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Seeing Through the Fog: Using Scattered Light to Peer Deeper into Biological Tissue
Get PDFOptical scattering is a fundamental problem in biomedical optics and limits most optical techniques to shallow operating depths less than 1 millimeter. However, although the scattering behavior of tissue scrambles the information it contains, it does not destroy it. Therefore, if you can unscramble the scattered light, it increases the accessible imaging depths up the absorption limit of light (several centimeters deep).
One such way to beat optical scattering is using wavefront shaping. Borrowing ideas from adaptive optics in astronomy and phased arrays in radar and ultrasonic imaging, the basic concept of wavefront shaping is to control the phase and amplitude of the light field in order to harness scattered light. Using wavefront shaping techniques, scattered light can be used to form focal spots or transmit information through or inside optically scattering media. Furthermore, even without correcting for scattering directly by shaping the input light field, the properties of the scattered light can be analyzed to recover information about the structure and dynamic properties of a sample using methods from diffuse optics.
The main contributions of this thesis are along these two lines of research: moving wavefront shaping toward more practical applications and developing new techniques to recover useful physiological information from scattered light. This is developed through three main projects: (1) an investigation of how dynamic samples impact the scattering process and the practical implications of these dynamics on wavefront shaping systems, (2) the development of a wavefront shaping system combining light and ultrasound to focus light inside acute brain slices to improve light delivery for optogenetics, (3) a novel method to sensitively detect the dynamics of scattered light and use it to tease out information about the flow of blood within the tissue sample of interest.</p
Bioanalytical Tools to Develop Rapid Diagnostics and Study Physiology
Get PDFThis work details the development of bioanalytical tools for use in rapid diagnostics (Chapters 2-4) and in the study of physiology (Chapters 5-6). This research harness the power of real-time, singlemolecule microfluidics to study loop-mediated isothermal amplification in urinary tract infections (Chapter 2), chlamydia (Chapter 3), and gonorrhea (Chapter 4). In Chapter 5, non-reactive beads are designed and optimized to study the impact of polymers on murine gastrointestinal mucosa. Chapter 6 details the implementation of a mass spectrometry method to quantify bile acids and investigate their interaction with the microbiota in the murine gastro-intestinal tract.</p
Iron-Oxide Geochronology to Constrain the Formation of Soils and Paleosols
Get PDFIn this thesis, I show how iron-oxide geochronology can be applied to soils and paleosols and I explore new applications of the (U-Th)/He and cosmogenic 3He dating methods. In Chapters II and III, I apply the (U-Th)/He method to goethite pisoliths in paleosols and fissure fillings of the Bohnerz deposits, which are a widespread erosional lag deposit in Central Europe. I show that this deposit formed between ~55 Ma and 2 Ma and not in the late Cretaceous-early Eocene, as was previously claimed. A map compiled from published sources shows the paleo-extent of the Bohnerz deposit, which were developed on every limestone plateau in Central Europe. Concentrations of cosmogenic 3He measured on pisoliths in paleosols, demonstrate that ancient cosmic-ray exposure occurred over at least 5 Ma, possibly 10-20 Ma. This shows that the Bohnerz deposits represent surfaces, which were stable for tens of millions of years before burial. Even today, these surfaces experience extremely low erosion rates of ~0.1 m/Ma, comparable to some of the Earth's most stable surfaces in arid environments. The hiatus represented by the Bohnerz unconformity lasts 125-150 Ma, yet only 30-40% of that duration is recorded in Bohnerz deposits. Pisoliths of the Bohnerz deposits are a continuous record of Central European continental climate for most of the Cenozoic.
In Chapter IV, I develop a laboratory technique to enable (U-Th)/He dating of hematite samples by the single-aliquot method. Highly retentive hematite samples have to be heated to >1000 °C to fully degas helium, but U is lost from the sample at around 980 °C. Through infrared spectroscopy and trace element analysis of heated samples, I show that U-loss correlates with a phase change from hematite to magnetite. Delaying this phase change to higher temperatures extends the usable range of temperatures to which samples can be safely heated without U-loss. This is achieved by degassing samples in a partial oxygen pressure of around 100 mbar, which permits degassing of samples up to 1150 °C without loss of U. I demonstrate that precise and accurate (U-Th)/He ages can be obtained for hematite samples, which agree with established two-aliquot ages. I show how this method can be implemented and automated.
In Chapter V, I extend the use of 3He cosmogenic dating to fine-grained iron-oxide particles, which are abundant in most types of modern soils. Diffusion modeling predicts that hematite particles down to ~10 nm should quantitatively retain helium for at least 1 Ma at Earth-surface conditions. In order to test whether pedogenic iron-oxides can be used for geochronology, I study a vertical profile of a relict soil developed on a fanglomerate terrace at Whitewater Hill, California. Profiles of 10Be and 26Al in detrital quartz agree well with an exponential decrease in cosmogenic nuclide production and they yield an exposure age of 52.4±2.2 ka assuming no erosion. The vertical profile of 3He concentrations in pedogenic iron-oxides shows a decrease with depth, but concentrations are higher than expected in the 40-100 cm depth range. This indicates vertical movement of iron-oxides in the soil, which is a well-known soil formation process. These observations are more consistent with a soil age of 208±44 ka. This approach yields information on both age constraints and the formation and migration of pedogenic iron-oxides in the soil column.</p
Fabrication, Mechanical Characterization, and Modeling of 3D Architected Materials upon Static and Dynamic Loading
Get PDFArchitected materials have been ubiquitous in nature, enabling unique properties that are unachievable by monolithic, homogeneous materials. Inspired by natural processes, man-made three-dimensional (3D) architected materials have been reported to enable novel mechanical properties such as high stiffness- and strength-to-density ratios, extreme resilience, or high energy absorption. Furthermore, advanced fabrication techniques have enabled architected materials with feature sizes at the nanometer-scale, which exploit material size effects to approach theoretical bounds. However, most architected materials have relied on symmetry, periodicity, and lack of defects to achieve the desired mechanical response, resulting in sub-optimal mechanical response under the presence of inevitable defects. Additionally, most of these nano- and micro-architected materials have only been studied in the static regime, leaving the dynamic parameter space unexplored.
In this work, we address these issues by: (i) proposing numerical and theoretical tools that predict the behavior of architected materials with non-ideal geometries, (ii) presenting a pathway for scalable fabrication of tunable nano-architected materials, and (iii) exploring the response of nano- and micro-architected materials under three types of dynamic loading. We first explore lattice architectures with features at the micro- and millimeter scales and provide an extension to the classical stiffness scaling laws, enabled by reduced-order numerical models and experiments at both scales. After discussing the effect of nodes (i.e., junctions) on the mechanical response of lattice architectures, we propose alternative node-less geometries that eliminate the stress concentrations associated with nodes to provide extreme resilience. Using natural processes such as spinodal decomposition, we present pathways to fabricate a version of these materials with samples sizes on the order of cubic centimeters while achieving feature sizes on the order of tens of nanometers. In the dynamic regime, we design, fabricate, and test micro-architected materials with tunable vibrational band gaps through the use of architectural reconfiguration and local resonance. Lastly, we present methods to fabricate carbon-based materials at the nano- and centimeter scales and test them under supersonic impact and blast conditions, respectively. Our work provides explorations into pathways that could enable the use of nano- and micro-architected materials for applications that go beyond small-volume, quasi-static mechanical regimes.</p
Evolution and Characterization of Carbene Transferases for Cyclopropanation and Carbon–Silicon Bond Formation
Get PDFHeme proteins have recently been demonstrated to catalyze cyclopropanation reactions via a putative carbene transfer mechanism. Carbene transfer reactions are not known to occur in natural biological systems, but are highly useful synthetic reactions. There is growing interest in developing new "carbene transferases" that bring new chemical reactions into the realm of biology, and growing interest in engineering these enzymes for use in organic synthesis. Additionally, the mechanistic details of iron porphyrin-catalyzed carbene transfer reactions are largely unknown, especially with regards to how the enzyme environment influences the outcome of a carbene transfer reaction. This thesis details both the engineering of carbene transferases with novel catalytic capabilities and investigations into how these enzymes catalyze carbene transfer reactions. Chapter 1 introduces heme protein-catalyzed carbene transfer reactions and describes the directed evolution of new enzymes that allow access to a range of useful cyclopropane products. Chapter 2 describes the evolution of an enzyme that performs carbene transfer to silicon–hydrogen bonds, resulting in a highly efficient and selective carbon–silicon bond-forming enzyme, the first of its kind. Chapter 3 focuses on the characterization of a key reactive intermediate, the iron-porphyrin carbene, in the active site of the evolved carbon–silicon bond-forming enzyme. This study provides an explanation of the remarkable enantioselectivity of the enzyme and provides a foundation from which to investigate the enzyme reaction mechanism. The mechanism of carbon– silicon bond formation is elucidated in Chapter 4, and the is then used to explain how the enzyme achieves chemoselectivity, which in turn guides the evolution of enzyme variants with altered chemoselectivity. Finally, two off-cycle catalytic pathways that cause inactivation of the carbene transferase are characterized, and methods to prevent and/or circumvent inactivation are investigated (Chapter 5). Overall, the work presented here expands the repertoire of enzyme-catalyzed reactions and facilitates the continuing development of new carbene transferases by developing our mechanistic understanding of this novel class of enzymes.</p
Engineering Acoustic Protein Nanostructures for Non-Invasive Molecular Imaging using Ultrasound
Get PDFVisualizing biomolecular and cellular processes in real time within deep tissues is fundamental to our understanding of the normal and pathological activity underlying health and disease. Ultrasound provides the ability to non-invasively image deep inside biological tissues with high spatial and temporal resolution. However, this technology has limited capacity to monitor molecular and cellular processes, due to the lack of appropriate intra-cellular and endogenously producible nanoscale contrast agents, which can directly couple sound waves to the activity or concentration of physiologically relevant molecules. This problem could in principle be solved by developing genetically encodable ultrasound sensors – biomolecules that can get illuminated in ultrasound imaging in response to specific cellular or molecular activity. This thesis describes the engineering and characterization of acoustic protein nanostructures called 'gas vesicles', or 'GVs', to accomplish this task.
GVs are protein-shelled gas-filled nanostructures produced by buoyant microbes, and were recently shown to be capable of scattering sound waves to produce ultrasound contrast. Owing to this property, they were initially conceptualized as a new class of ultrasound contrast agents. However, little was known about their tunability to enable molecular ultrasound imaging for a wide range of applications. In this thesis, we leveraged the genetic encodability of GVs to modify them at the level of their DNA sequence and constituent proteins, and thereby tune their mechanical, acoustic, surface and targeting properties. We accomplished this by establishing a facile and modular molecular engineering platform, to produce GVs that provide enhanced nonlinear signals for sensitive and specific detection in deep tissues, target specific cell types such as cancer and immune cells, and also provide distinct acoustic collapse spectra for multiplexed imaging. We then extended this platform to build GV-based biosensors that modulate their nonlinear ultrasound signals in response to changes in the activity or concentration of specific molecules in their environment. Specifically, we engineered acoustic sensors for three different types of enzymes and for calcium – whose activity or flux underlie a wide range of important cellular processes. Furthermore, we succeeded in transferring the genetic code of gas vesicles from their species of origin into a variety of other microbes that do not naturally produce them, in order to unlock their potential as ultrasound reporter genes. Our results establish GVs as reliable acoustic biomolecules, and thereby extend the capabilities of ultrasound for molecular and cellular imaging in a manner analogous to green fluorescent protein (GFP) and its derivatives in optical microscopy. When combined with the advantages of ultrasound for non-invasive imaging, this work facilitates novel technology to significantly enhance our understanding of molecular and cellular processes in basic biology, as well as enable improved diagnosis, monitoring and treatment of diseases.</p
Deployable Piezoelectric Thin Shell Structures: Concepts, Characterization and Vibration Control
Get PDFThe thesis presents three interconnected technology paths to the design and realization of novel deployable active thin shell structures. The baseline concept envisioned is built upon a deployable ultra-thin piezoelectric active thin shell architecture, with segmented tessellations. This vision is motivated by the need to deploy and control large, curved and precise surfaces for a variety of applications including future space telescopes, and is made possible by recent progress in ultra-thin high-performance composites and active material technologies. The thesis uses a combination of heuristic design, theoretical analysis, numerical modeling and novel experimental techniques to construct and validate proposed concepts for deployable piezoelectric thin shells.
Specifically, the thesis answers the following questions: i) How to design and manufacture precise, foldable and curved piezoelectric shells. ii) How to deploy these shells reliably and maintain shape correctability in the deployed state. iii) How to synthesize large, curved deployable surfaces with the aforementioned advantages. iv) How to characterize and predict the nonlinear behavior of piezoelectric materials and thin structures under high electric field actuation and large bending deformations. v) How to improve the shape stability of piezoelectric active thin shells under dynamic disturbances without introducing external sensors.
First, the thesis proposes new methodologies and design criteria to synthesize deployable, modular edge-supported thin shells based on a combination of origami-inspired folding patterns and spatial mechanisms. In contrast to traditional deployable surface designs, which attach rigid shells to deployable trusses, the proposed methodology enables concurrent folding of flat or curved shells along with the support structures. Starting from a basic module, a variety of deployable surface concepts are proposed through tessellations of the module.
A piezoelectric material unimorph architecture is further introduced, providing global curvature and shape correction capabilities. All components of the basic concept are validated through model prototyping and material folding tests, and it is discovered that both the ultra thin carbon fiber composites and piezoelectric ceramic materials can achieve a small folding radius without failure. A composite, doubly-curved foldable shell is also designed and manufactured while still maintaining low shape error. These efforts have led to a new family of deployable piezoelectric thin shell structures that integrate low areal density, high shape accuracy, and structural foldability to an unprecedented degree.
The thesis then tackles the challenge of estimating the actuation response and residual structural deformation of unimorph active thin shells under high electric field and large bending motion. A rate-independent, full field phenomenological constitutive model for a polycrystalline piezoelectric material is characterized experimentally. It successfully captures both the observed ferroelectric and ferroelastic domain switching effects. To overcome the difficulty of testing ultra thin piezoelectric plates, a set of novel characterization techniques is developed and implemented to measure the dielectric and mechanical responses of this material. The characterized material constitutive relation is implemented in an efficient model for estimating the structural response of unimorph thin shells under general electric and mechanical loading. The complete set of governing equations is integrated with a Backward-Euler algorithm, reproducing the measured responses of both the material and the structure under complex loading sequences.
Active vibration damping based on self-sensing piezoelectric thin shells is then analyzed and demonstrated on testbed. The self-sensing architecture removes redundant external sensors by making dual use of the piezoelectric layer of the active shell. An adaptive identification method with the associated hardware to track the evolution of field dependent piezoelectric capacitance is implemented, and a new identification strategy is proposed. Closed loop damping with in-situ capacitance adaptation is conducted in bench tests on self-sensing cantilever beams and achieves -12~dB attenuation at the resonance frequency. A highly efficient modeling technique for general self-sensing piezoelectric thin shell structures is proposed which is able to construct closed loop dynamic models based on the vibration eigenmodes and actuation responses obtained from commercial finite element software. These validated modeling techniques are extended to a multi-electrode doubly curved thin shell, where the improvements of shape stability under closed loop damping are evaluated through simulations. It is discovered that the electrode pattern of the self-sensing piezoelectric layer determines the damping performance under the specific boundary conditions of the shell.</p
Aerosol Particle Measurements: Strategies for Health-Relevant Data Collection and Analysis
Get PDFParticulate matter (PM) is an important component of outdoor and indoor air pollution that can cause significant harm to human health. The present work, organized into two parts, introduces strategies for optimizing the collection and analysis of airborne particle measurements to inform PM health-effect research.
Part I focuses on the fundamental aerosol data analysis task of interpreting indirect measurements of particle size to reveal the distribution of sizes of particles in a sampled aerosol. An approach to this aerosol data inversion problem is developed that shows improved particle size distribution recovery compared to other common approaches described in the literature. This inverse solution method incorporates cubic spline interpolation to represent the particle size distribution within a discrete linear model of the inverse problem while placing no constraints on the number or spacing of solution points. The inverse problem setup can then interface with three established numerical methods for solution computation. The accuracy of this procedure is demonstrated through analysis of test-case data for differential mobility analyzer systems. Source code and supporting documentation are also provided to encourage researchers to use and adapt this inversion algorithm for analyzing data collected from existing as well as potential future measurement systems.
Part II of this work focuses on the retrieval of health-relevant information from aerosol particle measurement data. The inversion analysis introduced in Part I is incorporated into an extended analysis procedure for evaluating the metrics of PM exposure and respiratory dose that can be obtained from different measurement systems. Applying this evaluation procedure to a range of existing and potential future measurement techniques reveals that full characterization of particle size distributions need not be time and resource intensive and should be pursued for the great benefits this information would provide to health studies. Not only can size distribution information permit lung tissue dose estimates through a set of relatively simple calculations, but a single set of size distribution data can be analyzed and reanalyzed to provide dose estimates for human populations of interest by applying the appropriate respiratory tract deposition profiles. The measurement evaluation procedure developed here reveals target criteria for the particle characterization necessary to provide sufficient exposure and dose information for health studies. The intent is not to eliminate the current measurements and standards, but to help direct future developments in health-related aerosol particle measurement design.</p
Engineering and Application of cGAL, a GAL4 Bipartite Expression System for Caenorhabditis elegans
Get PDFThe core objectives of genetics are to dissect and understand the function of genes, the consequence of their perturbation on an organism, and how their collective action influences an organism’s biology. For genetic model organisms, transgenesis is a tool that allows researchers to introduce synthetic genetic constructs to determine where a gene acts, when it is required, and infer its function. Caenorhabditis elegans is a powerful genetic model organism, with a variety of transgenesis methods available to researchers. Each has its own advantages in speed, efficiency, control of copy number, and control of integration site. However, all methods suffer from issues of reproducibility, reusability, and labor cost. Bipartite systems offer solutions to these issues- they separate the promoter element from the gene product producing strains in which one sex contains the promoter (‘driver’ strain) and the other contains the gene (‘effector’ strain). Crossing driver and effector strains reunites promoter and gene in the progeny, which are assayed and analyzed for gene function. This separation of drivers from effectors allows for a variety of benefits. Driver and effector strains can be combinatorially reused, meaning less time-consuming strain construction. Reusing strains allows for more reproducibility and consistency between experiments and between laboratories. Additionally, novel genes and promoters can be crossed to existing strains for novel transgenic patterns requiring minimal effort. Thus, bipartite systems greatly increase the rigor and pace of genetic analysis. This thesis details the engineering of cGAL, a GAL4-based bipartite system for C. elegans. It uses a novel GAL4 gene from Saccharomyces cerevisiae, a yeast whose optimal growth temperature is similar to that of C. elegans. This thesis also describes an intein-based split bipartite system that offers more refined spatiotemporal control, by allowing two promoters to dictate gene expression instead of one. This split method is used to analyze rhythmic feeding in C. elegans. Finally, engineering of cGAL using single copy methodology is detailed, with a discussion of future improvements to, and usage of, single copy cGAL. This development of a new bipartite system will greatly accelerate genetic analysis for the C. elegans, improve reproducibility for the field, and generate a valuable resource for the community
Quantum Groups and Integralities in Chern-Simons Theory
Get PDFIn this dissertation, we investigate integralities in Chern-Simons theory. The integralities of interest arise from non-local observables (Wilson lines) in Chern-Simons theory and the partition function itself. In the associated supersymmetric gauge theories (via 3d-3d correspondence), they encode certain BPS spectrum, which are often identified with homological invariants of links and three-manifolds. In this dissertation, we observe that all of them are equipped with non-trivial algebraic structures, such as quantum group actions, modularity, and logarithmic vertex algebras. In the first half of this dissertation, we identify quantum group representations with the dynamics of line operators and their lift to surface operators. In the second half, Chern-Simons partition functions on Seifert manifolds are studied in detail, and its ``hidden'' integralities are identified with quantum modular forms and the characters of logarithmic vertex operator algebra. From the latter, we also observe that quantum group actions control the ``dynamics'' of characters