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Out of the growing body of evidence demonstrating the role of higher-order chromatin organization within the nucleus in regulating the functions of the linear sequence of DNA emerges the genome as a physical entity. DNA packs into hierarchical levels of chromatin condensation, which then tailor accessibility to the linear sequence for nuclear processes while also serving as a central feature of nuclear organization. Further, varying condensation state alters the physical properties of the chromatin fiber. These may then exert or facilitate forces aiding in the spatial organization within the nucleus. Yet, this complex concept of nuclear structure even neglects the dynamic aspects of the genome continuously fluctuating and undergoing structural remodeling within the nucleus. Thus, while chromatin position within the nucleus is critical for biological functions including transcription, we must reconcile a particular position of a gene locus with the dynamic and physical nature of chromatin. Here we characterize the physical aspects of the genome associated with its dynamic properties that aid in regulation. We focus on developing techniques that measure the evolution of physical properties associated with nuclear processes. We leverage these techniques, capable of quantifying and spatially resolving its structural state within the nucleus and elucidating the underlying physics of its dynamics, to illuminate physical features associated with cellular processes. Specifically, we investigate the nuclear structural changes associated with growth factor stimulation on primary human cells known to impact large scale gene expression pathways. We also demonstrate dysfunction associated with these physical mechanisms accompany disease pathologies. Thus, we unify the biological understanding of cellular processes within the context of physical features of genome structure, organization and dynamics that are critical to human health and disease.

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In recent years, localization-based super-resolution imaging has been developed to overcome the diffraction limit of far-field fluorescence microscopy. Photoswitchable probes are a hallmark of this technique. Their fluorescence can be modulated between an emissive and dark state whereby the sequential, nanoscale measurement of individual fluorophore positions can be used to reconstruct an image at higher spatial resolution. Despite the importance of photoswitchable probes for localization-based super-resolution imaging, both a mechanistic and quantitative understanding of the essential photoswitching properties is lacking for most fluorophores. In this thesis, we begin to address this need. Furthermore, we demonstrate the development of new probes and methodologies for both multicolor and live-cell super-resolution imaging. Chapter 2 describes our mechanistic insights into the photoswitching of a common class of dyes called carbocyanines. Red carbocyanines, such as Cy5, enter a long-lived dark state upon illumination with red light in the presence of a primary thiol. We show that the dark state is a covalent conjugate between the thiol and dye and that this dark state recovers by illumination with ultraviolet light. We also speculate on possible reactivation mechanisms. Our mechanistic studies may ultimately lead to the creation of new probes with improved photoswitching properties. Chapter 3 details our quantitative characterization of the photoswitching properties of 26 organic dyes, including carbocyanines and several other structural classes. We define the essential properties of photoswitchable probes, including photons per switching event, on/off duty cycle, photostability, and number of switching cycles, and demonstrate how these properties dictate super-resolution image quality. This rigorous evaluation will enable more effective use of probes. In Chapters 4 and 5, we focus on expanding the super-resolution toolbox with novel strategies for multicolor and live-cell imaging. Chapter 4 discusses two approaches we have developed for multicolor super-resolution imaging, which distinguish probes based on either the color of activation or emission light. These tools allow multiple cellular targets to be resolved with high spatial resolution. Lastly, Chapter 5 introduces a method for targeted cellular labeling with photoswitchable probes using a small peptide tag, as well as a new sulfonate-protection strategy for intracellular delivery of high performing photoswitchable dyes.

DNA replication is a fundamental cellular process. However, the structure and dynamics of the eukaryotic DNA replication machinery remain poorly understood. A soluble extract system prepared from Xenopus eggs recapitulates eukaryotic DNA replication outside of a cell on a variety of DNA templates. This system has been used to reveal many aspects of DNA replication using a variety of ensemble biochemical techniques. Single-molecule fluorescence imaging is a powerful tool to dissect biochemical mechanisms. By immobilizing or confining a substrate, its interaction with individual, soluble, fluorescently-labeled reactants can be imaged over time and without the need for synchrony. These molecular movies reveal binding parameters of the reactant and any population heterogeneity. Moreover, if the experiments are imaged in wide-field format, the location or motion of the labeled species along the substrate can be followed with nanometer accuracy. This dissertation describes the use and development of novel single-molecule fluorescence imaging techniques to study eukaryotic DNA replication. A biophysical characterization of a replication fork protein, PCNA, revealed both helical and non-helical sliding modes along DNA. Previous experiments demonstrate that the egg extracts efficiently replicate surface-immobilized linear DNA. This finding suggested replication of DNA could be followed as motion of the replication fork along the extended DNA. However, individual proteins bound at the replication fork could not be visualized in the wide-field due to the background from the high concentration of the fluorescent protein needed to compete with the extract’s endogenous protein. To overcome this concentration barrier, I have developed a wide-field technique that enables sensitive detection of single molecules at micromolar concentrations of the labeled protein of interest. The acronym for this method, PhADE, denotes three essential steps: (1) Localized PhotoActivation of fluorescence at the immobilized substrate, (2) Diffusion of unbound fluorescent molecules to reduce the background and (3) Excitation and imaging of the substrate-bound molecules. PhADE imaging of flap endonuclease I (Fen1) during replication revealed the time-evolved pattern of replication initiation, elongation and termination and the kinetics of Fen1 exchange during Okazaki fragment maturation. In the future, PhADE will enable the elucidation of the dynamic events at the eukaryotic DNA replication fork. PhADE will also be broadly applicable to the investigation of other complex biochemical process and low affinity interactions. It will be especially useful to those researchers wishing to correlate motion with binding events.

Imaging systems often make use of macroscopic lenses to manipulate light. Modern microfabrication techniques, however, have opened up a pathway to the development of novel arrayed imaging systems. In such systems, centimeter-scale areas can contain thousands to millions of micro-scale optical elements, presenting exciting opportunities for new imaging applications. We show two such applications in this thesis: pressure sensing in microfluidics and high throughput fluorescence microscopy for high content screening. Conversely, we show that arrayed elements are not always needed for three dimensional light field imaging. / Engineering and Applied Sciences

Grinding/polishing and indentation induced residual stresses were measured by confocal Cr³⁺ fluorescence microscopy with high spatial resolution (~2 μm),obtaining local stress variation information rather than the mean stress averaged over a large sampling volume as is measured by other techniques. Due to the translucency of alumina materials, a substantial portion of the fluorescence signal comes from beneath the surface of the specimen. A probe response function (PRF) was developed taking account of microscope resolution, refraction, absorption and scattering, to quantitatively describe where the collected signal came from. It described the fluorescence intensity variations against defocus distance very well for a range of materials including sapphire, ruby, polycrystalline alumina and AI₂O₃/SiC nanocomposites. Large variations in the residual stresses on ground and polished surfaces were observed, owing to the surface fracture and pullouts. The broad peaks and narrow peaks separated from the spectra collected near the ground/polished surfaces physically represented the two distinct regions in the ground region: a plastically deformed surface layer and the elastically deformed material underneath. A model for the residual stress field taking into account the pullout was proposed using an array of virtual dislocations. The model agreed with the experimental results well when the PRF was included. Tensile stresses were detected on the ground surfaces of polycrystalline aluminas and 2 vol.% SiC nanocomposite, but not on the polished surfaces of polycrystalline aluminas or ground surfaces of 5 and 10 vol.% SiC nanocomposites. This was explained in terms of difference in the amount of pullouts on the surfaces. The depth of deformation was deeper in the ground polycrystalline alumina compared to the polished condition; the depth of deformation in alumina and the AI₂O>sub>3</sub>/SiC nanocomposites were similar (~1 μm) while the compressive stresses in the nanocomposites were greater owing to the reduction in pullout. The main difference between ground alumina and AI₂O₃/SiC nanocomposites was the brittle fracture behavior rather than the plastic deformation. Line scans and area mapping were carried out on 1 kg loaded Vickers indentations of alumina-based ceramics. Tensile stresses were found at the tips of radial cracks and lateral cracks and compressive stresses were found around the indent impression. The line scan results in the elastic regions agreed qualitatively with Yoffe's model and the quantitative discrepancy was attributed mainly to the cracking that relaxed the stresses. The differences in residual stresses between alumina and AI₂O₃/SiC nanocomposites were small if measured with high spatial resolution but it would be exaggerated with lower resolution.

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published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Μελέτη διφωτονικά διεγερμένου φθορισμού για τον υπολογισμό των ενεργών διατομών διφωτονικής απορρόφησης οργανικών μορίων. / Study of the two photon excitation fluorescence for the estimation of two photon cross sections of organic molecules