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Endoscopic Optical Coherence Tomography: Design and Application
Get PDFThis thesis presents an investigation on endoscopic optical coherence tomography (OCT). As a noninvasive imaging modality, OCT emerges as an increasingly important diagnostic tool for many clinical applications. Despite of many of its merits, such as high resolution and depth resolvability, a major limitation is the relatively shallow penetration depth in tissue (about 2∼3 mm). This is mainly due to tissue scattering and absorption. To overcome this limitation, people have been developing many different endoscopic OCT systems. By utilizing a minimally invasive endoscope, the OCT probing beam can be brought to the close vicinity of the tissue of interest and bypass the scattering of intervening tissues so that it can collect the reflected light signal from desired depth and provide a clear image representing the physiological structure of the region, which can not be disclosed by traditional OCT. In this thesis, three endoscope designs have been studied. While they rely on vastly different principles, they all converge to solve this long-standing problem.
A hand-held endoscope with manual scanning is first explored. When a user is holding a hand- held endoscope to examine samples, the movement of the device provides a natural scanning. We proposed and implemented an optical tracking system to estimate and record the trajectory of the device. By registering the OCT axial scan with the spatial information obtained from the tracking system, one can use this system to simply ‘paint’ a desired volume and get any arbitrary scanning pattern by manually waving the endoscope over the region of interest. The accuracy of the tracking system was measured to be about 10 microns, which is comparable to the lateral resolution of most OCT system. Targeted phantom sample and biological samples were manually scanned and the reconstructed images verified the method.
Next, we investigated a mechanical way to steer the beam in an OCT endoscope, which is termed as Paired-angle-rotation scanning (PARS). This concept was proposed by my colleague and we further developed this technology by enhancing the longevity of the device, reducing the diameter of the probe, and shrinking down the form factor of the hand-piece. Several families of probes have been designed and fabricated with various optical performances. They have been applied to different applications, including the collector channel examination for glaucoma stent implantation, and vitreous remnant detection during live animal vitrectomy.
Lastly a novel non-moving scanning method has been devised. This approach is based on the EO effect of a KTN crystal. With Ohmic contact of the electrodes, the KTN crystal can exhibit a special mode of EO effect, termed as space-charge-controlled electro-optic effect, where the carrier electron will be injected into the material via the Ohmic contact. By applying a high voltage across the material, a linear phase profile can be built under this mode, which in turn deflects the light beam passing through. We constructed a relay telescope to adapt the KTN deflector into a bench top OCT scanning system. One of major technical challenges for this system is the strong chromatic dispersion of KTN crystal within the wavelength band of OCT system. We investigated its impact on the acquired OCT images and proposed a new approach to estimate and compensate the actual dispersion. Comparing with traditional methods, the new method is more computational efficient and accurate. Some biological samples were scanned by this KTN based system. The acquired images justified the feasibility of the usage of this system into a endoscopy setting.
My research above all aims to provide solutions to implement an OCT endoscope. As technology evolves from manual, to mechanical, and to electrical approaches, different solutions are presented. Since all have their own advantages and disadvantages, one has to determine the actual requirements and select the best fit for a specific application.</p
Measurement of Thermo-Optic Properties of Thin Film Dielectric Coatings
Get PDFWe have carried out an experiment to test the theory of the thermo-optic response of a dielectric stack mirror coating and to measure parameters of interest in calculating thermo-optic noise. Specifically, we measured the coefficient of thermal expansion and the change of index of refraction with temperature (dn/dT ) for thin film silica (SiO2) and tantala (Ta2O5) in mirror coatings. These measurements were achieved by driving thermal fluctuations in such mirrors in one arm of a small Michelson interferometer. We report on the results of that experiment along with its potential implications for future gravitational wave detectors, and suggest next steps for this important line of investigation
A Novel Method for Studying Nucleated Pathways in Membranes: Development and Applications for Gene Delivery
Get PDFThe development of a safe, selective, and efficient gene delivery system is key to the success of human gene therapy. In polymer-based gene delivery systems, biocompatible polymers electrostatically bind and condense the genetic material into protective nanoparticles. These nanoparticles must subsequently overcome several challenges, which remain poorly understood. In particular, once internalized by the cell, the nanoparticles are trapped inside a membrane-bound compartment called the endosome. In the proton sponge hypothesis, the buffering capacity of the polymers leads to an increase in osmotic pressure that eventually ruptures the endosomal membrane and releases the trapped nanoparticles.
To obtain a mechanistic understanding of the endosomal escape, we first develop a coarse-grained model to study the equilibrium interaction between a positively charged nanoparticle and a lipid membrane. Results indicate the existence of a pore with an inserted particle, whose metastability depends on the membrane tension and particle properties (size and charge). These pores are subsequently shown to lower the critical tension necessary for membrane rupture, thus possibly enhancing the release of the trapped genetic material from the endosome.
Next, we address the actual escape pathway, which is likely a thermally nucleated process and cannot be simulated directly or studied by equilibrium methods. Hence, we develop a novel method for studying minimum free energy paths in membranes. Our results indicate that thermally nucleated rupture may be an important factor for the low rupture strains observed in lipid membranes. Under the moderate tensions found in this regime, there are multiple pathways for crossing the membrane: (1) particle-assisted membrane rupture, (2) particle insertion into a metastable pore followed by translocation and membrane resealing, and (3) particle insertion into a metastable pore followed by membrane rupture. This suggests a direct role of the nanoparticle in the endosomal escape not previously envisioned in the proton sponge hypothesis, and illustrates the importance of having an induced tension on the membrane.
Finally, the methodology developed in this work represents the most advanced theoretical technique for describing nucleation pathways in soft condensed matter systems that also include hard-particle degrees of freedom. We expect the method to be useful for studying a wide range of nucleation phenomena beyond membrane systems, for example, in nanoparticle polymer composites.</p
Hele-Shaw Flow Near Cusp Singularities
Get PDFThis thesis discusses the radial version of the Hele-Shaw problem. Different from the channel version, traveling-wave solutions do not exist in this version. Under algebraic potentials, in the case that the droplets expand, in finite time, cusps will appear on the boundary and classical solutions may not exist afterwards. Physicists have suggested that for (2p+1,2)-cusps, that near cusp singularities of Hele-Shaw flow, after scaling X, Y by some powers of time t respectively, the main part of Y(X, t) is a one-parameter family and does not depend on time t. They have also suggested that the solutions of the Hele-Shaw problem are connected with dispersionless KdV (dKdV) hierarchy. In this study, we rigorously proved that this is the case for (3,2)-cusps when the droplets are simply connected and the external potentials are algebraic. We gave exact solutions and showed that the main parts of the exact solutions are some special solutions of the dispersionless string equation. More over, borrowed from the physical paper\cite{Teo} with a little more details, we showed the arguments of how these special solutions are related to dKdV hierarchy
Mechanism of Intestinal Colonization by Symbiotic Bacteria
Get PDFAll animals live in symbiosis. Shaped by eons of co-evolution, host-bacterial associations have developed into prosperous relationships creating mechanisms for mutual benefits to both microbe and host. No better example exists in biology than the astounding numbers of bacteria harbored by the lower gastrointestinal tract of mammals. This community of symbionts establishes a life-long habitat within the distal gut and profoundly impacts host health. Although many recent investigations have led to determination of the microbiota composition, molecular mechanisms mediating establishment and maintenance of the microbial community within the gut is poorly described.
We use gnotobiotic mice to elucidate mechanisms of colonization by Bacteroides, one of the most numerically prominent genera in the intestine. We generate mutant strains of Bacteroides fragilis that lack the ability to express multiple capsular polysaccharides and demonstrate defect in colonization in competition with wild-type strain, suggesting a role for surface sugar architecture during host-symbiont mutualism. Through a functional in vivo genetic screen of colonization, we identify a novel operon from the genome of B. fragilis that is highly conserved among many sequenced intestinal Bacteroides and that mediate a species-specific colonization profile. We have named this genetic locus the commensal colonization factor (ccf). B. fragilis deleted in the ccf genes exhibit colonization defects in both germ-free and complex microbiota harboring mice. The ccf genes of B. fragilis are up-regulated during gut colonization, preferentially at the mucosal surface, supporting an in vivo function. Indeed, deletion of ccf genes leads to reduced mucosal association and a defect in bacterial occupation of colonic crypts of mice. The ability of B. fragilis to repopulate the gut after antibiotic perturbation or gastroenteritis requires expression of ccf, suggesting the niche within colonic crypts represents a colonization reservoir for the gut microbiota following environmental stress. These findings suggest a novel and evolutionarily conserved mechanism for persistent gut colonization by the Bacteroides species.</p
Studies of the N-End Rule Pathway in Saccharomyces cerevisiae
Get PDFMany intracellular proteins are either conditionally or constitutively short-lived, with in vivo half-lives that can be as brief as a few minutes. The regulated and processive degradation of intracellular proteins is carried out largely by the ubiquitin (Ub)-proteasome system (UPS). In eukaryotes, the N-end rule pathway is a part of the UPS. The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. E3 Ub ligases of the N-end rule pathway are called N-recognins. They bind to primary destabilizing N-terminal residues of N-end rule substrates. The N-end rule pathway comprises two major branches, the Arg/N-end rule pathway and the Ac/N-end rule pathway.
The Arg/N-end rule branch involves the N-terminal arginylation of protein substrates and also the targeting of specific unmodified N-terminal residues by E3 N-recognins. The S. cerevisiae Arg/N-end rule pathway contains a single N-recognin, Ubr1. The Ub-fusion degradation (UFD) pathway is also a part of the UPS. This pathway recognizes a "nonremovable" N-terminal Ub moiety of a Ub fusion as a primary degron. My collaborator, Cheol-Sang Hwang, and I demonstrated that the RING-type Ubr1 E3 and the HECT-type Ufd4 E3 interact, both physically and functionally. We showed that the Ubr1-Ufd4 complex targets the S. cerevisiae Mgt1 DNA repair enzyme through a degron near its N-terminus, in addition to mediating the Arg/N-end rule pathway and a part of the UFD pathway as well. We also further characterized the physical interaction between Ubr1 and Ufd4.
I also report the discovery of the other branch of the N-end rule pathway, the Ac/N-end rule pathway, which recognizes N-terminally acetylated residues as N-degrons, termed Ac/N-degrons. We showed that Ac/N-degrons are recognized by the Doa10 E3 Ub ligase and apparently by other E3s as well. Given the prevalence of Ac/N-degrons, as nearly 90% of human proteins are Nt-acetylated, we also demonstrated the physiological role of Ac/N-degrons in protein quality, including the regulation of input stoichiometries of subunits in oligomeric proteins.</p
Improving the Biological Activity of Pyrrole-Imidazole Polyamides
Get PDFDNA is nature’s blueprint, holding within it the genetic code that defines the structure and function of an organism. A complex network of DNA-binding proteins called transcription factors can largely control the flow of information from DNA, so modulating the function of transcription factors is a promising approach for treating many diseases. Pyrrole-imidazole (Py-Im) polyamides are a class of DNA-binding oligomers, which can be synthetically programmed to bind a target sequence of DNA. Due to their unique shape complementarity and a series of favorable hydrogen bonding interactions that occur upon DNA-binding, Py-Im polyamides can bind to the minor groove of DNA with affinities comparable to transcription factors. Previous studies have demonstrated that these cell-permeable small molecules can enter cell nuclei and disrupt the transcription factor-DNA interface, thereby repressing transcription. As the use of Py-Im polyamides has significant potential as a type of modular therapeutic platform, the need for polyamides with extremely favorable biological properties and high potency will be essential. Described herein, a variety of studies have been performed aimed at improving the biological activity of Py-Im polyamides. To improve the biological potency and cellular uptake of these compounds, we have developed a next-generation class of polyamides bearing aryl-turn moieties, a simple structural modification that allows significant improvements in cellular uptake. This strategy was also applied to a panel of high-affinity cyclic Py-Im polyamides, again demonstrating the remarkable effect minor structural changes can have on biological activity. The solubility properties of Py-Im polyamides and use of formulating reagents with their treatment have also been examined. Finally, we describe the study of Py-Im polyamides as a potential artificial transcription factor
Essays on Behavioral and Neuro-economics
Get PDFThis thesis examines foundational questions in behavioral economics—also called psychology and economics—and the neural foundations of varied sources of utility. We have three primary aims: First, to provide the field of behavioral economics with psychological theories of behavior that are derived from neuroscience and to use those theories to identify novel evidence for behavioral biases. Second, we provide neural and micro foundations of behavioral preferences that give rise to well-documented empirical phenomena in behavioral economics. Finally, we show how a deep understanding of the neural foundations of these behavioral preferences can feed back into our theories of social preferences and reference-dependent utility.
The first chapter focuses on classical conditioning and its application in identifying the psychological underpinnings of a pricing phenomenon. We return to classical conditioning again in the third chapter where we use fMRI to identify varied sources of utility—here, reference dependent versus direct utility—and cross-validate our interpretation with a conditioning experiment. The second chapter engages social preferences and, more broadly, causative utility (wherein the decision-maker derives utility from making or avoiding particular choices).</p
Proton Transfers at the Air-Water Interface
Get PDFProton transfer reactions at the interface of water with hydrophobic media, such as air or lipids, are ubiquitous on our planet. These reactions orchestrate a host of vital phenomena in the environment including, for example, acidification of clouds, enzymatic catalysis, chemistries of aerosol and atmospheric gases, and bioenergetic transduction. Despite their importance, however, quantitative details underlying these interactions have remained unclear. Deeper insight into these interfacial reactions is also required in addressing challenges in green chemistry, improved water quality, self-assembly of materials, the next generation of micro-nanofluidics, adhesives, coatings, catalysts, and electrodes. This thesis describes experimental and theoretical investigation of proton transfer reactions at the air-water interface as a function of hydration gradients, electrochemical potential, and electrostatics. Since emerging insights hold at the lipid-water interface as well, this work is also expected to aid understanding of complex biological phenomena associated with proton migration across membranes.
Based on our current understanding, it is known that the physicochemical properties of the gas-phase water are drastically different from those of bulk water. For example, the gas-phase hydronium ion, H3O+(g), can protonate most (non-alkane) organic species, whereas H3O+(aq) can neutralize only relatively strong bases. Thus, to be able to understand and engineer water-hydrophobe interfaces, it is imperative to investigate this fluctuating region of molecular thickness wherein the ‘function’ of chemical species transitions from one phase to another via steep gradients in hydration, dielectric constant, and density. Aqueous interfaces are difficult to approach by current experimental techniques because designing experiments to specifically sample interfacial layers (< 1 nm thick) is an arduous task. While recent advances in surface-specific spectroscopies have provided valuable information regarding the structure of aqueous interfaces, but structure alone is inadequate to decipher the function. By similar analogy, theoretical predictions based on classical molecular dynamics have remained limited in their scope.
Recently, we have adapted an analytical electrospray ionization mass spectrometer (ESIMS) for probing reactions at the gas-liquid interface in real time. This technique is direct, surface-specific,and provides unambiguous mass-to-charge ratios of interfacial species. With this innovation, we have been able to investigate the following:
1. How do anions mediate proton transfers at the air-water interface?
2. What is the basis for the negative surface potential at the air-water interface?
3. What is the mechanism for catalysis ‘on-water’?
In addition to our experiments with the ESIMS, we applied quantum mechanics and molecular dynamics to simulate our experiments toward gaining insight at the molecular scale. Our results unambiguously demonstrated the role of electrostatic-reorganization of interfacial water during proton transfer events. With our experimental and theoretical results on the ‘superacidity’ of the surface of mildly acidic water, we also explored implications on atmospheric chemistry and green chemistry. Our most recent results explained the basis for the negative charge of the air-water interface and showed that the water-hydrophobe interface could serve as a site for enhanced autodissociation of water compared to the condensed phase.</p
Physical Investigations of Small Particles: (I) Aerosol Particle Charging and Flux Enhancement and (II) Whispering Gallery Mode Sensing
Get PDFPart I
Particles are a key feature of planetary atmospheres. On Earth they represent the greatest source of uncertainty in the global energy budget. This uncertainty can be addressed by making more measurement, by improving the theoretical analysis of measurements, and by better modeling basic particle nucleation and initial particle growth within an atmosphere. This work will focus on the latter two methods of improvement.
Uncertainty in measurements is largely due to particle charging. Accurate descriptions of particle charging are challenging because one deals with particles in a gas as opposed to a vacuum, so different length scales come into play. Previous studies have considered the effects of transition between the continuum and kinetic regime and the effects of two and three body interactions within the kinetic regime. These studies, however, use questionable assumptions about the charging process which resulted in skewed observations, and bias in the proposed dynamics of aerosol particles. These assumptions affect both the ions and particles in the system. Ions are assumed to be point monopoles that have a single characteristic speed rather than follow a distribution. Particles are assumed to be perfect conductors that have up to five elementary charges on them. The effects of three body interaction, ion-molecule-particle, are also overestimated. By revising this theory so that the basic physical attributes of both ions and particles and their interactions are better represented, we are able to make more accurate predictions of particle charging in both the kinetic and continuum regimes.
The same revised theory that was used above to model ion charging can also be applied to the flux of neutral vapor phase molecules to a particle or initial cluster. Using these results we can model the vapor flux to a neutral or charged particle due to diffusion and electromagnetic interactions. In many classical theories currently applied to these models, the finite size of the molecule and the electromagnetic interaction between the molecule and particle, especially for the neutral particle case, are completely ignored, or, as is often the case for a permanent dipole vapor species, strongly underestimated. Comparing our model to these classical models we determine an “enhancement factor” to characterize how important the addition of these physical parameters and processes is to the understanding of particle nucleation and growth.
Part II
Whispering gallery mode (WGM) optical biosensors are capable of extraordinarily sensitive specific and non-specific detection of species suspended in a gas or fluid. Recent experimental results suggest that these devices may attain single-molecule sensitivity to protein solutions in the form of stepwise shifts in their resonance wavelength, \lambda_{R}, but present sensor models predict much smaller steps than were reported. This study examines the physical interaction between a WGM sensor and a molecule adsorbed to its surface, exploring assumptions made in previous efforts to model WGM sensor behavior, and describing computational schemes that model the experiments for which single protein sensitivity was reported. The resulting model is used to simulate sensor performance, within constraints imposed by the limited material property data. On this basis, we conclude that nonlinear optical effects would be needed to attain the reported sensitivity, and that, in the experiments for which extreme sensitivity was reported, a bound protein experiences optical energy fluxes too high for such effects to be ignored.</p