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Background: The Centers for Disease Control and Prevention (CDC) reports that men have recently experienced a rise in rates of sexually transmitted infections (STIs)--particularly chlamydia, gonorrhea, and syphilis. While STI testing has improved in cost, portability, and accuracy, STI notification still relies on telephone calls. Prompt STI notification is critical to reduce the number of days between STI test and STI treatment in order to stop future STI transmissions and prevent long-term health complications. In 2014, AIDS Healthcare Foundation (AHF) began implementing Healthvana, an online patient engagement platform and smartphone application, to notify AHF Wellness Center clients of STI test results. The primary study objective of this dissertation is to determine whether Healthvana reduced the number of days between STI test, notification, and treatment for male clients. The secondary study objective of this dissertation is to better understand the characteristics of clients who opted in to Healthvana. Methods: STI test, notification, and treatment data as well as risk assessment data from 18 AHF Wellness Centers between January 1, 2014 and December 31, 2015 were retrospectively collected. Male clients were included in the cross-sectional analysis if they were tested, notified, and treated for STIs (n = 1,460). Ordinary least squares and Poisson regressions were utilized to analyze differences in the mean number of days between STI test, notification, and treatment. Logistic regressions were utilized to analyze the difference between clients who did not opt in to Healthvana and clients who opted in to Healthvana. Results: The mean number of days between STI test and notification was reduced by 22 percent following Healthvana implementation, from nine days to seven days (p < 0.001). Overall, Healthvana implementation reduced the total days between STI test and treatment by two days, from 13 days to 11 days (p = 0.022). Clients under the age of 25 years experienced greater odds of opting in to Healthvana than clients who were 30-39 years old (p = 0.027) and clients who were aged 40 years and older (p = 0.004). Healthvana implementation at AHF Wellness Centers demonstrates a significant reduction in the number of days to STI treatment. [The dissertation citations contained here are published with the permission of ProQuest LLC. Further reproduction is prohibited without permission. Copies of dissertations may be obtained by Telephone (800) 1-800-521-0600. Web page: http://www.proquest.com/en-US/products/dissertations/individuals.shtml.]

Mechanical forces in the cell’s natural environment have a crucial impact on growth and behavior, from single-cell gene expression and cell division to the spatial patterning of complex organ architectures. Few areas of biology can be understood without taking into account how both individual cells and networks of cells sense and transduce physical stresses as part of a tightly-controlled and highly integrated, hierarchical collective. However, the field is currently held back by the limitations of the available methods to apply physiologically relevant stress profiles on cells, particularly with sub-cellular resolution, in controlled in vitro experiments. Here, we present a new active cell culture material that allows highly localized, directional, and reversible deformation of the cell growth substrate, with control at scales ranging from the entire surface to the subcellular, and response times on the order of a few seconds. This material has a hybrid architecture composed of a polymeric array of microstructure actuators that serve as anchorage points for adherent cells, embedded in a temperature-responsive poly(N-isopropylacrylamide) hydrogel layer. By incorporating gold nanorods as a phototransducer component into the hydrogel, we are able to remotely control with focused near-infrared light the localized and highly directional actuation of the supported microstructures over a range of scales down to 10 um, with response times under 5 s. Additionally, the surface cues provided by this hybrid material provide opportunities for controlling the cell attachment and alignment patterns. Thus, by triggering surface deformations at selected locations, we applied uniaxial mechanical stresses directly on defined portions of single cells within a population, with resolution over the cell distortion close to ~3 um, cell strains between 0 and ~45% reached locally in different regions within the same cell, cycling frequencies on the order of ~0.1 Hz, and no losses in cell viability. These capabilities are not matched by any other method, and this versatile material has the potential to bridge the performance gap between the existing single cell micro-manipulation and 2D cell sheet mechanical stimulation techniques., Chemistry and Chemical Biology

This thesis presents five contributions to machine learning, with themes of differentiability and Bayesian inference. We present Firefly Monte Carlo, an auxiliary variable Markov chain Monte Carlo algorithm that only queries a potentially small subset of data at each iteration yet simulates from the exact posterior distribution. We describe the design and implementation of Autograd, a software package for efficiently computing derivatives of functions written in Python/Numpy using reverse accumulation mode differentiation. Using Autograd, we develop a convolutional neural network that takes arbitrary graphs, such as organic molecules, as input. This generalizes standard molecular feature representations and allows end-to-end adaptation of the feature extraction pipeline to particular tasks. We show how to compute gradients of cross-validation loss with respect to hyperparameters of learning algorithms, with both time and memory efficiency, by chaining gradients backwards through an exactly reversed optimization procedure. Finally, by accounting for the entropy destroyed by optimization, we show that early stopping and ensembling, popular tricks for avoiding overfitting, can be interpreted as variational Bayesian inference., Physics

Nature demonstrates the efficiency of hierarchically integrated components that work in cooperation to produce a variety of useful phenomena in organisms, such as movement or shape change. A key feature of these components is the response of multiple reconfigurable constituents to certain stimuli, partaking in a cascade of events that lead to specific outcomes. Artificial systems strive for such efficiencies and can be greatly enhanced by the assimilation of responsive soft materials that respond to various and distinct stimuli. Here, I describe the combination of reversibly dynamic, stimuli-responsive and flexible components that are affected by its environment towards directed outputs, at the premise of which is a cross-linked hydrogel network capable of volume change in different aqueous solutions. In the first part, I will elaborate on the integration of hydrogel embedded around flexible microstructures in an aqueous microfluidic system. Chemo-mechanical activation and cooperation of the stimuli-responses of the integrated soft components and functionalities in the microfluidic can produce specific outputs, including oscillatory behavior through feedback loops that is able to maintain local temperature within a narrow range. I will show that functionalization of the system with aptamer enables the separation of target biomolecules from a solution mixture. I will then describe a hydrogel modified with spiropyran, a molecule that isomerizes between two forms with orthogonal properties in response to multiple stimuli, including light. In the absence of light activation, the volume change of the hydrogel can influence spiropyran isomerization mechano-chemically in different aqueous solutions. Integration of the optically responsive molecule with a volume-changing hydrogel is explored further in the context of nonlinear dynamic optics. In particular, the reversible self-trapping, or lensing, of laser light in spiropyran-modified hydrogels is investigated. Furthermore, propagating two laser beams through the system is found to have highly interesting implications in studying the mechanical stresses within the hydrogel network. From this work we come closer to the nature-inspired, adaptable reconfigurability that leads to more efficient systems with applications ranging from biomedicine to optics, while also promoting methods for fundamental studies on polymer mechanics as well as molecular-level reconfigurability that is important to all sciences., Chemistry and Chemical Biology

Fluorescent-labeling imaging techniques have been widely used in live cell studies; however, the labeling processes can be laborious and challenging for non-transfectable cells and clinical cells, and labels can interfere with protein functions. In this thesis, we present the first demonstration of label-free detection and quantification of intracellular biomarker dynamics with a nanoscale localized-surface-plasmon fiber-tip-probe (LSP-FTP). Our results have established the FTP technique as a new tool to study the time dynamics of proteins and protein phosphorylations in single live cell, and could be generalized to study other types of primary cells in response to external triggers, including drugs. Chapter 2 focuses on the fabrication methods of fiber tip probes (FTPs). FTP is a lab-on-a-fiber probe wet-etched at nanoscale dimensions. The base material of FTPs may comprise any solid materials as long as they have matching etchants. FTPs take various configurations and may comprise of single-ended fiber, double-ended fiber, or an end-portion or mid-portion nanowire with various aspect ratios. Structures of FTPs can be tuned at nanoscale accuracy with extreme surface smoothness. These FTPs can be mass-fabricated and multiplexed at a single fabrication step and at low cost. Chapter 3 discusses the procedures and results on measuring intracellular biomarker dynamics in intact live cells. We demonstrated label-free detection of tumor suppressor p53 dynamics in single HeLa cells under ultraviolet radiation and under treatment with neocarzinostatin (NCS). Also, we employed the FTP technique to prove that \textbeta -Amyloid generation precedes Tau phosphorylation by continually monitoring intracellular levels of \textbeta -Amyloid and phosphorylated Tau in live human neuroblastoma cells (SY5Y). Chapter 4 discusses other potential applications of FTPs. An FTP with a nanodiamond at its tip can be used to measure various intracellular properties such as electromagnetic fields, temperature, or pressure. Manipulation of electron spins at nitrogen-vacancy centers in a nanodiamond allows these measurements. An FTP with a plasmonic particle at its tip can be used for surface-enhanced Raman spectroscopy (SERS) and potentially allow single molecular detection. A mechanical FTP with an end-portion nanowire (a few hundred micrometers long and sub-micron thick) functions as a cantilever sensor, and we can detect changes in its vibration modes induced by binding of analytes to a reaction entity immobilized relative to the nanowire. Free-standing FTP waveguides with a sub-micron portion in diameter can be used for efficient light coupling and device characterizations., Chemical Physics

The ability of graphene and carbon nanotubes to generate an electric potential from flowing fluids has attracted much interest. The effect is thought to occur because certain ionic and molecular species bind to the surface of these materials more strongly than others. Although several physical models have been proposed, none has yet been rigorous enough to be confirmed by experiment. In this work, I describe an electromechanical device, i.e. a supercapacitive electrical energy generator (SCEEG) made with graphene electrodes, that generates electricity from oscillating droplets of electrolyte solutions and ionic liquids. I provide a theoretical model of how the graphene SCEEG (G-SCEEG) works that quantitatively agrees with experimental results. More importantly, the model allows one to characterize electrochemically-useful properties of the interaction between graphene and hydrated ions from the device’s experimentally measured output. The identity of the ions that preferentially adsorb to graphene as well as the effective surface density and effective binding energy of these ions can all be determined using the G-SCEEG. Additionally, both the capacitance per unit area and the surface potential of the graphene-solution interface can also be determined. Although this work focuses on a SCEEG device whose electrodes are made from graphene, the electrodes can, in principle, be made from any conductive material, e.g. metals, such as gold, and semiconductors, such as indium-tin-oxide. All SCEEGs described in this thesis, however, work by exchanging charge between two supercapacitors, i.e. capacitors that spontaneously form at the interface between an electrode and an electrolyte solution (or ionic liquid) as a result of ionic adsorption. Charge exchange occurs not by charging and discharging these interfacial capacitors but instead by increasing and decreasing their capacitance. This is accomplished mechanically as a moving droplet wets and dewets the electrodes. The changing interfacial capacitance acts as a source of current; the flow of current through the device’s internal impedance creates an electric potential; and, when connected to an external load, the SCEEG is then capable of generating electric power. I show that a G-SCEEG can generate a peak power of up to 7 μW from the oscillatory motion of two 20 μL droplets of 6.0 M HCl. I demonstrate that the device can be successfully modeled as a source of alternating current that is in parallel with a time-varying internal impedance. Using this model and the G-SCEEG’s output, I determine that the chloride anions in 6.0 M HCl adsorb to graphene with the greatest effective surface density (5.6 x 10^12 ions/cm^2) and induce in it the largest charge density (900 nC/cm^2) of all the solutions I studied. These chloride anions adsorb to graphene with an effective binding energy of 350 meV, in turn producing an electric potential of 690 mV at the graphene-solution interface. I also find that the density and the sign of the surface charge induced in graphene are dependent on the ionic species present in each solution, the concentration and pH of the solution, and the presence of multi-layers in the graphene electrodes with the solution’s pH providing the greatest effect. Finally, I describe in this dissertation the study of the Raman spectral properties of suspended mono- and multi-layer graphene membranes and the use of graphene and graphite to electrostatically trap DNA from an aqueous solution. I conclude by explaining the unique methods I developed during the course of this work including the synthesis of large-grain graphene using chemical vapor deposition (CVD) and the transfer of large areas of CVD-grown graphene to hydrophobic substrates., Biophysics

In the first part of this dissertation, we investigate on the presence of quantum effects in the exciton dynamics of the Fenna-Matthews-Olson photosynthetic complex of green sulfur bacteria using an atomistic Quantum Mechanics / Molecular Mechanics (QM/MM) model combined with open quantum systems methods. Subsequently, we explore the theoretical connection between the atomistic QM/MM approach and the open quantum system methods and propose the correct theoretical expressions to maintain consistency when using both approaches contemporarily. In particular we show that when using the correct prefactor to extract the spectral density - the strength of coupling between excitation and other degrees of freedom - the atomistic results are in good agreement with experimental predictions. We then describe a first atomistic study of the full light-harvesting complex of green sulfur bacteria. The various units are treated atomistically and the full system's exciton dynamics is obtained using a Markovian open quantum system master equation. To conclude the first part, we describe a Machine Learning algorithm which we developed and implemented to learn time-dependent density functional theory energies by using trained neural networks and supplying these with coulomb matrices extracted from molecular dynamics simulations. This approach provides a much more rapid solution to obtaining a QM/MM Hamiltonian and subsequently extracting dynamics. It is particularly useful when multiple identical molecules are found in similar environments as one can train the network on a single molecule and predict all others. We applied this method to the Fenna-Matthews-Olson complex. In the second part of this dissertation we focus on model systems and synthetic aggregates. In particular, we investigate the exciton dynamics in thin-film J-aggregates using a Markovian stochastic Schrödinger equation approach. We derive expressions to obtain diffusion constants from the dynamics and compare a series of different thin-film J-aggregates. The parameters of the model are obtained atomistically. From this model we obtain information on the parameters which lead to optimal exciton diffusion. This can guide the design of new exciton transfer materials., Chemistry and Chemical Biology

Paper (and other cellulose-based materials such as cotton thread and fabrics) are underexploited as materials for the construction of “high-tech” and “lab-on-a-chip” devices. One major drawback of paper is its tendency to absorb water from the environment and, with wetting, to change its mechanical properties; other challenges relate to control over the attachment of molecules (e.g. antibodies, DNA) and cells on its surface, and to the addition of electronic function. The goal of this thesis is to develop paper as a substrate for a range of applications— microfluidics, substrates for electronic systems and MEMS, low-cost diagnostics, cell biology, and optics. The approach involves chemically modifying the surface of the paper to provide new functions without altering any of its defining properties: mechanical flexibility, foldability, light weight, gas permeability, and low cost. The first part of my thesis describes the modification of paper by silanization with organosilanes such as alkyl- and fluoroalkyl trichlorosilanes in the gas phase. Here, silanization is used to lower the surface free energy of the paper and to minimize the tendency of paper to absorb liquids and vapors, and especially water. Chapter 1 and Appendix 3 demonstrate that the combination of long fluoroalkyl chains of grafted siloxanes with the micro-scale roughness and porosity of paper yielded a material that is omniphobic (both hydrophobic and oleophobic), while preserving the properties of mechanical flexibility and low resistance to transport of gas of the untreated paper. Appendix 3 shows that features of omniphobic paper can be used to construct microtiter plates and liquid-filled gas sensors using standard paper folding techniques, while Appendix 4 shows that new type of microfluidic device fabricated by carving microchannels into the surface of omniphobic paper. The resulting devices have open, unobstructed channels (with dimensions as small as 45 μm) and thus exhibit fluid dynamics similar to conventional PDMS-based microfluidics, but are much lighter and have the potential to be much less expensive than PDMS-based devices. The second part of my thesis is focused on engineering the surface of paper to enable efficient immobilization of capture and target molecules for bioanalysis. In one approach, described in Appendix 5, we exploit the ease with which the surface chemistry of paper (i.e. the surface of the cellulose fibers making up the paper) can be modified, in order to enhance the immobilization of antibodies and antigens on the surface of the paper via hydrophobic interactions, while preventing the wicking of the fluids into the paper substrate. As an application in low-cost diagnostics, we describe a low-cost electrochemical device for ELISA intended for use in resource-limited settings. In a second approach, described in Chapter 2, we developed of an efficient procedure for assembling microarrays of ssDNA and proteins on paper, at the lowest practical cost. This method starts with the synthesis of DNA oligonucleotides covalently linked to paper, and proceeds to generate ssDNA arrays that, through hybridization with complementary strands of DNA, are capable of simultaneously capturing DNA, DNA-conjugated protein antigens, and DNA-conjugated antibodies. The third part of my thesis describes the simple, inexpensive fabrication of electrodes for paper-based electrochemical systems. A first method describes, in Appendix 6, the development of inkjet printing as a method for high resolution printing of conductive patterns on omniphobic “RF” paper, both to extend its promise as a substrate for paper electronics, and to enable us to integrate it into our program in low-cost, paper based diagnostics. A second method, described in Chapter 3, circumvents the need for printing, and instead focuses on the fabrication and reconfiguration of simple, versatile, and inexpensive electroanalytical devices in which conventional stainless-steel pins—in unmodified form or after coating with a carbon paste—are used as electrodes., Chemistry and Chemical Biology

Many cellular signaling pathways exhibit a bowtie topology: multiple distinct signal inputs converge on a single master transcription factor, which controls the expression of downstream genes. Recent evidence suggests that information about signal inputs can be encoded by regulating the activation dynamics of the master transcription factor. However, it was unclear whether this is sufficient to obtain specificity in gene expression, such that each input induces a specific set of output genes. Using the budding yeast transcription factor Msn2 as a model system, we address this question. We systematically dissect how different promoters decode transcription factor translocation dynamics in single cells (Chapter 2). We find that promoters fall into four main classes depending on the threshold of signal required for activation and on the timescale of activation. Furthermore, we provide insight into the mechanistic basis for promoter class. We show that it is possible to differentially control expression of the different promoter classes by control of Msn2 dynamics. We find that slow promoters exhibit dramatically higher noise in gene expression, but are able to filter out Msn2 oscillations. This highlights a general trade-off: for promoters, implementing a high-pass temporal filter comes at the cost of much higher noise in gene expression. Applying tools from information theory and focusing on gene expression, we rigorously quantify the limits on information transduction through regulation of Msn2 dynamics (Chapter 3). Although we find that the amount of information transduced by Msn2 to target genes is only sufficient for reliable binary decisions, information transduction can be improved by modulating promoter cis-elements or by integrating information from multiple genes. We find amplitude-encoding to be more reliable than frequency-encoding. Taken together, our results suggest that information transduction through regulation of Msn2 dynamics is limited to reliable transduction of signal identity, but not signal intensity. The work we describe in this dissertation would not have been possible without the development of high-throughput microfluidic technologies (Chapter 4). We describe our development of a multiplexed microfluidic device, which we combine with four-color quantitative time-lapse microscopy to control nucleocytoplasmic shuttling of Msn2 and measure gene expression dynamics in single cells. We provide a detailed protocol for future studies. Our work demonstrates that it is possible to encode multiple distinct gene expression programs in the dynamics of a single transcription factor. Nonetheless, at the level of individual genes, noise in the decoding step places an upper limit on information transduction., Chemistry and Chemical Biology

The development of next-generation, nanoscale technologies that interface biological systems will pave the way towards new understanding of such complex systems. Nanowires – one-dimensional nanoscale structures – have shown unique potential as an ideal physical interface to biological systems. Herein, we focus on the development of nanowire-based devices that can enable a wide variety of biological studies. First, we built upon standard nanofabrication techniques to optimize nanowire devices, resulting in perfectly ordered arrays of both opaque (Silicon) and transparent (Silicon dioxide) nanowires with user defined structural profile, densities, and overall patterns, as well as high sample consistency and large scale production. The high-precision and well-controlled fabrication method in conjunction with additional technologies laid the foundation for the generation of highly specialized platforms for imaging, electrochemical interrogation, and molecular biology. Next, we utilized nanowires as the fundamental structure in the development of integrated nanoelectronic platforms to directly interrogate the electrical activity of biological systems. Initially, we generated a scalable intracellular electrode platform based on vertical nanowires that allows for parallel electrical interfacing to multiple mammalian neurons. Our prototype device consisted of 16 individually addressable stimulation/recording sites, each containing an array of 9 electrically active silicon nanowires. We showed that these vertical nanowire electrode arrays could intracellularly record and stimulate neuronal activity in dissociated cultures of rat cortical neurons similar to patch clamp electrodes. In addition, we used our intracellular electrode platform to measure multiple individual synaptic connections, which enables the reconstruction of the functional connectivity maps of neuronal circuits. In order to expand and improve the capability of this functional prototype device we designed and fabricated a new hybrid chip that combines a front-side nanowire-based interface for neuronal recording with backside complementary metal oxide semiconductor (CMOS) circuits for on-chip multiplexing, voltage control for stimulation, signal amplification, and signal processing. Individual chips contain 1024 stimulation/recording sites enabling large-scale interfacing of neuronal networks with single cell resolution. Through electrical and electrochemical characterization of the devices, we demonstrated their enhanced functionality at a massively parallel scale. In our initial cell experiments, we achieved intracellular stimulations and recordings of changes in the membrane potential in a variety of cells including: HEK293T, cardiomyocytes, and rat cortical neurons. This demonstrated the device capability for single-cell-resolution recording/stimulation which when extended to a large number of neurons in a massively parallel fashion will enable the functional mapping of a complex neuronal network., Physics

Cellular lipid membranes can – and often do – support a transmembrane electric field, serving as biological capacitors that maintain a voltage difference between their two sides. It isn't hard to see why these voltage gradients matter; the electrical spiking of neurons gives rise to our thoughts and actions, and the voltage dynamics of cardiomyocytes keep our hearts beating. Studies of bioelectricity have historically relied on electrode-based techniques to perturb and measure membrane potential, but these techniques have inherent limitations. I present new optogenetic methods of studying membrane potential that will broaden the scope of electrophysiological investigations by complementing traditional approaches. I introduce the microbial rhodopsin Archaerhodopsin-3 (Arch), a transmembrane protein from Halorubrum sodomense. The fluorescence of Arch is a function of membrane potential, allowing it to serve as an optical voltage reporter. We use time-dependent pump-probe spectroscopy to interrogate the light- and voltage- dependent conformational dynamics of this protein, to elucidate the mechanism of voltage-dependent fluorescence in Arch. I then present two new methods for imaging voltage using engineered variants of Arch. Both techniques take advantage of the unique photophysical properties of Arch(D95X) mutants. The first method, Flash Memory, records a photochemical imprint of the activity state -- firing or not firing -- of a neuron at a user-selected moment in time. The Flash Memory technique decouples the recording of neural activity from its readout, and can potentially allow us to take large-scale snapshots of voltage (e.g. maps of activity in a whole mouse brain). The second method allows for the quantitative optical measurement of membrane potential. This technique overcomes the problems that typically hinder intensity-based measurements by encoding a measurement of voltage in the time domain. Finally, I present a method to visualize cellular responses to changes in membrane potential. I engineer mutants of Channelrhodopsin-2 (ChR2), a light-gated cation channel from Chlamydomonas reinhardtii that is used for optical control of neural activity, and use these optogenetic actuators in conjunction with GFP-based sensors to study the activity-dependent behavior of cultured neurons.

My work has focused on the development and application of fluorescent voltage-sensitive proteins based on microbial rhodopsins. These probes led to the discovery of electrical activity in the bacterium Escherichia coli, the first robust optical recordings of action potentials (APs) in mammalian neurons using a genetically encoded voltage reporter, and the development of a genetically targetable all-optical electrophysiology system. I first introduce an engineered fluorescent voltage sensor based on green-absorbing proteorhodopsin. Expression of the proteorhodopsin optical proton sensor (PROPS) in E. coli revealed electrical spiking at up to 1 hertz. Spiking was sensitive to chemical and physical perturbations and coincided with rapid efflux of a small-molecule fluorophore, suggesting that bacterial efflux machinery may be electrically regulated. I then present another microbial rhodopsin, Archaerhodopsin 3 (Arch), whose endogenous fluorescence exhibited a twofold increase in brightness between -150 mV and +150 mV and a sub-millisecond response time. In rat hippocampal neurons, Arch detected single electrically triggered APs with an optical signal-to-noise ratio > 10. A mutant, Arch(D95N), lacked endogenous proton pumping and had 50% greater sensitivity than the wild type but had a slower response (41 ms). Nonetheless, Arch(D95N) also resolved individual APs. Finally, I introduce evolved archaerhodopsin-based voltage indicators and a spectrally orthogonal channelrhodopsin actuator, which together enabled all-optical electrophysiology. A directed evolution screen yielded two mutants, QuasAr1 and QuasAr2, that showed improved brightness and voltage sensitivity relative to previous archaerhodopsin-based sensors, and microsecond response times. An engineered channelrhodopsin actuator, CheRiff, showed high light sensitivity and rapid kinetics. A coexpression vector, Optopatch, enabled cross-talk-free genetically targeted all-optical electrophysiology. In cultured neurons, the Optopatch system probed membrane voltage across temporal and spatial scales, from the sub-cellular and sub-millisecond dynamics of AP propagation, to the simultaneous measurement of firing patterns of many neurons in a circuit. In brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. In human stem cell-derived neurons, Optopatch measurements revealed homeostatic tuning of intrinsic excitability, a subtle form of plasticity that had yet to be observed in human neurons. The suite of tools and techniques presented here enable high-throughput, genetically targeted, and spatially resolved electrophysiology without the use of conventional electrodes., Engineering and Applied Sciences

I present applications of imaging and spectroscopy to understand mechanical, chemical, and electrical dynamics in biological materials. The first part describes the development and characterization of a protein-based fluorescent calcium and voltage indicator (CaViar). The far-red fluorescence of CaViar faithfully tracks the cardiac action potential in cardiomyocytes. CaViar's green fluorescence reports the resulting calcium transients. I demonstrated the applicability of CaViar in vivo with transgenic zebrafish designed to express CaViar in their hearts. Spinning disk confocal imaging allowed detailed three-dimensional mapping of simultaneous voltage and calcium dynamics throughout the heart of zebrafish embryos, in vivo, as a function of developmental stage. I tested the effect of channel blockers on voltage and calcium dynamics and discovered a chamber-specific transition from a calcium-dependent to a sodium-dependent action potential. I also describe a new measurement technique using a fluorescent voltage indicator to report absolute voltage via the indicator's temporal response., Physics

This thesis presents the development and application of nanoscale techniques to trap and, Chemistry and Chemical Biology

I present two applications of fluorescence spectroscopy in biophysics. The first is an instrument, the anti-Brownian electrokinetic (ABEL) trap, which is capable of trapping individual small molecules in aqueous solution at room temperature. The second is an investigation of the bending mechanics of double-stranded DNA using a novel DNA structure called a "molecular vise". Both projects take advantage of the sensitivity and specificity of fluorescence spectroscopy, and both benefit from the interplay of experimental work with theoretical and computational modeling. The ABEL trap uses fluorescence microscopy to track a freely diffusing particle, and applies real-time electrokinetic feedback forces to oppose observed motion. Small molecules are difficult to trap because they diffuse quickly and because their fluorescence emission is typically weak. I describe the experimental and algorithmic approaches that enabled small-molecule fluorophores to be trapped at room temperature. I additionally derive and discuss the theory of the molecules' behavior in the trap; this mathematical work informed the design of the trapping algorithm and additionally enabled trapped molecules to be distinguished on the basis of their diffusion coefficient and electrokinetic mobility. Molecular vises are DNA hairpins that use the free energy of hybridization to exert a compressive force on a sub-persistence length segment of double-stranded DNA. In response to the applied force, this "target strand" may either remain straight or bend, depending on its flexibility and length. Experimentally, the conformation can be monitored via Förster resonance energy transfer (FRET) between appended fluorophores. The experimental results quantitatively matched the predictions of the classic wormlike chain (WLC) model of DNA elasticity at low-to-moderate salt concentrations. Higher ionic strength induced an apparent softening of the DNA which was best accounted for by a high-curvature "kinked" state. The molecular vise is exquisitely sensitive to the sequence-dependent linear and nonlinear elastic properties of dsDNA and provides a platform for studying the effects of chemical modifications and small-molecule or protein binding on these properties.

This thesis describes work related to the theme of sculpted electromagnetic fields - engineered fields with particular spatial patterns - and their interactions with molecules. We are motivated by the following questions: what are ways of detecting spatial patterns in electromagnetic fields? What are possible applications of spatially engineered fields? Are there molecular transitions that are dark to plane waves but that can be probed by sculpted fields? The first part of this thesis is in the area of magnetic field effects in chemistry. We focus on magnetic field modulated fluorescence, which provides a convenient method for imaging magnetic field strength. We proposed and demonstrated a fluorescence technique that allows imaging through strongly scattering media. We achieve this by exploiting the fact that most materials do not scatter magnetic field. This allows us to project a magnetic field pattern beyond the scattering surface. The magnetic field dependent fluorescence then allows us to map out the object of interest. We constructed a setup that demonstrates 2D imaging using this technique. We synthesized new molecular systems to enhance the sensitivity to magnetic field. We characterized and compared these molecules with steady state fluorescence spectroscopy, transient fluorescence and transient absorption measurements. The results reveal patterns that point to directions for engineering chemical systems to further enhance their magnetic field sensing properties. The second portion of this thesis is a theoretical study of the molecular multipole transitions and their couplings to local electromagnetic quantities. Using a semiclassical approach, we performed a multipole expansion of molecular transitions driven by monochromatic radiation. We derived the local electromagnetic quantities that couple to different multipole transitions and observables such as circular dichroism and magnetic circular dichroism. It was observed that certain transitions are dark to plane waves, but could be probed by simple spatial arrangements such as superpositions of plane waves. Experiments for their detection are also proposed., Engineering and Applied Sciences

This thesis conducts a systematic study on the chirality of light and its interaction with chiral matter. In the theory section, we introduce a measure of local density of chirality, applying to arbitrary electromagnetic fields. This optical chirality suggests the existence of superchiral modes, which are more selective than circularly polarized light (CPL) in preferentially exciting single enantiomers in certain regions of space. Experimentally, we demonstrate an 11-fold enhancement over CPL in discriminating chiral fluorophores of single handedness in a precisely sculpted superchiral field. This result agrees to within 15% with theoretical predictions. Any chiral configuration of point charges is beyond the scope of our theory on optical chirality. To address chiroptical excitations at nanoscale, we develop a model of twisted dipolar oscillators. We design a simple tunable chiral nanostructure and observe localized chiroptical “hot spots” with dramatically enhanced circular differential scattering. Our work on superchiral light and 3D chiral metamaterials establishes optical chirality as a fundamental and tunable property of light, with implications ranging from plasmonic sensors, absolute asymmetric synthesis to new strategies for fabricating three-dimensional chiral metamaterials. This thesis is organized as such: Chapter 1 provides a background on previous studies of chiroptical phenomena, and recent efforts in preparing chiral metamaterials. Chapter 2 derives theory on optical chirality, superchiral modes and coupled-dipolar oscillators at nanoscale. Chapter 3 introduces material, apparatus, and pitfalls in chiroptical experiments. Chapter 4 is an overview of the experimental procedure and results on generating and observing superchiral enhancement. Chapter 5 describes the experiments on using spectroscopic polarization microscopy to study chiral 3D chiral metamaterials. Finally in Chapter 6, I discuss quantization of optical chirality and perspectives on future directions., Physics