The first optical detection of a single molecule (SM) at cryogenic temperatures 30 years ago laid the groundwork for the routine detection of SMs today at biologically relevant temperatures, thus uncovering hidden heterogeneity that might be obscured by ensemble techniques. In addition to enabling studies of the intricate photochemistry and photophysics of fluorescent labels at the SM level, SM fluorescence has also proven useful for the imaging and tracking of cellular structures and biomolecules in a non-invasive manner with high sensitivity. The ability to genetically express fluorescent protein fusions in live cells has allowed specific labeling, and thus imaging and tracking, of dynamic processes and structures in cells. This dissertation describes applications of SM-based single-particle tracking (SPT) and super-resolution (SR) microscopy for the study of spatial organization and dynamics of bacterial proteins in two and three spatial dimensions. In an SPT experiment, the position of a SM emitter at very low concentration is measured over time to generate a trajectory, allowing for observation and quantification of labeled biomolecule dynamics at the SM level. In a SR microscopy experiment, the diffraction-limited (DL) resolution of a conventional fluorescence microscope (~200 nm in xy) is circumvented by temporally separating the emission of many SM emitters decorating a structure through control of their emissive state. A "super-resolved" image, with a factor of ~5-10 resolution improvement over a conventional DL fluorescence image, is generated by estimating the positions of many non-moving SM emitters over many frames and building up an image reconstruction in a pointillist manner. Chapter 1 of this dissertation provides an introduction to fluorescence, SM imaging, SM-based SR microscopy, and SPT. Chapter 1 also gives a brief introduction to Caulobacter crescentus, the bacterium used as the model organism in the SM studies in Chapters 4 and 5. Chapter 2 describes the experimental methods used to perform quantitative SM imaging of bacterial cells. The combination of SM imaging with point spread function (PSF) engineering has enabled the accurate and precise localization of SMs in three dimensions (3D) by the intentional introduction of specifically chosen aberrations in the emission path of an SM microscope. Throughout this dissertation, the double-helix (DH) PSF, a rotating PSF composed of two lobes whose angle encodes axial position, was used to estimate 3D SM positions. Chapter 2 describes the implementation of the DH-PSF via optical Fourier processing, and Chapter 3 describes the robust and comprehensible two-color Easy-DHPSF v2 software for localizing molecules in 3D and for registering localizations from two spectral channels into the same coordinate system with nanoscale accuracy. The resolution improvement gained from SM-based techniques is particularly useful for bacteria, the sizes of which are on the order of the DL. 3D SM-based SR and SPT have enabled the observation of structures and dynamics at length scales below the DL. Caulobacter is a useful biological target where understanding of the mechanisms for asymmetric cell division need to be explored and quantified. Central to Caulobacter's asymmetric division is the dynamic spatiotemporal regulation of gene expression and protein localization. Chapters 4 and 5 describes research performed in collaboration with Prof. Lucy Shapiro's laboratory (Department of Developmental Biology, Stanford School of Medicine) to study gene expression and signaling proteins in Caulobacter. Chapter 4 describes work studying the spatial organization and dynamics of ribosomes and a RNA-degrading enzyme RNase E using 3D SR microscopy and SPT. Results showed that the organization and dynamics of RNase E and ribosomes are closely related to the transcriptional activity of the cell. Finally, Chapter 5 describes SPT studies of the membrane-bound histidine kinase and stalked cell fate determinant DivJ in an effort to probe the physical properties of the Caulobacter stalked pole. Preliminary SPT results suggest that disrupting the physical properties and interactions at the stalked pole has an influence on DivJ diffusion and signaling.

Single-molecule fluorescence methods have become a mature and powerful tool to unravel the fundamental mechanisms of biological phenomena. These approaches enable researchers to investigate individual components on the nanoscale for heterogeneous mixtures and to monitor the dynamics of single biomolecules. Unfortunately, traditional burst-based methods are often limited by short observation time, and the immobilization of single biomolecules that enables long observation time is often undesirably perturbative. To address these limitations, a solution-based technique known as the Anti-Brownian ELectrokinetic (ABEL) trap has been developed. In the ABEL trap, high-speed feedback electronics sense position and compensate Brownian motion of a single molecule by applying electrokinetic forces, which keep the molecule in the trapping region for an extended period of time. This dissertation describes my work on the investigation of single-molecule interactions and photodynamics using the ABEL trap technology. First, I show how to measure and monitor single-molecule rotational diffusivity by combining the ABEL trap and maximum likelihood analysis of time-resolved fluorescence anisotropy based on the information inherent in each detected photon. The rotational diffusivity is extremely sensitive to the size of the molecule, and thus can be used to detect and monitor size-changing events, such as association and dissociation. We demonstrate this approach by resolving a mixture of single- and double-stranded fluorescently-labeled DNA molecules at equilibrium, freely rotating in a native solution environment. Next, we investigate photosynthetic photoprotection mechanisms at the level of individual antenna complexes (LHCII), the fundamental units which green plants use to harvest sunlight. We access the intrinsic conformational dynamics of individual LHCII complexes. In addition to an unquenched state, two partially quenched states of LHCII are observed. One of the quenched conformations significantly increases in relative population under environmental conditions mimicking high light. Our results suggest that there are at least two distinct quenching sites with different molecular compositions, meaning multiple dissipative pathways in the LHCII complex. Last, we describe the theoretical basis of a new method to measure the diffusion coefficient of diffusing objects on the surface of individual nanoscale lipid vesicles. We discuss how to probe diffusion in these highly confined environments using FRET pairs and fluorescence intensity correlation, and illustrate that this method is essentially a fluorescence correlation spectroscopy (FCS) experiment with a nanometer-sized probing volume.

Since the first optical detection of a single molecule 29 years ago, the development of single-molecule microscopy and spectroscopy has revolutionized the study of complex chemical systems. As reviewed in Chapter 1, by imaging and computationally localizing individual fluorescent dyes or proteins within a sample, their positions can be localized with typical precisions (10-40 nm) an order of magnitude or better than the optical diffraction limit of visible light (~250 nm laterally and ~500 nm axially). This technique is critical to super-resolution fluorescence microscopy and single-molecule tracking, which are now regularly used to measure the nanoscale structures, biomolecular motions, and stochastic chemical processes underlying the biology of cells. This dissertation comprises two intertwined single-molecule imaging projects: 1) optical and analytical methods development for three-dimensional (3D) single-molecule tracking and super-resolution microscopy, and 2) the application of these methods to understand the nanoscale organization and dynamics of proteins at the poles of the bacterium Caulobacter crescentus. Without modification, a single-molecule microscope only improves imaging resolution in the lateral (xy) dimension, but biological cells are intrinsically 3D. To improve the imaging resolution in z, the detection path of a standard widefield microscope can be modified using Fourier processing to encode z position in the pattern of light formed by each fluorescent emitter and detected on the camera. Chapter 2 reviews the development of a two-color 3D single-molecule microscope that uses the double-helix point spread function pattern to encode 3D position, while Chapter 3 describes how to correctly align and to calibrate the fine aberrations of such a microscope to achieve nanoscale imaging accuracy in multiple color channels simultaneously. The bacterium Caulobacter crescentus is a model organism for the study of cell polarization and asymmetric cell division. Chapters 4 and 5 describe work performed in collaboration with Prof. Lucy Shapiro and her laboratory in the Department of Developmental Biology in the Stanford University School of Medicine to study how the tips, or poles, of Caulobacter cells use proteins to act as nanoscale spatial landmarks that polarize cells and induce spatially organized development. The polar organizing protein PopZ is one such critical landmark, and Chapter 4 describes results obtained from 3D super-resolution imaging of PopZ. Such imaging showed that PopZ forms 150-200 nm space-filling polar microdomains of roughly uniform density, and that proteins of the chromosome partitioning machinery (ParA and ParB) exhibit different spatial behaviors (recruitment vs. tethering) relative to the PopZ microdomain depending on their biochemistry and role in the chromosome replication process. Chapter 5 discusses the combination of single-molecule tracking and super-resolution imaging to study the polar localization of the signaling molecules of that activate the master regulator protein CtrA. Precise 3D imaging and tracking showed that PopZ acts as a selectively permeable localization hub that slows the motion of signaling proteins. In combination with reaction-diffusion modeling and transcriptional assays, these microscopic measurements indicated that the PopZ microdomain acts to sequester the CtrA signaling pathway within the pole and spatially pattern transcriptional output within the predivisional Caulobacter cell.

The H + H2 reaction and its isotopic variants have served for many years as a benchmark reaction system for studying bimolecular reaction dynamics. It is well-established that the minimum energy path for the H + H2 reaction is where the three atoms line up to form a collinear transition state. This path, commonly known as the direct recoil mechanism or Spiral mechanism, is very classical in nature and shows its signature in the angular distribution of the products which is a universal probe to the collision dynamics of a chemical reaction. The signature of the direct recoil mechanism is generally a single backscattered peak for low impact parameter or head-on collisions in the differential cross sections (DCSs) of the product states and a more sideways/forward scattered peak for high impact parameter or glancing collisions. While this behavior has been seen repeatedly in state-of-the-art experiments as well as high dimensional QM and classical theories, there have also been exceptions when the H + H2 system did not behave so classically and provided us with new insights to this reaction and to chemical reactions in general. This dissertation primarily focuses on two studies which bring forward new insights to this simplest chemical reaction: (i) scattering dynamics of H + D2 ⟶HD (v'=1, j') + D reaction at 1.97 eV, and (ii) probing the dynamics of H + D2 reaction beyond the energy of the conical intersection of H3 surface at a collision energy of 3.26 eV. Using the PHOTOLOC (photoinitiated reactions analyzed via law of cosines) technique developed by Zare and coworkers, 4 we have performed state-to-state measurements of the H + D2(v, j=0-2)⟶ HD(v', j') + D reaction at varying collision energies and investigated how energetics play a crucial role in the dynamics of this reaction. What has been observed for the HD product states is that the scattering dynamics do not show signatures of the conventional minimum energy path, vide supra. Instead of showing the conventional backward/sideways scattered single peak, the experimental DCSs showed multiple oscillatory structures. These structures are well reproduced by time-independent quantum mechanical (TIQM) calculations, but, somewhat surprisingly, not by the quasiclassical approach. Analysis of the classical results reveals that for these HD (v', j') states, several classical scattering mechanisms occur simultaneously. However, no clear evidence of these mechanisms is obvious in the quasiclassical state-to-state DCSs because their outcomes overlap in the range of scattering angles where they could be observable. It turns out that, analogous to the observations in a double-slit experiment, quantum interferences between the various mechanisms change and govern the angular distribution of the HD products. Owing to its quantum nature, this effect cannot be described in terms of the classical motion of the nuclei, and the QCT method fails. These mechanisms are characterized by different values of the total angular momentum J, which makes it possible to investigate theoretically the effect of 'closing' or 'opening' one of the slits in the two-slit experiment and determine the origin of the interferences. What is interesting is that while these different classical mechanisms and the interferences arising from them start to show for HD (v'=1, j') at energies (1.97 eV) way below the conical intersection (CI) of the H3 surface (2.74 eV), it is only at collision energies much higher than the CI that the higher vibrational manifolds of HD (v'=3, 4; j') show similar behavior. This can be explained with a total energy perspective and its distribution between the kinetic, internal and potential energies required to cross the barrier. At Ecoll = 1.97 eV, while the HD(v'=1, j') state has enough total energy available for the reaction to go through pathways with higher energy barriers, the relatively higher internal energies for the v' = 3 and 4 manifolds leaves less energy for the reaction and therefore the only feasible pathway for these states is the minimum energy path. At higher energies, for example, at 3.26 eV, there is enough energy available for even the higher vibrational states to undergo multiple mechanisms.

Previous studies in the Boxer lab characterized two variants of split green fluorescent protein (GFP), which appeared to behave distinctly with respect to interaction with their complementary peptides in the presence of light. Truncated green fluorescent protein (GFP) with its 11th β-strand removed (i.e. the strand 11 system) can recover the absorbance and fluorescence properties of the whole protein when refolded in the presence of synthetic strand 11 peptide. When refolded on its own, the same truncated protein is no longer receptive to binding exogenous strand 11; however, light irradiation generates a species which is capable of binding the complementary peptide. Meanwhile, the truncated GFP with its 10th β-strand removed (i.e. the strand 10 system) can readily bind to synthetic strand 10 peptide to recover the absorbance and the fluorescence of the whole protein. It was found that strand 10 spontaneously dissociates from this bound complex very slowly, and this process is greatly accelerated by light irradiation. In short, the strand 11 system undergoes photoassociation, and the strand 10 system undergoes photodissociation, but the origin of this difference was unclear despite extensive characterization of the two species. Our work attempted to reconcile the apparent differences in the photophysical properties of the two systems by directly investigating the structure of the strand 11 system at atomic level resolution using two techniques: solution state NMR and X-ray crystallography. Although we fell short of obtaining a full NMR structure, we were able to assign backbone chemical shifts for most of the residues in two variants of the strand 11 system, and probed the solvent accessibility of different residues by hydrogen-deuterium exchange. In the process of preparing this protein for crystallography, we found that the attached N-terminal His-tag, which is generally assumed to have negligible impact on the properties of the fused protein, was necessary to preserve the stability and spectral properties of the truncated protein. In the crystal structure of this construct, we found that the N-terminal His-tag and several neighboring residues play a highly unusual structural and functional role in stabilizing the truncated GFP by substituting as a surrogate β-strand in the groove vacated by the native strand. This finding provides serves to reconcile many of the apparent differences between the peptide binding and photodissociation properties of split proteins involving β-strands 10 and 11. We show that these truncated GFPs can bind other non-native sequences, and this promiscuity invites the possibility for rational design of sequences optimized for strand binding and photodissociation, both useful for optogenetic applications.

The bacterium Caulobacter crescentus divides asymmetrically. This lends to its suitability as a model organism for studying the bacterial cell cycle as well as the asymmetry inherent in all life. Central to Caulobacter's cell cycle is its single circular chromosome, which encodes genetic elements whose expression patterns are coordinated in a temporally oscillating transcriptional network. But there is more to the bacterial chromosome than genetic elements like genes and their promoters; in reality, the chromosome is a DNA polymer with physical constraints, capabilities, and complexities outside of its role as a template for transcription. For instance, DNA in bacteria is supercoiled and compacted by three orders of magnitude. At the ~1Mb level, the E. coli nucleoid is characterized by insulated macrodomains and flexible unstructured regions. At the ~10 kb level the bacterial nucleoid is arranged into so-called microdomains by nucleoid-associated proteins (NAPs). This structuring of DNA functions in segregation, replication, decatenation, and double- and single-strand break repair. In the work presented in this thesis I leverage four Next Generation Sequencing (NGS)-based technologies to examine the regulation and function of four physical and non-genic features of the Caulobacter chromosome: its covalent modifications, essential intergenic regions, and global accessibility, as well as a novel, essential nucleoid-associated protein. First, in Chapter 2, I explore the covalent modification systems in Caulobacter using SMRT sequencing technologies. By measuring the methylation state of every base pair in the chromosome at five times in the cell cycle, I demonstrate that DNA methylation by the cell cycle-regulated methyltransferase CcrM is in fact dynamic, changing from fully methylated to hemimethylated at the time of replication fork passage. Importantly, the master transcriptional regulators ctrA and dnaA have promoters that become activated or repressed respectively once hemimethylated. From this perspective, the methylation data supports a model in which the replicating chromosome acts like a clock, with the cell cycle timing of ctrA activation and dnaA repression precisely synchronized with replication fork passage. I then use the SMRT sequencing data to predict which additional promoters besides PctrA and PdnaA are possibly controlled by methylation state. In addition, I also report that some of the CcrM target sites were found to be constitutively unmethylated. Work by our colleagues has shown that at one such unmethylated region of the chromosome called Gap 7, the DNA-binding proteins MucR1/2 block CcrM from its cognate site. In Chapter 3, I investigate Caulobacter's essential intergenic regions, called Gap regions. These non-genic elements of unknown function appeared as essential regions of the chromosome during Transposon-sequencing (Tn-seq) experiments. By performing molecular genetics experiments I demonstrated that at least some Gaps are nonessential. Furthermore, I deduce that at least two of those nonessential Gaps appeared in the Tn-seq screen because they are specifically protected from transposase, the enzyme that carries out transposition, in vivo. I end this chapter by exploring the effects of placing the intergenic regions encompassing Gaps onto high copy plasmids. These experiments demonstrate that such perturbations actually cause elongation and division defects in Caulobacter in a growth rate-dependent manner. I predict that some of these transposase-protected regions may be blocked by the binding of specific proteins that function in the Caulobacter cell cycle. In Chapter 4, I build further support for these conclusions by developing and performing ATAC-seq in Caulobacter. By measuring transposase accessibility of the entire chromosome in vivo, ATAC-seq more definitively and globally divides Gaps into non-disruptable regions, which are relatively protected from transposase enzyme, and true candidate essential regions. Thus, uncharacterized non-genic elements of the chromosome may perform essential functions in Caulobacter. I also performed ATAC-seq on a strain lacking the novel nucleoid-associated protein GapR, which binds over many Gaps in vivo, to test whether this protein is responsible for forming Gap regions where it binds the chromosome. While GapR does not affect local DNA accessibility, global analysis of ATAC-seq data provides evidence that GapR plays key roles in shaping megabase-scale properties of the Caulobacter nucleoid. In WT (wild type) cells, this global accessibility pattern is reminiscent of the macrodomain-level structure of the E. coli chromosome, which features insulated origin and terminus-proximal regions as well as two highly flexible origin-flanking regions. These domains compose the largest scale of chromosome organization known in prokaryotes and are thought to help control the fidelity of chromosome segregation. Such macrodomains have not been observed explicitly before in Caulobacter. Overall, ATAC-seq shows that the chromosome is varied in accessibility along its length, reflecting how transcription, local DNA structure, NAPs like GapR, and possibly other protein-DNA interactions, alongside other unknown factors, may together yield globally varying enzyme access to the nucleoid. In Chapter 5, I focus on GapR, a novel, conserved and essential DNA binding protein introduced above. ChIP-seq experiments revealed that GapR binds AT-rich DNA globally throughout the chromosome, including over a majority of Gap regions. Although GapR shares many similarities with the E. coli NAP H-NS, there are two important differences between these proteins. First, whereas H-NS represses transcription of many genes in E. coli, depletion-RNAseq experiments in which gene expression changes were measured after proteolytic removal of GapR from Caulobacter demonstrated that GapR does not control transcription. In addition, although GapR is essential in Caulobacter, hns is dispensable in E. coli. In the final Chapter I will explore efforts to elucidate the essential function of GapR. Perspectives on additional functions of GapR proposed by our colleagues, including the regulation of initiation of chromosome segregation, are also discussed. Altogether, the work in this chapter provides evidence that NAPs can perform multiple diverse functions, including those that help fine-tune cell cycle progression. These experiments highlight an integrated view of the bacterial cell cycle in which both genic as well as non-genic and physical elements of the chromosome play key roles. Further, they herald novel applications of NGS techniques as promising tools for microbiologists.

Since the first optical detection of single molecules in 1989, there has been a rapid growth in applications, especially in biophysics. Single-molecule techniques are well suited for investigating the heterogeneity of populations, which is of particular interest in living systems that are defined by their complexity. Single-molecule fluorescence microscopy incorporates three main advantages of fluorescence imaging: biocompatibility, target specificity, and extreme sensitivity, and then improves upon the spatial precision by an order of magnitude relative to traditional, diffraction-limited imaging (down to tens of nanometers). This dissertation describes the application of single-molecule tracking to the investigation of the Hedgehog signaling pathway in mammalian cells. Hedgehog signaling is an essential developmental signaling pathway. Aberrant signaling leads to birth defects and has been implicated in a wide variety of cancers. Despite its oncological importance, the precise molecular mechanisms of signal transduction remain unclear. Previous work showed that pathway transduction involves the translocation of signaling proteins into and out of the primary cilium, a small organelle that protrudes from the cell surface. By tracking individual pathway proteins Patched1 and Smoothened with high spatial and temporal resolution (30 nanometers, 100 Hz), we have discovered and characterized changes in their dynamic motions within the cilium when the pathway is active or inactive at the most basic, functional level. In particular, we have found that the propensity for these proteins to bind at the base and tip of the cilium is a function of the pathway activation state. Moreover, aspects of the behavior of Smoothened are anticorrelated with the behavior of Patched1. These results have helped elucidate pathway function, and are applicable to the development of specific anti-cancer drugs that aim to disrupt particular steps of Hedgehog signaling. Chapter 1 provides an introduction to single-molecule imaging and an overview of Hedgehog signaling. Chapter 2 details the construction and operation of a fluorescence microscope with single-molecule sensitivity and outlines the various methodology utilized in later chapters. Chapters 3 and 4 describe our investigation of the Hedgehog signaling transducers Smoothened and Patched1, respectively. Chapter 5 summarizes our results and highlights several promising directions for further investigation. Finally, Chapter 6 contains the source code and manuals for the tools developed in these projects.

The movement of molecules through a condensed phase is governed by an extremely complex hierarchy of considerations and constraints on motion. This is particularly true when electric fields from ions and hydrogen bonding is involved, let alone heterogeneous structuring of the liquid. This thesis seeks, principally, to quantify the timescales of probe molecule motions and to then improve the understanding of how these motions occur. The ultimate goal is to predict how changes in the environment will affect motion both of the liquid and any dissolved solutes. The nature of molecular motions in the condensed phase was investigated using ultrafast two-dimensional infrared spectroscopy. This enabled non-perturbative interrogation of the systems in question as well as the time resolution necessary to detect changes in motion. Both the reorientation of the molecules and the spectral diffusion (which quantifies structural fluctuations) were used to better understand the motions in a number of chemical systems, focusing on the effect of salts. Both aqueous solutions of salts and room temperature ionic liquids (which are a subset of salts that remain molten at room temperature) with dissolved solutes were investigated. A number of noteworthy results were obtained and some interesting conclusions were drawn about aqueous salt solutions. It was found that, in water, some salts (particularly those with a high charge density) can slow the movement of water molecules considerably. This is primarily dependent on the anion, to which the water is directly hydrogen bonded, but the cation can play a role. However, certain salts (including borohydride) leave the dynamics of water unperturbed even when extremely concentrated. This is facilitated by the formation of hydrogen bonds with water molecules such that the hydrogen bond network remains fairly intact despite the presence of the ions. The structuring of the liquid may also play a role -- ionic liquids, which exist as liquids even without the addition of water and have internal structuring, have different dynamics than typical salts when the same amount of water is added to both. Studies were conducted on ionic liquid solutions to determine the effect of structuring. By changing the alkyl chain length of the cation, the ionic liquid adopts different configurations. This cause the dynamics of solutes to change in ways that would not necessarily be expected given the change in bulk properties like viscosity. The region of the ionic liquid that is solvating the probe molecule is found to determine the dynamics rather than the liquid as a whole. However, as was found with dissolved carbon dioxide as it is of note for carbon capture applications, the long range structuring of the ionic liquid can play a role for a subset of the motions that the probe experiences.

The properties of liquid systems confined on nanometer length scales may differ considerably from those of bulk liquids, with consequences for the dynamics of processes taking place in those systems. Proton transfer dynamics in nanoconfined aqueous environments are a topic of considerable scientific interest. A significant area of research within that topic is the influence of the confining interface on dynamics within the confined environment. Similarly, liquids that display relatively long-range structure (nanometer size scales) may show the effects of that structure in the dynamics of dissolved solutes. For example, room temperature ionic liquids (RTIL), a class of compounds that has attracted considerable interest for applications in catalysis, synthesis, and energy storage, have been shown to exhibit complicated nanoscale liquid structure with the formation of hydrophilic and hydrophobic domains. The orientational relaxation dynamics of the fluorescent probe perylene dissolved in solutions of various concentrations of lithium salts in RTILs were studied by measurement of fluorescence anisotropy. Increasing concentrations of lithium salt were found to cause both the in-plane and out-of-plane rotational diffusion of perylene to become slower as the liquid's bulk viscosity increased. However, the rotational diffusion did not slow sufficiently to track the viscosity and both of the corresponding viscosity-independent molecular friction coefficients decreased. This reveals that lithium ions, which are solvated by the ionic regions of the RTILs, change the structure of the alkyl regions of the RTIL. Another series of experiments studied excited-state proton transfer kinetics in both ionic and neutral reverse micelles. TCSPC measurements of the population decay were conducted on the fluorescent photoacid 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) in bulk water, ionic reverse micelles, and non-ionic reverse micelles, and the orientational dynamics of dissolved MPTS, the methoxy derivative of HPTS, in those systems were also investigated by time dependent fluorescence anisotropy measurements. The orientational diffusion data suggest that in ionic reverse micelles, the probe molecules are located in the water core of the reverse micelle, while in non-ionic reverse micelles, the data suggest the probes are located at the water-surfactant interface. HPTS proton transfer in ionic reverse micelles followed kinetics qualitatively like those in bulk water, albeit slower, with the power law time dependence associated with recombination of the proton with the dissociated photoacid having a smaller exponent, suggesting a modified diffusion-controlled process. In neutral reverse micelles, proton transfer kinetics did not show discernible power law behavior and were best represented by a two component model with one relatively water-like population, and a population with a faster fluorescence lifetime and negligible proton transfer. Finally, the analysis of proton transfer dynamics in bulk water resulted in a t-1.1 power law for proton recombination as opposed to the t-1.5 power law predicted by theory and some previously reported observations. The reliability of these newer observations is justified, and possible ways in which existing theory may be incomplete are discussed.

Transcription is considered the first step in gene expression. During the transcription initiation phase, a cell decides where and when transcription will occur on its template DNA using a complex network of regulation. Understanding the initiation process is therefore fundamental to any broader understanding of gene regulation. Despite being an active research area for past few decades, fundamental questions regarding mechanistic aspects of initiation remain unanswered. Here, we present findings related to the transcription initiation processes in prokaryotes and eukaryotes, based on single-molecule, real-time observations of RNA polymerase (RNAP) activity using optical tweezers. The only protein complex required to initiate transcription in bacteria is the RNAP holoenzyme itself, which is composed of RNAP core enzyme and a σ factor. The RNAP holoenzyme is able to make multiple contacts with promoter DNA sequences. These contacts modulate both the frequency of transcription, and duration of subsequent steps in the transcription initiation process. Here, we describe a novel single-molecule optical trapping assay that enables us to map the contacts of the Escherichia coli (E. coli) RNAP holoenzyme on the promoter DNA. Using this assay, we were able to observe the initiation process in real time. Through the study, my colleague and I were able to identify strong contacts between the RNAP holoenzyme and promoter DNA at various promoter sequence elements. By monitoring the initiation process from holoenzyme binding to promoter escape, we were also able to observe the remodeling of key contacts between the RNAP holoenzyme and promoter DNA. In contrast to bacterial transcription initiation, the eukaryotic transcription initiation is less studied. A major difference between prokaryotic and eukaryotic transcription initiation is that more proteins are essential to initiate transcription in eukaryotes. To form a minimal, transcription-competent pre-initiation complex (PIC), five general transcription factors (GTFs) are required, in addition to a 12-subunit RNAPII (pol II). Previous biochemical and structural studies have shown that the initiation of pol II transcription proceeds in the following stages: 1) assembly of pol II with GTFs and promoter DNA in a "closed" complex, within which the DNA still remains fully annealed; 2) unwinding about 15 bp of the promoter DNA to form an "open" complex; 3) scanning the downstream DNA sequence for a transcription start site; 4) synthesis of a short RNA transcript, believed to be about 10 nucleotides; 5) and promoter escape. In my thesis, I present findings based on real-time observations of initiation using a reconstituted 32-protein, 1.5 megadalton PIC derived from Saccharomyces cerevisiae. Measurements were made on detailed aspects of the motions of transcription factor IIH (TFIIH), as well as those of pol II, throughout the initiation process. In addition to characterizing the physical properties of TFIIH, including velocity and processivity, our findings establish TFIIH as the motor responsible for start-site scanning. Contrary to expectations, scanning driven by TFIIH entailed the rapid opening of a large transcription bubble, averaging 85 bp, accompanied by the synthesis of a transcript up to the entire length of the extended transcription bubble, followed by promoter escape.

Single molecules were first detected at low temperatures twenty-six years ago in the laboratory of W. E. Moerner. Subsequent technological advances have allowed researchers to study single molecules at room temperatures and within living cells, providing novel biological insight about underlying spatial and dynamical heterogeneity. By combining single molecule detection with the ability to control the emissive state of the fluorescent label (also called "active control"), a suite of super-resolution imaging techniques has been developed. These single-molecule-based super-resolution imaging strategies leverage the fluorescence microscope's ability to non-invasively study multiple targets within living cells, while bridging the resolution gap between optical and electron microscopies. In large part, future advances to improve single molecule and super-resolution imaging require better fluorophore and labeling technologies. Utilizing fluorophore with higher photon yields will increase the resolution of super-resolution images and the data acquisition speed. Additionally, a greater library fluorophores with different of colors and sensing capabilities will enable application to more imaging targets. Currently, many single molecule and super-resolution experiments within living systems use fluorescent proteins because the labeling of target proteins is more straightforward. However, the limited photon yield of fluorescent proteins often results in tantalizingly fuzzy super-resolution images. Imaging the same targets, labeled instead with brighter organic emitters, could provide more image detail, but better fluorogenic and genetically encoded labeling schemes must be developed and discovered. The first chapter of this dissertation will introduce and discuss the historical context and basic principles of single molecule and super-resolution imaging. Chapter 2 will then describe the general experimental procedures necessary for quantitative single molecule and super-resolution imaging, including quantifying the number of photons detected (and emitted) from a single molecule, as well as the preparation of bacterial samples for fluorescence microscopy. Later chapters apply these fundamental experimental measurements to study bacterial biology and fluorophore photophysics. Chapters 3 and 4 concern the development and characterization of organic emitters suitable for single molecule or super-resolution imaging, work achieved with the synthetic collaboration of organic chemists in the laboratory of Professor Robert J. Twieg at Kent State University. Chapter 3 discusses the optimization of rhodamine spirolactam photoswitching such that activation could occur at visible wavelengths. The optimized rhodamine spirolactams were then covalently attached to the surface of bacterial cells and imaged with three-dimensional super-resolution. Images of the bacterial cell surface demonstrates a marked improvement in labeling uniformity, specificity, and density compared to previous methods which labeled the surface with the transient binding of a membrane sensitive dye. Chapter 4 introduces a novel enzyme-based strategy to control the fluorescence from nitro-aryl fluorogens. A proof-of-principle experiment demonstrated that endogenous nitroreductase enzymes within bacterial cells could catalyze the fluorescence-activating reaction, thus generating free fluorophores, which were detectable on the single-molecule-level within the cell. Lastly, chapter 5 summarizes three-dimensional imaging experiments (performed in collaboration with the laboratory of Professor Lucy Shapiro in the Department of Developmental Biology at Stanford University) of components of the bacterial gene expression machinery labeled with fluorescent proteins. Super-resolution imaging is ideally suited to the small size scale of bacterial cells, and a wealth of biological insights remains to be discovered. Simultaneously improving fluorophore photon yield, specificity, and active control strategies will have a profound impact on super-resolution precision and speed.

Single fluorescent molecules and particles can be localized in space with precision on the order of tens of nanometers (i.e. "super localized") using state-of-the-art microscopy techniques. The ability to probe complex environments at the sub-diffraction size scale has proven invaluable in revealing fundamental heterogeneity and improving overall understanding across the applied physical sciences. Super-localization microscopy is at the heart of both single-molecule super-resolution microscopy and single-particle tracking. The work presented in this dissertation concerns the application of super-localization microscopy to problems of biophysical interest, as well as theoretical and experimental advances in the methodology of this class of techniques. While the most common methods of super-localized position estimation ensure high localization precision, they might not always ensure high accuracy. In particular, the anisotropy of single-molecule dipole emission can result in mislocalizations of hundreds of nanometers, depending on the orientation of the molecule and its distance from the focal plane. In this dissertation I discuss different ways to correct this potential source of error. On the one hand, a theory based on a wobbling-in-a-cone model is presented that shows how this error is mitigated by molecular rotational mobility. On the other hand, for the worst-case scenario of a rotationally fixed emitter, an experimental approach based on Fourier optics is also discussed that allows for estimation of molecular orientation and enables active correction of mislocalization effects. The last third of this dissertation discusses applications of super-localization microscopy to three-dimensional tracking of fluorescently labeled genetic loci in budding yeast. In order to localize loci in the axial dimension, I used a Fourier optics approach to engineer the point spread function of the microscope into a Double-Helix Point Spread Function (DH-PSF). With this method, many single copies of a specific locus were analyzed, each with 3D spatial precision on the order of 10 nm at a rate of 10 Hz. A two-color implementation of the microscope allowed measurement of the correlations of 3D motion between pairs of loci under variable transcriptional pressure. I also discuss the importance of properly accounting for the inescapable effects of static and dynamic tracking errors caused by finite photon statistics and motion blur, respectively. These errors affect the statistics of the estimated motion and distort common metrics for characterizing stochastic motion such as the mean-squared displacement (MSD) and velocity autocorrelation (VAC). Analytical expressions for the MSD and VAC in the presence of these errors are given, along with applications to chromosomal locus tracking.

Neurons are highly polarized cells with their axon often extending over large distances away from the cell bodies (up to 1 meter in axonal length with a cell body less than 50 micrometer in diameter). Given that the majority of neuronal proteins and materials are synthesized in the cell body, such a long axon precludes effective diffusion of soma-produced proteins to their presynaptic destinations at the axonal terminals. As a result, neurons rely extensively on active axonal transport to deliver newly synthesized synaptic proteins, ion channels, lipids, and mitochondria to their axonal destinations via anterograde transport. On the other hand, retrograde axonal transport is responsible for carrying molecules and organelles destined for degradation from the axonal terminals back to the cell body. A highly efficient and tightly regulated machinery is thus required for a robust long-range transport of materials to ensure the neurons' proper growth, maintenance and survival. This thesis is a quantitative study of the underlying mechanism of axonal transport, with a specific focus on the retrograde axonal transport machinery in neurons. In our experimental setup, the axonal transport of cargos can be directly visualized in real-time using a neuronal microfluidic platform and fluorescence microscopy technique. We observe that cargos have high tendency to slow down their transport speed when crossing various obstacles along the axon such as non-moving cargos and stationary mitochondria. Single molecule study of retrograde nerve growth factor transport reveals that mechanical tug-of-war and intracellular motor regulation are complementary features of the near-unidirectional endosome directionality. Specifically, a stochastic mechanical simulation suggests that the endosomes are driven on average by 5-6 active dyneins and 1-2 down-regulated kinesins. This result is further supported by a study of the dynamics of endosomes detaching under load in axons, showcasing the cooperativity of multiple dyneins and the subdued activity of kinesins. Lastly, we present a quantitative characterization of the complex behavior of light-sensitive cryptochrome 2 (CRY2) protein under blue light. The results contribute to the understanding of the light-inducible CRY2 system and can be used as a guide to establish new optogenetic strategies to probe cellular processes in live cells.

More than two centuries old, battery technology has never attracted so much attention as it is today from all over academia, industry, government, and general public. Its extended application in our daily life, from portable electronic devices, electric vehicles, to power grid storage is driving the urgent need for major breakthroughs, in energy density, cycle life, and cost. One of the materials of choice is silicon. Silicon anodes have an order of magnitude higher capacity than state-of-the-art graphite anodes, providing great promise for use not only in Li-ion, but also in next generation high energy Li-S and Li-O2 batteries. However, Si anodes of conventional structure have very short cycle life, because the volume change of Si upon cycling leads to fracture and unstable interfaces. In this dissertation, I employed nanoscale materials design to overcome these problems, by rationally making accurate void space available inside the structure, and limiting the surfaces that are exposed to the electrolyte. The first-generation "yolk-shell" anode prevents fracture, stabilizes the interface, and significantly extends cycle life at small mass loading. Then I designed a second-generation "pomegranate" anode that has reduced interface side-reaction, increased energy density, and enhanced electrode-level conductivity. This design performs excellently even at mass loading as high as commercial batteries. Moreover, its fabrication is highly scalable. Next, I developed a method that produces the key source material for the above designs, Si nanoparticles, from rice husks, an agricultural byproduct with extremely high annual yield, and low cost. Finally, a prelithiation method has been developed for silicon anodes so that it could be paired with high-energy sulfur cathodes to make a full battery. Such a combination can give almost 400% the energy of state-of-the-art Li-ion batteries, enabling the next generation of battery technology.

A majority of biological microscopy investigations involve the focusing of visible light with conventional lenses. Fluorescence microscopy is one of the most widely used tools in biology but its resolution has historically suffered from the diffraction limit to about 200 nm laterally and 800 nm axially. In the past decade, this resolution problem has been overcome by the rapidly emerging field of super-resolution microscopy. The first demonstrated super-resolution technique, STimulated Emission Depletion (STED) Microscopy, is the topic of this Dissertation. This Dissertation has two primary areas of focus: the design optimization of a STED microscope, covered in Chapters 2-4, and its application to super-resolution imaging in cells and tissues, covered in Chapters 4-6. Chapter 2 describes the STED apparatus and experimental methods used. This chapter covers the guiding principles behind the design of a STED microscope, which forms a basis for understanding the logic underlying the homebuilt STED microscope which was constructed for this research. This STED microscope has a typical resolution of approximately 60 nm (full-width-at-half-maximum) or 25 nm (sigma) and has the sensitivity to image single fluorophores. In Chapter 3, a framework for evaluating and optimizing STED performance in the presence of several key tradeoffs is presented. Chapter 4 describes both developments in STED Microscopy required to utilize far-red-emitting dyes and the challenges associated with performing super-resolution imaging in intact Drosophila tissue. In Chapter 5, the optimization of labeling density revealed the 9-fold symmetry of a centriole protein structure, an important organelle in cell development. In Chapter 6, Huntingtin protein aggregates are resolved beyond the diffraction limit in a cell model of the neurodegenerative Huntington's disease.

Single-molecule, super-resolution fluorescence microscopy is a powerful technique for studying biological systems because it reveals details beyond the optical diffraction limit (on the 20-100 nm scale) such as structural and conformational heterogeneity. Further, single-molecule imaging measures distributions of behaviors directly through the interrogation of many individual molecules and reports on the nanoscale environment of molecules. Sub-diffraction imaging adds increased resolution to the advantages of fluorescence imaging over the techniques of atomic force microscopy and electron microscopy for studying biological structures, which include imaging of large fields of view in aqueous environments, specific identification of protein(s) of interest by fluorescent labeling, low perturbation of the system, and the ability to image living systems in near real-time (limited by the time required for super-resolution sequential imaging). This dissertation describes the application of single-molecule and super-resolution fluorescence imaging to studying the huntingtin (Htt) protein aggregates that are a hallmark of Huntington's disease and that have been implicated in the pathogenesis of the disease. The intricate nanostructures formed by fibrillar Htt aggregates in vitro and the sub-diffraction widths of individual fibers mark the amyloids as important targets for high-resolution optical imaging. The characterization of Htt aggregate species is critical for understanding the mechanism of Huntington's disease and identifying potential therapies. Following an introduction to single-molecule, super-resolution imaging and Huntington's disease in Chapter 1, Chapter 2 describes the single-molecule methods, experimental techniques, Htt protein sample preparations, and data analysis performed in this dissertation. Chapter 3 discusses the development of super-resolution imaging of Htt protein aggregates and the validation of the images by atomic force microscopy. Chapter 4 continues the study of Htt by one- and two-color super-resolution with imaging of Htt protein aggregates over time from the initial protein monomers to the large aggregate assemblies of amyloid fibers. In Chapter 5, I detail our progress to-date in studying the earliest stages of Htt aggregation using zero-mode waveguide technology. Chapter 6 concludes the dissertation with a discussion of the results from additional projects comprising the effect of chaperonin proteins on Htt aggregation, extension of super-resolution Htt imaging to three dimensions, and cellular imaging of Htt aggregates. The future directions for these exciting projects are summarized with the expectation that research efforts directed in these areas will contribute to our understanding of Htt aggregation and Huntington's disease.

The increasing demand for energy together with the growing concerns about air pollution and global warming has stimulated intensive research on energy processes ranging from production, conversion, storage, transmission and consumption. Energy storage is to become more and more important with the gradual shift from fossil fuels to renewable energy sources which are temporally intermittent and geographically localized. On the other hand, electric vehicles are now a trend in the automobile industry with the goal to cut emission and reduce oil consumption. It is thus crucial to develop electrochemical energy conversion and storage devices such as batteries and supercapacitors with high specific energy and power, long cycle life, low cost and safety. We aim to design and synthesize novel nanostructured electrode materials and electrocatalysts by using chemically derived graphene sheets as growth substrates for electrochemical functional materials. The unique chemical interactions between graphene and the active nanomaterials affect the morphology and size of the nanomaterials, enhance electron transport, stabilize the nanomaterials during cycling, and generate synergistic effects in electrocatalysis, leading to superior electrochemical performance. We have grown nanocrystals of hydroxides, oxides, chalcogenides and phosphates with controlled morphology, sizes and structures on graphene, affording materials that can be readily integrated in current lithium ion batteries, alkaline batteries and supercapacitors to boost their performance, as well as materials that support rising technologies such as Li-S and Li-air batteries. The novel materials we have studied also allow for deepening our understanding in materials chemistry and electrochemistry.

The Enriched Xenon Observatory (EXO) is a series of experiments seeking to measure the neutrino mass through observation of neutrinoless double beta decay (0nbb). The next generation of 0nbb experiments aims to probe Majorana neutrino masses at or below 10 meV. To reach this sensitivity, ton-scale detectors are needed with lower radioactive backgrounds than the best ones operating today. The EXO collaboration is developing a novel strategy for a virtually background-free search for the 0nbb of Xe-136, based around detecting individual Ba-136 ions resulting from such decays. This dissertation details the efforts to develop a barium tagging technique which uses resonance ionization spectroscopy (RIS) to selectively and efficiently ionize barium atoms for injection and detection in an ion trap. A simple radionuclide-driven single-ion source has been developed to push the technology to high efficiency with a small number of ions.

Vesicle fusion is a central process in transport and communication in biology. In neuronal transmission, synaptic vesicles carrying neurotransmitters dock and fuse to the plasma membrane of the neuron, a process mediated by a combination of several membrane anchored and soluble proteins. Fusion results in the merger of the two apposing lipid bilayers, leading to the exchange of both the lipidic and aqueous components. The fusion reaction is thought to proceed through several stages: first, the membranes are brought into close proximity (docking), second, the outer leaflets mix, but the inner leaflets and contents remain separate (hemi-fusion), and finally, the inner leaflet and contents exchange (full fusion). Due to the complex nature of the fusion reaction and the multitude of proteins involved, the mechanism of the fusion reaction is not well understood. Simplified model systems for vesicle fusion can bring insight into the mechanism by studying the fusion reaction in a more defined and controllable system. This thesis describes a DNA-based model for the protein fusion machinery. Previously, DNA-lipids were used to tether lipid vesicles to glass-supported lipid bilayers. These vesicles could be observed by fluorescence microscopy, and are laterally mobile along the plane parallel to the supported bilayer. DNA-mediated docking between vesicles was characterized, but fusion was not observed due to the fact that the DNA partners were both coupled at the 5' end, so antiparallel hybridization holds the membranes apart. In this work, a new synthesis of DNA-lipid conjugates is described which allows coupling at both the 3' and 5' end of the DNA. Incorporation of complementary DNA-lipids coupled at opposite ends mediates fusion between lipid vesicles. Vesicle fusion was measured in bulk fluorescence assays (Chapter 2 and 3), by both lipid mixing and content mixing assays. The rate of vesicle fusion showed a strong dependence on the number of DNA per vesicle, as well as the sequence of the DNA. Consistent with previous results measured for the docking reaction, fusion was faster for a repeating DNA sequence than for a non-repeating sequence that required full overlap of the strands for hybridization. The role of membrane proximity on the rate of vesicle fusion was investigated in Chapter 3 by insertion of a short spacer sequence at the membrane-proximal end of fusion sequences. The length of the spacer sequence was varied between two and 24 bases, corresponding to length scales of approximately 1-12 nm. Fusion, as measured in bulk assays by lipid and content mixing, decreases systematically as the membranes are held progressively further apart, demonstrating a clear dependence of the rate of the fusion reaction on membrane proximity. While the bulk vesicle fusion assays showed that DNA-lipids can mediate vesicle fusion, these ensemble measurements convolve the multiple steps (docking, hemifusion, and full fusion) of the fusion reaction, complicating any kinetic analysis. In order to image individual vesicle fusion events between tethered vesicles, a new tethering strategy was developed (Chapter 4). This strategy exploits the dependence of DNA hybridization on salt by covalently attaching lipid vesicles to a glass-supported lipid bilayer, then triggering DNA-mediated docking and fusion by spiking the salt concentration. The kinetics of individual vesicle fusion events were subsequently measured using a FRET-based lipid mixing assay for many vesicles (Chapter 6). An analysis of the distribution of waiting times from docking to fusion indicated that this transition occurs in a single step. A second model membrane architecture was used to study individual fusion events between vesicles and a planar bilayer (Chapters 5 and 6). This architecture uses a DNA-tethered planar free-standing bilayer as the target membrane. The kinetics of individual vesicle fusion events to this membrane patch were also consistent with a single step process, as for vesicle to vesicle fusion. In this system, it was also possible to observe content transfer of vesicles containing a self-quenched aqueous dye (Chapter 5). By analyzing the diffusion profile of the dye, it was shown that the dye indeed is transferred into the region below the planar membrane patch, and is not released into the solution above the patch due to vesicle rupture or leakage.

Since the first successful detection single molecules over two decades ago, single-molecule spectroscopy has developed into a burgeoning field with a wealth of experiments at room temperature and inside living cells. Probing asynchronous and heterogeneous populations in situ, one molecule at a time, is not only desirable, but critical for many biological questions. Further, super-resolution imaging based on sequential imaging of sparse subsets of single molecules, has seen explosive growth within the last five years. This dissertation describes both the application of live-cell single-molecule imaging as an answer to important biological questions, and development and validation of fluorescent probes for targeted super-resolution imaging.