.</p> / Doctor of Philosophy (PhD)

<p>Bid is a member of the Bcl-2 family of proteins, which are known as the regula- tors of apoptosis. Bid recruits Bax, another Bcl-2 family protein, which forms large oligomers that permeabilize the mitochonrdial outer membrane during apoptosis. In this thesis, Bid complexes embedded in a mitochondria-like supported lipid bilayer were investigated using single molecule fluorescence techniques. The bilayer, con- taining a lipophilic tracer, was formed on a mica surface and ATTO647 labelled Bid was added to it. For experiments where the effect of Bax on Bid complexes was investigated, a wild type Bax or a HiLyte488 labelled Bax was added as well. The protein-bilayer sample was imaged using total internal reflection fluorescence (TIRF). The formation of a fluid bilayer was confirmed by the observation of the lateral diffusion of DiD. Single particle tracking of the lipid molecules was used to measure the diffusion coefficent of DiD which was determined to be 2.2μm2 /s. The TIRF images also revealed two populations of Bid complexes, immobile and mobile. The diffusion coefficient of the observed Bid complexes was determined to be about three times slower than that of DiD (0.8±0.5μm2 /s). This provides evidence that mobile Bid is embedded in the bilayer. Image analysis of immobile Bid complexes showed a step-wise decrease in the fluorescence intensity due to photobleaching. The oligomeric distribution of the immobile Bid complexes was determined from the num- ber of steps, which corresponds to the number of particles in each complex. From these distributions it was concluded that the imaged immobile Bid existed mainly as monomers. However dimer and trimer complexes of Bid were also observed. The detected oligomeric distribution was not affected by the presence of either wild type Bax or Hilyte488 Bax. However Bid was imaged for the first time participating in Bax complexes. The acquired results somewhat differ from what had been observed in confocal imaging of the same samples, where mostly larger Bid complexes (dimers and up) were detected. We attribute the difference to the superior sensitivity of the TIRF method presented here.</p> / Master of Science (MSc)

We consider modified versions of a cell population model for periodic chemotherapy treatment of tumors. These models contain both proliferating and quiescent cells, and treats the transitions between the two types of cells. Our models are defined by replacing the loss term function, μ(t, c), in Webb's model by a constant. This corresponds to having a constant infusion of the drug rather than having a periodic chemotherapy treatment. We compare our results with those of Webb who shows that shorter periods of treatment are advantageous.

The application of auto focus, using an optical beam deflection technique, to existing live cell interferometry measurements was developed and examined. The benefit to relevant experiments, currently being performed, is shown as well as its performance across various magnifications. Enough information is given so that the system can be reproduced to fit any end users needs.

Nanopore-based resistive pulse sensing represents an important class of single-molecule measurements. It provides information about many molecules of interest (i.e. DNA, proteins, peptides, clusters, polymers, etc.) without the need for labeling. Two experiments that are especially well suited for studying with nanopore sensors are DNA sequencing and DNA-protein force measurements. This thesis will describe progress that has been made in both areas. DNA sequencing has become an active area of research for stochastic single-molecule sensing, with many researchers striving for the ultimate goal of single-molecule de novo DNA sequencing. One intriguing method towards that goal involves the use of a DNA exonuclease or polymerase enzyme, which when attached close to the mouth of a pore, leads to cleavage of individual DNA nucleotide bases for loading into the pore for sensing. Though this method seems promising, the end goal has been elusive because the nucleotide motion is dominated by diffusion over the relevant length scales. This limits the likelihood of the cleaved nucleotide entering the pore to be characterized. The first part of this thesis will describe a method for addressing this problem, where it is shown that increasing the nucleotide capture probability can be achieved by lowering the bulk diffusion coefficient relative to the pore diffusion coefficient. The second part of this thesis will describe the design and implementation of a new type of sensor that combines a biological nanopore experimental apparatus with optical tweezers. The goal of this apparatus is to develop a means to independently measure the force on a charged molecule inside of the pore. The setup will be thoroughly described, and preliminary results showing that it is possible to optically trap a micron sized bead within a few microns of an isolated biological nanopore while simultaneously making current measurements through that pore will be presented. This will enable force measurements on DNA molecules tethered to the bead, which opens the door for the study of molecular force interactions between DNA and biological nanopores, DNA-bound protein interactions that cause diseased states, and controlled translocation of DNA through biological nanopores.

Photorefraction is a technique that has been used in the past two decades for pediatric vision screening. The technique uses a digital or photographic camera to capture the examinee‟s retinal reflex from a light source that is located near the camera‟s lens. It has the advantages of being objective, binocular and low cost, which make it a good candidate for pediatric screening when compared to other methods. Although many children have been screened using this technique in the U.S., its sensitivity and other disadvantages make it unacceptable for continued use. The Adaptive Photorefraction system (APS) was developed at the Center for Laser Applications (CLA) at the University of Tennessee Space Institute (UTSI) to correct the problems in the existing PS devices. APS was designed to determine quantitatively binocular refractive errors and strabismus and to accomplish these tasks objectively, without the need of medical professionals, and it is capable of performing these objectives and reporting the digitally recorded results within one- to-two minutes. In this dissertation, two APS prototypes were constructed, and measurements were performed using both an artificial eye and human subjects. Binocular measurements of refractive error were determined, and the effects of the variation of pupil-size and gaze angle were determined. After initial corrections for ocular scattering effects, measurement of the binocular refractive error of forty human subjects was achieved, and in the myopic region with uncertainty of the method was 0.6 diopter. Ocular alignment determinations were achieved, and using a novel cover-uncover test, strabismus detection was demonstrated.