BIOMEDICAL ENGINEERING THE EFFECT OF PULSE STRUCTURE ON SOFT TISSUE LASER ABLATION AT MID-INFRARED WAVELENGTHS MARK ANDREW MACKANOS Dissertation under the direction of Professor E. Duco Jansen A series of experimental investigations have demonstrated that targeting a mid-infrared Mark-III Free-Electron Laser to wavelengths near 6.45 Ým results in tissue ablation with minimal collateral damage and substantial efficiency useful for human surgery. Thermodynamic reasoning suggests that the minimal collateral damage at this wavelength is due to the differential absorption of protein and water; which causes compromised tissue integrity by laser heating of the non-aqueous components prior to explosive vaporization. These properties are advantageous for surgery because they reduce the structural integrity of the tissue, thus reducing amount of energy needed for ablation. While the FEL, based on these findings, has been used successfully in eight human surgeries to date, it is unlikely that this laser will become broadly used clinically due to its expense and difficult implementation. Recent developments in conventional laser technology have provided access to this wavelength. While the average and peak power of these sources are still not equivalent to the FEL, recent data indicates that ablation studies are feasible. The research described here investigates the role of pulse structure with regards to soft tissue ablation to determine the feasibility of these sources as potential FEL replacements for clinical applications. Relevant parameters including the threshold radiant exposure and ablated crater depth were studied for comparison of the native FEL micropulse with a stretched FEL micropulse and a ZnGeP2 OPO. Brightfield imaging was used to analyze the effect of pulse structure on the dynamics of ablation, while histology on cornea and dermis was performed to study pulse effects on thermal damage. Mass spectrometry was also used to investigate whether non-linear effects are involved with the FEL micropulse in changing the chemical structure of proteins prior to ablation. The results of this analysis show that the micropulse structure of the FEL does not play a role in the efficient ablation of soft tissue with minimal collateral damage that has been shown previously, and alternative sources remain viable alternatives to the FEL.

This dissertation covers research regarding the use of laser range scanning (LRS) during neurosurgery. Impetus for this work stems from the desire to provide relevant intraoperative data regarding the position and motion of the brain relative to preoperative image tomograms. LRS provides an excellent technology for providing fast, accurate, and well-resolved surface data of the exposed brain. Methods described in this dissertation represent a novel visualization system capable of providing real-time cortical surface characterization. More specifically, techniques have been developed that register intraoperative LRS data to preoperative MR tomograms and quantify the motion of the brain using serial LRS acquisitions. The results generated from these techniques are presented as graphical renderings that provide correspondence between the exposed cortical surface and anatomical structures in the preoperative tomograms. In vivo validation shows that cortical surface registration and motion tracking can be achieved to millimetric accuracy. The impact of these results allows enhanced recognition of cortical structures while providing meaningful assessment of brain deformation during surgery. In summary, this research provides a comprehensive examination of LRS for use within the operating theater and constitutes a significant step toward the use of intraoperative cortical surface data in image-guided neuronavigation.

Pancreatic islet transplantation is a promising treatment for type 1 diabetes. However, current efforts to study islet transplantations are hampered by the lack of a non-invasive method of imaging islets and quantifying islet mass post transplantation. Transplanted pancreatic islets can be imaged and quantified non-invasively using in vivo bioluminescence imaging (BLI). Pancreatic islets transfected with the firefly reporter gene, luciferase, emit light that can be quantified using photon-counting measurements. Pancreatic islet number is linearly related to light emission both in vitro and in vivo. Application of bioluminescence imaging for this application can be greatly enhanced by relating light emission to the number of islets surviving post-transplantation. Determining this relationship requires detailed knowledge of the factors that influence photon-counting measurements. Bioluminescence was modeled using constant light emitting phosphorescent beads implanted at the two common sites of islet transplantation: the renal capsule and liver. This model was used to quantify light attenuation by tissues overlying the islet transplantations. The ratio of implanted light emission to in vitro light emission was found to be .2394+/- 0.0261 for renal implantation and 0.0645 +/- 0.0140 for hepatic implants. Mathematical modeling of light propagation using Monte Carlo simulation is in excellent agreement with these experimental results. Monte Carlo modeling yields an in vivo to in vitro luminescence ratio for renal and hepatic sources to be 0.2860 and 0.0495, respectively. Surgical artifacts were found to influence bioluminescence measurements. Surgical scar tissue leads to lower light emission the week immediately post-op, but this attenuation is negligible two weeks after surgery. The orientation of the subject also influences quantification of bioluminescence. Rotation of 50 degrees from flat can lead to a 73% decrease in light transmission for renal implants and 52% decrease for hepatic implants. The rate of luminescence decrease with increasing angle depends on the surface light is projected upon. Flatter surfaces lead to a slower decrease in luminescence while higher curvature leads to more rapid decrease in luminescence. Spot size of bioluminescence was found to increase with increasing tissue depth. The spot size of hepatic implants was found to be 17% larger than renal implants, as measured by full width at half maximum measurements. Constant light emission modeling of transplanted islet bioluminescence permits quantification of actual islet number from photon counting measurements and insight into factors which influence these measurements.

A new inter-modality surface registration algorithm that uses textured point clouds and mutual information is presented within the context of model-updated image guided procedures. The algorithm has been developed to capitalize on the information generated by a laser range scanner. The current iteration of the algorithm is optimized for cortical surface registration. Intra-modality validation for the algorithm is provided in both physical and imaging phantoms. The physical phantom is generated using a laser range scanner that reports texture coordinates. The imaging phantom is generated from gadolinium enhanced MR volumes of the brain. Simulated inter-modality registration experiments on a cortical surface are also presented. Results of the experiments show successful registration accuracies on the order of the resolution of the surfaces (i.e. submillimetric). The results demonstrate that the registration algorithm and laser range scanner have potential application in deformation tracking during surgery and model-updated image-guided procedures.

Cell encapsulation is a novel therapeutic approach for the treatment of Type I Diabetes Mellitus that circumvents both the immunosuppression and limited allograft donor source dilemma. Current methods for scoring the biocompatibility of the alginate-based capsules that sequester Islets of Langerhans include fabrication and implantation into the peritoneal cavity of mice, incubation for specified periods of time, retrieval via peritoneal lavage, and observation of the number of cells or cell layers surrounding the capsules. This method allows only one data point to be obtained per animal. In this experiment we propose to measure biocompatibility real time and in vivo This new method of monitoring immune response using bioluminescent technology and a Nuclear Factor-kappa Beta sensitive transgenic mouse model allows unlimited data points to be acquired per animal, reduces the number of animals required to obtain statistically significant immune response data over time, and in turn reduces error associated with animal variability. NF-kB is a transcription factor that plays a critical upstream role in the coordination of the inflammatory and would healing cascades by initiating the transcription of many cytokines, chemokines, adhesion molecules, and proinflammatory genes. Five types of capsules were monitored over 6 six weeks after transplantation into the dorsal-cervical fat pad, a capsule group, a bead group, a non-coated capsule group, a sham surgery group, and a control group. The bead, capsule, and non-coated capsule transplant groups allow the effects of capsule size and capsule wall composition on NF-kB activity to be monitored. This imaging modality was able to detect statistically significant differences in NF-kB activity between pre and post-operative data points per mouse. It was also able to discern with significance an unexpected increase in NF-kB activity due to capsule size instead of capsule wall composition over a six week time period.

Selective targeting of damaged or diseased cells is a concept with great potential to revolutionize the efficacy of systemically administered agents. Successful targeting preferentially delivers imaging agents and/or therapies to specific tissues, enhancing detection and diagnosis as well as therapies minimizing damage to nontarget cells. However, the current generation of targeted therapies has not yet generally achieved highly specific targeting of tumors and emerging neoplasia through a single recognition mechanism. Effective performance of targeted systems depends upon recognition of unique characteristics on the cellular surface. HT-1080 cells in vitro present 3,840,000 ± 70,000 CD13 receptors per cell, a level presumably suitable for effective targeting. Receptor presentation can be further modulated by exposure to factors such as ionizing radiation and various cytokines, presenting opportunities to modify receptor expression for optimized delivery of targeted constructs. Single modality targeted nanoscale quantum dots (QDs) were synthesized to enable specific interaction with target cells. Nonspecific interaction was limited by functionalizing the QD surface with a passive PEG coating. To facilitate specific binding to target receptors, QDs were surface-functionalized with targeting peptides. The resulting constructs (QD-PEG-NGR) bound to the surface of HT-1080 cells at a level of 15,410 ± 980/cell, enabling high contrast imaging and the potential for significant therapeutic delivery. To overcome specific limitations that plague current single-modality targeting technologies, a multifunctional QD-based proximity-activated (PA) targeting system was developed. QDs were functionalized with a proteolytically sensitive PA coating designed to mask an underlying targeting ligand. Specific matrix metalloprotease-7 (MMP-7) activity resulted in maximal cleavage of 90.9 ± 15.4% of the available PA structures from the QD surface. Effective cleavage was measured at exposure times and enzyme concentrations consistent with estimated in vivo conditions. Multifunctional NPs offer opportunities unavailable with molecular structures or current single-modality targeted constructs. Effective targeting, imaging, and delivery to specific cells can be achieved with the two-component targeting methodology detailed in this work. Application of these targeting methodologies may offer a powerful system to significantly improve cancer treatment.

Fundamental clinical approaches for assessing patient vital signs have changed little since the first invasive blood pressure measurements were made over 100 years ago. Interpreting patient physiology remains largely a manual, intermittent process, despite evidence suggesting that automated processing of continuously-captured physiologic data will yield new, important measurements. These new vital signs may predict patient improvement or deterioration, and signal specific opportunities for early therapeutic intervention in clinically meaningful, cost-effective ways. However, tools and methods to discover, refine, and validate new vital signs in working clinical settings, across large patient populations, have been lacking. This work describes the SIMON (Signal Interpretation and Monitoring) system, and its application to the discovery, refinement, and validation of a prototype new vital sign, integer heart rate variability (HRV). SIMONs modular architecture enables a high degree of reliability and scalability for dense physiologic data capture, processing, and decision support tasks. The system has been in use continuously since 1998 in the Vanderbilt trauma intensive care unit (ICU), provides physiologic data reporting, display, and alerting capabilities, and has archived physiologic data from over 3500 patients. Its alphanumeric pager alerting functionality has been evaluated in the domain of intracranial pressure management. Additionally, a new measurement of HRV has been developed, refined, and validated in a population of over 1000 trauma patients. The result is not only a new predictor of mortality but also represents proof of concept that a working intensive care unit can serve as a rich, automatic source of data to discover new predictive patterns in patient physiology. Ultimately, study of HRV and other new vital signs may correlate failure of the autonomic nervous system or other neural and hormonal communication pathways with specific injuries, diseases, or patient characteristics. These studies could, in turn, illuminate regulatory mechanisms uniting systems, organs, cells, proteins, and genes. Such knowledge provides a basis for additional research, and informs design of the next generation of ICU monitors and decision support tools to improve quality and efficiency of medical care.

Recent studies have demonstrated that organotypic raft cultures serve as an excellent model for the optical behavior of actual tissues. Other studies in our lab have demonstrated that Raman spectroscopy can discriminate among basal cell carcinoma, squamous cell carcinoma (SCC), non-normal benign, and normal skin in vivo. The primary purpose of this work is to understand the biologic basis of skin Raman spectra and to determine the impact of the location of SCC cells within raft cultures on the spectral bands. Rafts were constructed with SCC cells in the stroma or in the epidermis, and measurements were taken at multiple time points with a Raman probe system. The data allowed tissue discrimination as before, and they showed that the location and concentration of SCC cells do not have a large influence on macroscopically-gathered data. In addition, data gathered from the epidermal vs. stromal layers, combined with histology, support the hypothesis that Raman spectroscopy can detect biochemical changes associated with malignancy before such changes are evident via histology.

Coronary heart disease is the single largest cause of mortality in the United States. Approximately 335,000 people die annually from sudden cardiac death, and the majority of these cases are believed to be from ventricular fibrillation. To effectively treat and prevent cardiac rhythm disturbances, the response of the heart to electrical stimulation must be understood. Although cardiac defibrillation therapy is an invaluable medical procedure, the mechanisms by which strong electrical shocks terminate potentially lethal fibrillation are still debated. Bidomain models of cardiac tissue successfully characterize many of the effects of electrical stimulation of the heart. In the bidomain, the intracellular and extracellular spaces are distinct and have differing electrical anisotropies. With unequal anisotropy ratios, bidomain theory predicts simultaneous positive and negative polarization in response to stimulation, in the form of virtual cathodes and anodes that lead to interesting cardiac activation dynamics. This research examined experimentally the response of cardiac tissue to electrical stimulation from a bidomain perspective. Changes in transmembrane potential during and following electrical stimulation were recorded optically using a voltage-sensitive fluorescent dye. Optical mapping allows noninvasive measurement with high spatiotemporal resolution and avoids electrical stimulus artifacts. We found that: 1. During unipolar anodal stimulation of diastolic tissue, the mechanism of excitation depends upon the extracellular potassium concentration. 2. With proper timing of unipolar stimulation close to refractoriness, damped waves with diminished amplitude and velocity either gradually die or sharply increase in amplitude after a delay to become a steadily propagating wave. 3. We confirmed bidomain model predictions that virtual electrodes from unipolar stimulation affect excitability through the cardiac cycle as shown by strength-interval curves. 4. Field stimulation of the diastolic heart revealed that increasing shock strength and duration do not necessarily result in faster activation because of virtual anode polarization. 5. Alternating regions of positive and negative virtual electrode polarization around an insulating heterogeneity occur during field stimulation and may affect plunge electrode measurements. An increased understanding of how cardiac tissue responds to electrical stimulation in various conditions will guide improvements in treatment and prevention of cardiac rhythm disorders.

Two attractive features of ELISA are the specificity of antibody-antigen recognition and the sensitivity achieved by enzymatic amplification. We describe the development of a non-enzymatic virus detection platform based on circumferential bands of antibody probes coupled to a 120 mm diameter polyester filament. Automated processing was achieved by sequential positioning of filament-coupled probes through a series of liquid filled glass microreaction chambers. Antibody regions were first positioned within a microcapillary tube containing a solution of M13KO7 virus before being moved through subsequent chambers, where the filament-coupled probes were washed, exposed to a fluorescently labeled detecting antibody, and washed again. Using anti-M13KO7 mAb coupled to a polyester filament, the presence of 8.3 x 108 M13KO7 virus particles produced a 30-fold increase in fluorescence over an immobilized negative control antibody. Similar to ELISA, this filament-based approach had a lower limit of sensitivity of ~1.7 x 107 virus particles. We then combined the automated filament processing with an integrated laser-based optical detector to enable real-time controlled detection of virions in solution. A 638 nm laser with a photomultiplier at a right angle provided continuous monitoring for the presence of the fluorescently labeled detecting antibody. A virus incubation time of 1 minute detected 1010 virions/mL. Repeated incubations of antibody regions in either the virus or labeled antibody chambers increased fluorescence roughly proportional to the incubation times. This technology was used to identify and characterize a reovirus strain. We developed a decision tree that tested for reovirus with increasing specificity at each level of the tree. Using three types of reovirus and one bacteriophage, our system correctly detected and identified all three reovirus strains at a concentration of 2 x 1012 virions/ml and M13K07 phage at 3 x 1011 virions/ml. Fluorescence from all peak regions was determined to be significantly higher that background regions (p < 0.05). Using online feedback to guide testing, this scheme could easily be expanded into a much more complicated system with numerous levels and branches. This platform may prove attractive for point-of-care settings, the detection of biohazardous materials, or other applications where sensitive, rapid, and automated molecular recognition is desired.