<p>An objective measure of muscular low back pain (LBP) symptoms eludes clinicians. This study assessed efficacy of magnetic resonance imaging (MRI) of the lumbar multifidus using diffusion tensor imaging (DTI), blood oxygen level dependent (BOLD) signal fractal dimension (FD) analysis and muscle cross sectional area (CSA) in LBP assessment. MRI results were compared to two questionnaires, the Oswestry disability index (ODI) and visual analog score (VAS).</p> <p>Right-left asymmetry in both DTI metrics and T2-weighted (T2W) CSA were greater in the injured. Also, asymmetry measures were correlated with body mass index (BMI) but not age, height, or level of physical activity (measured via Godin activity questionnaire). The relationship between asymmetry and LBP symptoms in T2W and DTI scans increased for subjects with BMI below 35kg/m².</p> <p>BOLD FD did not scale with LBP symptoms. However, FD analysis showed promise following therapeutic Swedish massage, hypothesized as being related to local perfusion changes, indicating that FD is sensitive to changes in the lumbar muscle, just not LBP symptoms. Thus the BOLD FD does change with treatment, just not with the symptoms of LBP.</p> <p>When combining data from multiple scan types, the symptoms of LBP correlated best with the unweighted mean of DTI fractional anisotropy (FA) and T2W CSA asymmetry, and the correlation was greatest (R²=0.88) when only <em>symptomatic (not both symptomatic and control)</em> subjects with BMIs from 18-25kg/m² were considered. From these results there appears to be clinical utility in characterizing the symptoms of non-acute LBP using DTI and CSA.</p> / Doctor of Philosophy (PhD)

There is a large body of evidence supporting the theory that cell physiology largely depends on the mechanical properties of its surroundings or micro-environment. More recently studies have shown that changes to intra-cellular mechanical properties can also have a meaningful impact on cell function and in some cases lead to the progression of ailments or disease. For example, small changes to the protein sequence of a structural nuclear envelope protein called lamin-A is known to cause a variety of neurological and musculoskeletal diseases referred to as laminopathies. Currently, there is little incite into how these mutations lead to disease progression due in part to an inability to measure protein-specific mechanical changes and how these alterations may relate to disruptions in intra-cellular signaling or function. \par To improve upon the ability to measure mechanical properties inside living cells, a previously validated, genetically-encoded resonant energy transfer (FRET)-force biosensor was modified to localize to the nuclear envelope. This biosensor integrated into the nuclear envelope protein Nesprin-2G and senses small deformations that are resolved by indirect measurements of spectroscopic fluctuations in the fluorescent emission of the sensor. To accurately measure these changes, a new spectral-imaging technique named SensorFRET was developed which can resolve small changes in the FRET sensor under varying levels of fluorescent intensity and with known absolute precision. Using SensorFRET, the Nesprin-2G biosensor (Nesprin-TS) reported changes in actomyosin contractility, nuclear shape, and nuclear deformation. Using Nesprin-TS, fibroblasts derived from patients with Hutchinson-Gilford progeria syndrome (HGPS) reported less force on Nesprin-2G molecules relative to healthy fibroblasts on average.\par To demonstrate how intra-cellular forces on the nucleus may impact normal cell physiology, bone-marrow derived mesenchymal stem cells (MSCs) were genetically modified such that the cytoskeleton was decoupled from the nucleus by saturating KASH binding proteins with a non-functional truncated protein called DN-KASH. MSCs treated with DN-KASH preferentially differentiated into osteocytes (bone cells) at a higher rate than MSCs exposed to osteogenic growth factors. This osteogenic preference after DN-KASH treatment was independent of the cell substrate topology and did not significantly alter integrin expression. However, this tendency to differentiate into osteocytes was dependent on substrate stiffness. Overall, the data imply that an intra-cellular force-dependent mechanism connected to the cell nucleus strongly influences MSC differentiation.

Positron Emission Tomography (PET) is frequently used for medical imaging. Maturity and flexibility of PET as an imaging technique has expanded its utility beyond the medical domain. It can be used as a tool for fluid flow studies in opaque fluids and for flow within complex geometry where conventional optical flow measurement approaches fail. This study explores the capabilities of PET as flow measurement tool suited to validation of computational fluid dynamic (CFD) predictions. The MicroPET P4 scanner was used to image the diffusion process in flow around a rod bundle geometry similar to that found in a nuclear reactor fuel assembly. The PET data are compared with results from COMSOL CFD simulation and dye injection images. PET image resolution, acquisition speed and sensitivity are also examined in the context of flow measurement.

<p>Developmental biology is a field in which explorations are made to answer how an organism transforms from a single cell to a complex system made up of trillions of highly organized and highly specified cells. This field, however, is not just for discovery, it is crucial for unlocking factors that lead to diseases, defects, or malformations. The one key ingredient that contributes to the success of studies in developmental biology is the technology that is available for use. Optical coherence tomography (OCT) is one such technology. OCT fills a niche between the high resolution of confocal microscopy and deep imaging penetration of ultrasound. Developmental studies of the chicken embryo heart are of great interest. Studies in mature hearts, zebrafish animal models, and to a more limited degree chicken embryos, indicate a relationship between blood flow and development. It is believed that at the earliest stages, when the heart is still a tube, the purpose of blood flow is not for convective transport of oxygen, nutrients and waster, bur rather to induce shear-related gene expressions to induce further development. Yet, to this date, the simple question of "what makes blood flow?" has not been answered. This is mainly due limited availability to adequate imaging and blood flow measurement tools. Earlier work has demonstrated the potential of OCT for use in studying chicken embryo heart development, however quantitative measurement techniques still needed to be developed. In this dissertation I present technological developments I have made towards building an OCT system to study chick embryo heart development. I will describe: 1) a swept-source OCT with extended imaging depth; 2) a spectral domain OCT system for non-invasive small animal imaging; 3) Doppler flow imaging and techniques for quantitative blood flow measurement in living chicken embryos; and 4) application of the OCT system that was developed in the Specific Aims 2-5 to test hypotheses generated by a finite element model which treats the embryonic chick heart tube as a modified peristaltic pump.</p> / Dissertation

This study combines noninvasive hybrid diffuse optical spectroscopies [near-infrared spectroscopy (NIRS) and diffuse correlation spectroscopy (DCS)] with occlusive calibration for continuous measurement of absolute blood flow (BF), tissue blood oxygenation (StO2), and oxygen consumption rate (VO2) in exercising skeletal muscle. Subjects performed rhythmic dynamic handgrip exercise, while an optical probe connected to a hybrid NIRS/DCS flow-oximeter directly monitored oxy-, deoxy-, and total hemoglobin concentrations ([HbO2], [Hb], and [tHb]), StO2, relative BF (rBF), and relative VO2 (rVO2) in the forearm flexor muscles. Absolute baseline BF and VO2 were obtained through venous and arterial occlusions, respectively, and used to calibrate continuous relative parameters. Previously known problems with muscle fiber motion artifact in optical measurements were mitigated with a novel dynamometer-based gating algorithm. Nine healthy young subjects were measured and results validated against previous literature findings. Ten older subjects with fibromyalgia and thirteen age-matched healthy controls were then successfully measured to observe differences in hemodynamic and metabolic response to exercise. This study demonstrates a novel application of NIRS/DCS technology to simultaneously evaluate quantitative hemodynamic and metabolic parameters in exercising skeletal muscle. This method has broad application to research and clinical assessment of disease (e.g. peripheral vascular disease, fibromyalgia), treatment evaluation, and sports medicine.

This research investigates various applications of diffuse correlation spectroscopy (DCS) on in-vivo human muscle tissue, both at rest and during dynamic exercise. Previously suspected muscle tissue relative blood flow (rBF) baseline shift during extended measurement with DCS and DCS-Near infrared spectroscopy (NIRS) hybrid optical systems are verified, quantified, and resolved by redesign of optical probe and alteration in optical probe attachment methodology during 40 minute supine bed rest baseline measurements. We then translate previously developed occlusion techniques, whereby rBF and relative oxygen consumption rV̇O2 are calibrated to initial resting absolute values by use of a venous occlusion (VO) and arterial occlusion (AO) protocol, respectively, to the lower leg (gastrocnemius) and these blood flows are cross validated at rest by strain gauge venous plethysmography (SGVP). Methods used to continuously observe 0.5Hz, 30% maximum voluntary isometric contraction (MVIC) plantar flexion exercise via dynamometer are adapted for our hybrid DCS-Imagent diffuse optical flow-oximeter in the medial gastrocnemius. We obtain healthy control muscle tissue hemodynamic profiles for key parameters BF, V̇O2, oxygen saturation (StO2), deoxyhemoglobin, oxyhemoglobin, and total hemoglobin concentrations ([Hb], [HbO2], and THC respectively), as well as systemic mean arterial pressure (MAP) and pulse rate (PR), at rest, during VO/AO, during dynamic exercise and during 15 minute recovery periods. Next, we began investigation of muscle tissue hemodynamic disease states by performing a feasibility pilot study using limited numbers of controls and peripheral arterial disease (PAD) patients using the translated methods/techniques to determine the ability of our technology to assess differences in these populations.

In the last fifteen years, a great deal of effort has been put forth, worldwide, for investigating and enhancing various aspects of optical coherence tomography (OCT). This thesis begins with a description of the technique of OCT, and an analysis of its underlying theory. The design and construction of an OCT system is described, with particular emphasis on a novel delay scanning method, and novel signal processing. Application of OCT to non-destructive characterisation of seeds, examination of skin lesions, measurement of fluid flow, and refractive index determination, are then demonstrated. Two technological enhancements to OCT are presented in this thesis. The first, an extended-range Fourier domain optical delay line (FDODL), extends the scan range of the traditional FDODL by a factor of almost 9, by scanning the galvanometer mirror around the region of zero tilt-angle. Polarisation optics are used to prevent light coupling back into the interferometer after only a single pass through the FDODL. A non-coplanar version of the FDODL is also presented, which overcomes the losses associated with the polarisation-based design, but trades off scan range to do so. Both versions of FDODL demonstrated excellent linearity and scan uniformity. The second technology presented here, bifocal optical coherence refractometry (BOCR), affords OCT the ability to measure refractive indices within turbid media. It achieves this by generating two confocal gates within the sample. From knowledge of the system parameters, and measurements of the confocal gate separation, the refractive index within the medium is evaluated to within ±0.01. Refractive index mapping is then demonstrated in a number of turbid samples. Three other applications of OCT are also demonstrated in this thesis. The first is the use of OCT to measure full thickness in lupin seeds. Although OCT could not penetrate the entire thickness of the hull, it is demonstrated that the variation in thickness of the two layers observed with OCT, explained 81% of the variation in thickness of the entire hull measured under a SEM. OCT was then applied, for what is believed to be the first time, in a large scale seed screening program. The second application is a preliminary investigation of the suitability of OCT to aid in the diagnosis of skin lesions. Although our system did not possess sufficient positioning accuracy to enable a direct one-to-one comparison between OCT and histology, a number of correspondences between OCT and histology images were demonstrated. The final application of OCT demonstrated here is a novel phase-locked-loop based demodulation scheme, to perform Doppler OCT. This demodulation scheme demonstrated a dynamic range of 98dB, a velocity range of ±20mm/s, and velocity resolution of 0.5mm/s. Using this system, laminar flow was demonstrated in milk flowing through a capillary tube.

[Truncated abstract] This thesis describes the development, clinical validation and initial application of a technique for taking measurements of the shape and dimensions of the human upper airway, called anatomical optical coherence tomography (aOCT). The technique uses a transparent catheter containing a rotating optical probe which is introduced transnasally and positioned in the airway and oesophagus. Optical coherence tomography is used to take calibrated cross-sectional images of the airway lumen as the probe rotates. The probe can also be advanced or withdrawn within the catheter during scanning to build up three-dimensional information. The catheter remains stationary so that the subject is not aware of the probe motion. The initial application of the system is research into obstructive sleep apnoea (OSA), a serious condition characterized by repetitive collapse of the upper airway during sleep and an independent risk factor for deaths by heart disease, strokes or car accidents. Measurement of upper airway size and shape is important for the investigation of the pathophysiology of OSA, and for the development and assesment of new treatments. . . We have used aOCT to capture three-dimensional data sets of the airway shape from upper oesophagus to the nasal cavity, undertaken measurements of compliance and other airway characteristics, and recorded dynamic airway shape during confirmed sleep apnoea events in a hospital sleep laboratory. We have shown that aOCT generates quantitative, real-time measurements of upper airway size and shape, allowing study over lengthy periods during both sleep and wakefulness. These features should make it useful for study of upper airway behavior to investigate OSA pathophysiology, and aid clinical management and treatment development.

For children with intractable seizures, surgical removal of epileptic foci, if identifiable and feasible, can be an effective way to reduce or eliminate seizures. The success of this type of surgery strongly hinges upon the ability to identify and demarcate those epileptic foci. The ultimate goal of this research project is to develop an effective technology for detection of unique in vivo pathophysiological characteristics of epileptic cortex and, subsequently, to use this technology to guide epilepsy surgery intraoperatively. In this PhD dissertation the feasibility of using optical spectroscopy to identify unique in vivo pathophysiological characteristics of epileptic cortex was evaluated and proven using the data collected from children undergoing epilepsy surgery. In this first in vivo human study, static diffuse reflectance and fluorescence spectra were measured from the epileptic cortex, defined by intraoperative ECoG, and its surrounding tissue from pediatric patients undergoing epilepsy surgery. When feasible, biopsy samples were taken from the investigated sites for the subsequent histological analysis. Using the histological data as the gold standard, spectral data was analyzed with statistical tools. The results of the analysis show that static diffuse reflectance spectroscopy and its combination with static fluorescence spectroscopy can be used to effectively differentiate between epileptic cortex with histopathological abnormalities and normal cortex in vivo with a high degree of accuracy. To maximize the efficiency of optical spectroscopy in detecting and localizing epileptic cortex intraoperatively, the static system was upgraded to investigate histopathological abnormalities deep within the epileptic cortex, as well as to detect unique temporal pathophysiological characteristics of epileptic cortex. Detection of deep abnormalities within the epileptic cortex prompted a redesign of the fiberoptic probe. A mechanical probe holder was also designed and constructed to maintain the probe contact pressure and contact point during the time dependent measurements. The dynamic diffuse reflectance spectroscopy system was used to characterize in vivo pediatric epileptic cortex. The results of the study show that some unique wavelength dependent temporal characteristics (e.g., multiple horizontal bands in the correlation coefficient map g(λref = 800 nm, λcomp,t)) can be found in the time dependent recordings of diffuse reflectance spectra from epileptic cortex defined by ECoG.

Blood vessel mimics (BVMs) are simple tissue engineered blood vessel constructs intended for preclinical testing of vascular devices. This thesis developed and implemented methods to characterize two of these components. The first aim of this thesis investigated the effect of cell culture duration and flow conditions on endothelial cell gene expression, especially regarding endothelial-to-mesenchymal transition (EndMT). A trend of decreased endothelial marker gene expression and increased mesenchymal marker gene expression would indicate EndMT. qPCR analysis revealed that increased cell culture duration did not result in EndMT, and in fact increased endothelial marker expression as cell culture duration increased. Disturbed flow conditions decreased endothelial marker and increased mesenchymal marker expression relative to static culture. The second aim of this thesis developed methods to determine cytotoxicity of, and endothelial cell adhesion to, novel BTEAC salt scaffolds. Immunostaining was used to visualize these scaffold effects. The cytotoxicity elution assay showed that BTEAC salt scaffolds were not more cytotoxic than the standard PLGA scaffold. Direct contact assays spanning several timepoints also found that BTEAC salt scaffolds were not more cytotoxic than standard scaffolds but had higher endothelial cell adhesion and coverage than standard scaffolds. Overall, this thesis developed and implemented methods to characterize the endothelial cells used in the BVM model.