Endoscopic orbital procedures are hindered by both the difficulty in differentiating between orbital structures and the loss of orbital landmarks during these procedures. These difficulties are due to the orbital fat that obstructs direct vision of the orbital structures. Image guidance can address these problems because real time image and physical space tracking information can be provided to the surgeons during the orbital procedure to help in the delivery of therapy to the orbit. Image guidance requires an image-space to physical-space registration and tracking in physical-space with a localizer. To effectively use a magnetic localizer for transorbital guidance, the error metrics must be characterized so that expected guidance errors can be determined. After characterizing the magnetic tracker, the registration of the physical-space to image-space needs to be addressed. Since the target in this research is the optic nerve, a structure which can be anywhere in the retroorbital pyramid, a new form of fiducial placement is created. In this method the retroorbital pyramid was sampled and a fiducial placement which minimized TRE throughout the possible location of the optic nerve head was determined. After characterizing the magnetic localizer and determining an optimal fiducial placement for the task of optic nerve drug delivery, the performance of the system had to be tested. An experimental protocol which allowed performance quantification in an application mimicking manner was developed. Performance metrics from that protocol were gathered on a number of surgeons.

Diffusion tensor imaging (DTI) is a magnetic resonance imaging (MRI) technique that provides information about the organization and structural integrity of tissue. It has become an increasingly popular tool for investigation of white matter tissue in the brain emph{in vivo}, with clinical applications ranging from myelin-related diseases, such as multiple sclerosis and Krabbe disease, to psychiatric disorders, such as schizophrenia and bipolar disorder. In studies comparing DTI data between groups, whole-brain, voxel-wise analyses are commonly performed. However, there is not enough information in the scalar images typically used for image co-registration to accurately align specific fiber pathways within large white matter structures. This misalignment potentially results in decreased sensitivity to detecting subtle changes within specific tracts and makes interpretation of results more difficult in studies of disorders where it is suspected that changes in white matter diffusion properties are pathway-specific. In an effort to overcome this limitation, several tract-based analysis methods that utilize fiber tractography to isolate specific tracts of interest have recently been proposed. However, the majority of these methods have been developed for particular white matter pathways and are not easily translated to other tracts, or compare the average of diffusion parameters over the entire tract, overlooking localized changes within the tracts. Proposed here is a new tract-based method that utilizes the image co-registration necessary for a voxel-wise analysis to automatically isolate white matter tracts-of-interest in each subject with fiber tractography and parameterize them so that spatially localized statistical comparisons between groups can be made. It was applied to studies of schizophrenia and Williams syndrome, and the results were compared to voxel-based analyses of these studies. Our results suggest that the new tract-based analysis method performs better than the voxel-based method in regions where image co-registration performs poorly and in regions where image co-registration appears to perform well but actually fails to align smaller tracts within larger white matter structures. This new tract-based analysis method, used in combination with a voxel-wise analysis, may be able to improve the interpretation of results from group comparisons of diffusion tensor imaging data.

The processes of tumor growth and treatment response are associated with the upregulation of numerous proteins [1, 2], yet current clinical imaging methods of cancer characterization monitor only gross morphology [3, 4]. This study combines in vivo diffusion weighted magnetic resonance imaging (MRI) with matrix-assisted laser desorption ionization (MALDI) analysis of healthy and tumorous ex vivo specimens in order to examine the proteomic influences on the apparent diffusion coefficient provided by MRI. Spatial co-registration of MALDI and ADC datasets enables examination of the statistical correlations between the two metrics [5] in the hopes of elucidating the proteomic signatures that give rise to particular ADC values. <p> ADC and MALDI data were acquired for two rats, one control and one in which a C6 glioma model of brain cancer was implanted. Principal component analysis was conducted to determine the degree of spatial correlation between the ADC and protein measurements. It was found that ADC and MALDI data correlated significantly (p-value of 0.05) in 44.0% of 114 regions of interest (ROIs) in the two rats. Protein profiles were identified which correlated with statistically similar ADCs in selected ROIs for each rat. The results of this study are consistent with the theory that protein expression in both healthy and tumorous rat brain tissue is a molecular-level source of contrast in diffusion-weighted MRI.

The failure of synthetic vascular grafts due to de-endothelialization of the lumen as a result of exposure to fluid-induced shear stress prevents the widespread use of such grafts as small-diameter vessel replacements. Physical surface modification, an approach that seeks to alter the topography of the luminal surface, has been investigated as a method of reducing de-endothelialization under physiological stresses. Based on prior experimental evidence supporting this approach, computation fluid dynamics was used to investigate the impact of selected channel geometry parameters (wall angle, channel width, depth, and radius of curvature) on fluid flow and the resulting wall shear stress. Optimization of these parameters was performed in order to determine if micro-topographical modification of the lumen wall could alter fluid flow in a manner such that favorable conditions for both endothelial cell retention and stimulation are produced. It was found that a 50% decrease in the wall angle, width, and depth causes a decrease in maximum wall shear stress of around 8%, 8%, and 15%, respectively. Additionally, increasing the radius of curvature at the top and bottom edges by 50% results in a 10% decrease in maximum wall shear stress. These results indicate that it may be possible to tune the lumen micro-topography in order to provide a desired range of stresses, and subsequently reduce thrombogenicity by enhancing endothelial cell retention and function.

The emerging field of molecular imaging (MI) aims to noninvasively, quantitatively and repetitively monitor biological processes in vivo to detect disease, probe its basis, and study relevant biochemical pathways at the molecular level. Since molecular targets undergo alterations prior to morphological or physical transformations, MI should aid in early detection and improved diagnosis of disease, resulting in improved clinical outcomes and enhanced long-term patient survival. In addition, the capability to monitor lesion physiology in vivo may facilitate therapeutic efficacy monitoring, speed drug discovery, and potentially lead to patient-specific treatment regimens. Optical MI, particularly in the near infrared (NIR) wavelength region, is an inexpensive technique that provides relatively high sensitivity without the use of ionizing radiation. Fluorescence imaging is rapid, allowing for dynamic, real-time monitoring of agent biodistribution and clearance profiles and is commonly performed concurrently on multiple animals in a relatively high-throughput manner. The ultimate success of optical MI depends on the development, characterization and optimization of probes as well as superior instrumentation to accurately detect, localize and quantify these unique MI compounds. The overall objectives of this dissertation were directed at quantitative in vitro and in vivo characterization of two novel MI agents developed in our laboratory: a peripheral benzodiazepine receptor (PBR)-targeted NIR MI agent (NIR-conPK11195) and a potential optical analogue to the 2-[18F]fluoro-2deoxy-D-glucose (18FDG) positron emission tomography agent (NIR-glucosamine). Specific Aims I and II demonstrate the utility of NIR-conPK11195 for breast cancer screening and monitoring as well as for studying breast cancer metastases to the brain, respectively. The dose-dependent and PBR-specific cellular uptake of NIR-conPK11195 can be quantified in live-cell competition assays, visualized by fluorescence microscopy, and monitored in vivo. In Specific Aim III, NIR-glucosamine appears to preferentially label tumor tissue in vivo, but with a potential size and/or vascularity requirement for appreciable tumor-specific contrast. Furthermore, several observations suggest that NIR-glucosamine does not follow the GLUT/hexokinase pathway and may label tumors in a non-specific manner.

Image-guided surgery (IGS) has emerged as a valuable tool for the interactive incorporation of pre-operative image data into the surgical setting. While a majority of research has focused on neurosurgical procedures, the feasibility of implementing IGS methods in hepatic procedures has become evident. However, a lack of robustness in the methods developed for performing image-guided liver surgery (IGLS) has impaired their utility. In order to improve the robustness of the algorithms used within IGLS, the incorporation of salient anatomical features that can be reliably identified on the hepatic anatomy is proposed within this work. More specifically, these anatomical features can be weighted within the performance of the image-to-physical space mapping, or registration, that is required for display of the intra-operative location of surgical instruments within the context of pre-operative image data. Additionally, the salient anatomical feature registration can be used to quantify the extent of soft tissue deformation that is known to compromise the intra-operative registration due to the use of rigid body assumptions in determining the mathematical mapping. The quantification and analysis of soft tissue deformation within the context of IGLS provides unique insight into the design of algorithms that can be used to compensate for the shift induced guidance errors. Based on the deformation studies, a novel atlas-based model updating method is proposed for the improvement of IGLS guidance accuracy. The atlas-based method relies on the utilization of pre-operatively computed model solutions to expedite the determination of a non-rigid image-to-physical space mapping. Further, the atlas-based method incorporates the hepatic salient anatomical features to improve the robustness of the method.

Non-specific antibody binding limits the sensitivity of antibody-based detection technologies. We explore the use of logical operations among DNA logic tags associated with antibodies to increase specificity and sensitivity. DNA sequences were developed to perform a logical NOT operation to subtract non-specific binding prior to PCR amplification. Antibody-associated tags A and A' were designed to be partially complementary and contain a restriction enzyme site. Tag A is associated with a specific antibody; tag A' is associated with an isotype matched control antibody. If the concentration of A is greater than A', hybridized AA' is enzymatically cleaved and remaining tag A is subsequently amplified during real-time PCR. Quartz crystal microbalance (QCM), DNA agarose gels, and PCR were used to experimentally characterize components of the NOT operation in respiratory syncytial virus (RSV) detection. QCM showed gold nanoparticles functionalized with both tag DNA and antibody can bind virus. Successful enzymatic cleavage of AA' was visualized on a DNA agarose gel. After cleavage, remaining tag A was amplified by the addition of primers and standard real-time PCR. In the presence of RSV, magnetic pulldown led to the delivery of both tag A and A'. When PCR was run after enzymatic cleavage, the PCR Ct was increased, indicating a decrease in initial tag A. Our results suggest that combining the careful design of DNA logic tags, their association with antibodies, and standard molecular biology techniques is a promising approach to increase the specificity and sensitivity of antibody-based detection methods.

BIOMEDICAL ENGINEERING QUANTUM DOT-BASED IN VIVO IMAGING OF INFLAMMATION ASHWATH JAYAGOPAL Thesis under the direction of Professor Frederick R. Haselton The recruitment of leukocytes to tissue and their specific interactions with adhesion molecules are essential processes which provide for a natural mechanism of localizing immune defense. However, inappropriate immune responses can manifest as harmful inflammation in a variety of diseases. Much information has been derived from immunohistochemistry and in vitro cell culture studies, which have identified various inflammatory mechanisms and mediators. A key challenge has been the in vivo investigation of detailed cellular and molecular events in real-time, such as leukocyte extravasation. A variety of cell adhesion molecules and cell types facilitate these activities, and the ability to monitor their recruitment and proliferation would likely have an impact on the development of diagnostic and therapeutic avenues. Current imaging techniques to characterize these events are limited by low signal to background ratios, invasiveness, and fading. In this study, we use fluorescence microscopy and quantum dot-antibody conjugates to specifically investigate the expression of the cell adhesion molecule VCAM-1 in diabetic rats. The retina was used to non-invasively probe inflammatory activity in the circulation, and to investigate the impact of diabetes-induced inflammation on ocular complications. We report elevated VCAM-1 levels in diabetic rats relative to untreated controls. In addition, we observed ex vivo and in vivo quantum dot-labeled leukocytes in the healthy and diseased retinal circulation. Features of this technique include stable, high intensity of labeled species and a narrow, size-tunable emission spectra. These studies demonstrate the promise of quantum dots in the in vivo visualization of molecular and cellular inflammatory mediators. Approved: Date: Frederick R. Haselton 7/29/05

Corneal wound healing is a complex, and highly coordinated process of cell-cell signaling, cell spreading, migration, proliferation, and cell-extracellular matrix interaction. The main limitation in natural wound healing is that cells can only fill in the wound void from the outside wound margin. We hypothesize that epithelial cells applied to the interior of a wound will attach to the exposed extracellular matrix of the wound, and accelerate the healing process. In order to deliver epithelial cells to wounds, we have developed a cell transfer contact lens. Our objective was to produce and evaluate transfer contact lens designs that allow for primary epithelial cell attachment and in vitro culture growth, and in addition after application result in the transfer of a portion of these cells to wound areas to aid in re-epithelialization of corneal injuries. In the process of meeting these design criteria, we explored the use of surface topographies and surface coatings to enhance cell transfer. We found that cell transfer from Polydimethylsiloxane contact lenses to extracellular matrix was a function of both cell motility and adhesion. In order to enhance cell transfer through the modulation cell motility and adhesion, we explored some of the underlying biological mechanisms of these two inter-related cellular behaviors. Though there were many potential molecular targets to augment cellular adhesion and motility, we specifically explored the regulation of cell-cell contacts, which have a profound impact on cell phenotype and migration. Normal corneal epithelial cells are motile cells that move slowly as a sheet. In contrast, fibroblasts move more quickly as individual cells. When epithelial cells exist as individual cells at very low cell density, they are more motile and more closely resemble fibroblast cells. Individual epithelial cells do not exhibit cell junction proteins. In this investigation we altered Bves, a protein we have shown is associated with tight junctions, in an effort to make epithelial cells become more like fibroblasts regardless of cell density. This transition from an epithelial cell type to a fibroblastic cell type is called epithelial-mesenchymal transition, and has broad reaching implications for other fields such as metastasis in cancer biology.

Cancer is a complex and adaptable disease, and knowledge of the mechanisms that cause its progression is vital to creating and monitoring anti-cancer therapies. As a tumor grows beyond a few mm3, blood vessels are recruited to provide additional nutrients in a process called angiogenesis. Some novel drug therapies specifically target this process, and the efficacy of these drugs can in principle be monitored by a technique called angiography. 3D angiography, a method of imaging the vasculature, can be performed by several imaging modalities typically with the use of a contrast agent. A technique is introduced which uses high resolution ultrasound in conjunction with an ultrasound contrast agent to produce 3D images of the vasculature. This method offers a faster, more accessible, and cheaper alternative to assess the efficacy of anti-angiogenic drugs in preclinical cancer models. In addition to vessel imaging, modeling the kinetic behavior of the contrast agent in the vasculature can elucidate parameters such as blood flow, which can also serve as an indicator of drug treatment efficacy. The repeatability of a commonly used mono-exponential model is assessed in order to determine thresholds for inter/intra-subject error. The 3D vessel imaging technique presented in this thesis correlated with other measures of blood flow (r = 0.55 ± 0.04, p < 0.01) and shows an increased sensitivity to microvasculature within tumors. Also, preliminary repeatability analysis (n = 6) on the modeling parameter which is proportional to contrast agent velocity shows a mean difference of 0.061 ± 0.298 between independent measurements, and the limits of agreement range from -0.536 and 0.656. The developments exhibited provide additional methods for monitoring longitudinal anti-angiogenic cancer treatments in preclinical models.