Inductively coupled plasma-mass spectrometers (ICP-MS) have become the workhorses of many analytical labs over the past few decades. Despite the instruments' high sensitivities and low detection limits there is still a demand for improvements in several aspects of their performance. One area of improvement is in the understanding of "space charge effects" Space charge effects are classified as problems associated with the ion beam. Problems are created when the mutual repulsions of the ions make consistent focusing of the ion beam difficult. This is particularly problematic with samples containing a low concentration analyte contained within a high salt solution matrix, resulting in lower instrument sensitivity and inaccurate results. The research presented here used laser-induced fluorescence (LIF) imaging to characterize the ion beam as it enters the mass analyzer of a commercial ICP-MS. To perform the LIF imaging a laser system with two ring cavities was constructed to frequency double a CW titanium-sapphire laser to the calcium ion transition at 393.4 nm or to the barium ion transition at 455.4 nm. Ion beam images for both elements were taken under different instrument modes and matrix compositions. The same trends in shift and distortion of the barium ion beam with the addition of a lead matrix was observed as in previous experiments with calcium. A shift in the focal point of the ion beams of both elements was also observed in normal sensitivity mode and with the instrument's collisional reaction interface (CRI). This work indicates that a shift in beam focusing is responsible for the change in ion transmission due to changes in matrix composition and instrument modes.

Two-photon absorbing (2PA) materials has been widely studied for their highly localized excitation and nonlinear excitation efficiency. Application of 2PA materials includes fluorescence imaging, microfabrication, 3D data storage, photodynamic therapy, etc. Many materials have good 2PA photophysical properties, among which, the fluorenyl structure and its derivatives have attracted attention with their high 2PA cross-section and high fluorescence quantum yield. Herein, several compounds with 2PA properties are discussed. All of these compounds contain one or two fluorenyl core units as part of the conjugated system. The aim of this dissertation is to discuss the application of these compounds according to their photophysical properties. In chapters 2 to 4, compounds were investigated for cell imaging and tissue imaging. In chapter 5, compounds were evaluated for photodynamic therapy effects on cancer cells. Chapters 2 and 3 detail compounds with quinolizinium and pyran as core structures, respectively. Fluorene was introduced into structures as substituents. Quinolizinium structures exhibited a large increase in fluorescence when binding with Bovine Serum Albumin (BSA). Further experiments in cell imaging demonstrated a fluorescence turn-on effect in cell membranes, indicating the possibility for these novel compounds to be promising membrane probes. Pyran structures were conjugated with arginylglycylaspartic acid peptide (RGD) to recognize integrin and introduced in cells and an animal model with tumors. Both probes showed specific targeting of tumor vasculature. Imaging reached penetration as deep as 350 µm in solid tumors and exhibited good resolution. These results suggest the RGD-conjugated pyran structure should be a good candidate probe for live tissue imaging. Chapter 4 applied a fluorene core structure conjugated with RGD as well. Application of this fluorenyl probe compound is in wound healing animal models. Fluorescence was collected from vasculature and fibroblasts up to ≈ 1600 µm within wound tissue in lesions made on the skin of mice. The resolution of images is also high enough to recognize cell types by immunohistochemical staining. This technology can be applied for reliable quantification and illustration of key biological processes taking place during tissue regeneration in the skin. Chapter 5 describes three fluorenyl core structures with photoacid generation properties. One of the structures showed excellent photo-induced toxicity. Cancer cells underwent necrotic cell death due to pH decrease in lysosomes and endosomes, suggesting a new mechanism for photodynamic therapy.

Small molecule organic dyes with absorption and emission in the near infrared region (NIR) attracted much attention for various applications such as dye sensitized solar cells, fluorescent guided surgery, stimuli responsive bioimaging and photodynamic therapy. Dyes with high absorption and emission in the NIR region are beneficial for stimuli responsive bioimaging due to the deeper penetration of NIR light, less cell damage, high resolution, and low background autofluorescence from biomolecules. Of the many small molecule dyes, xanthene-based dyes exhibit outstanding photophysical properties and good stimuli response for use in bioimaging applications. However, absorption and emission of the xanthene dyes lie in the visible region, which limit their applications in cellular imaging. Many of the NIR dyes have very poor fluorescence; consequently, an alternative approach to fluorescent imaging is photoacoustic imaging that uses sound waves to generate pictures of deep tissues. In this dissertation, we discuss the utility of xanthene based NIR dyes as photoacoustic imaging contrast agents for multiplex imaging and deep tissue nitric oxide sensing in the drug-induced liver injury. Chapter I discuss the fundamentals of fluorescence and photoacoustic imaging, background of the xanthene dyes and other fluorescent dyes, and the design strategies to develop NIR xanthene-based dyes. Chapter II is based on our approach to the design and synthesis of NIR xanthene-based dyes by C-H bond functionalization, with the first example being Rhodindolizine, which absorb and emits in NIR II or short-wave infrared (SWIR) region. In chapter III, we describe the design and synthesis of thienylpiperidine xanthene-based NIR and shortwave-infrared (SWIR) dyes for the photoacoustic imaging. One dye in particular (XanthCR-880) boasts a strong PA signal at 880 nm with good biological compatibility and photostability, yields multiplexed imaging with an aza-BODIPY reference dye, and is detected at a depth of 4 cm. In chapter IV, we report a series of SWIR dyes based on a dibenzazepine donor conjugated to thiophene (SCR-1, SCR-4), thienothiophene (SCR-2, SCR-5), and bithiophene (SCR-3, SCR-6). We leverage the fact that SCR-1 undergoes a bathochromic shift when aggregated to develop a ratiometric nanoparticle for nitric oxide (NO) (rNP-NO). rNP-NO was used to successfully perform in vivo studies to visualize pathological levels of nitric oxide in a drug-induced liver injury model via deep tissue SWIR photoacoustic (PA) imaging. Chapter V describes another series of xanthene-based dyes with a thiophene ᴫ spacer and several different donors. UV-Vis absorption studies were performed after converting the dyes to the opened form with trifluoracetic acid. These novel XanthCR-TD dyes exhibit absorption maxima in NIR I region from 700 - 900 nm.

Regenerative medicine involves formation of tissue or organ for replacement of a wounded or dysfunctional tissue. Healthy cells extracted from the patient are expanded and are seeded on a three-dimensional biodegradable scaffold. The structure is then placed in a bioreactor and is provided with nutrients for the cells, which proliferate and migrate throughout the scaffold to eventually form a desired to tissue that can be transplanted into the patient's body.  Inability to monitor this complex process of regeneration in real-time makes control and optimization of this process extremely difficult. Histology, the gold standard used for tissue structural assessment, is a static technique that only provides "snapshots" of the progress and requires the specimen to be sacrificed. This inefficiency severely limits our understanding of the biological processes associated with tissue growth during the in vitro pre-conditioning phase. Optical Coherence Tomography (OCT) enables imaging of cross sectional structure in biological tissues by measuring the echo time delay of backreflected light. OCT has recently emerged as an important method to assess the structures of physiological, pathological as well as tissue engineered blood vessels. The goal of the present study is to develop an imaging system for non-destructive monitoring of blood vessels maturing within a bioreactor. Non-destructive structural imaging of tissue-engineered blood vessels cultured in a novel bioreactor was performed using free-space and catheter-based OCT imaging, while monitoring of the endothelium development was performed using a fluorescence imaging system that utilizes a commercial OCT catheter. The project included execution of three specific aims. Firstly, we developed OCT instrumentation to determine geometrical and optical properties of porcine and human skin in real-time. The purpose of the second aim was to assess structural development of tissue-engineered blood vessels maturing in a bioreactor. We constructed a novel quartz-based bioreactor that will permit free space and catheter-based OCT imaging of vascular grafts. The grafts were made of biodegradable PCL-collagen and seeded with multipotent mesenchymal cells. We imaged the maturing grafts over 30 days to assess changes in graft wall thickness. We also monitored change in optical properties of the grafts based on free-space OCT scanning.   Finally, in order to visualize the proliferation of endothelial cells and development of the endothelium, we developed an imaging system that utilizes a commercial OCT catheter for single-cell-level imaging of the growing endothelium of a tissue-engineered blood vessel. We have developed two modules of an imaging system for non-destructive monitoring of maturing bioengineered vascular grafts. The first module provides the ability to non-destructively examine the structure of the grafts while the second module can track the progress of endothelialization. As both modules use the same endoscope for imaging, when operated in sequence, they will produce high-resolution, three-dimensional, structural details of the graft and two-dimensional spatial distribution of ECs on the lumen. This non-destructive, multi-modality imaging can be potentially used to monitor and assess the development of luminal bioengineered constructs such as colon or trachea. / Ph. D.

Thesis (MSc (Physics))--University of Stellenbosch, 2007. / Imaging and spectroscopy of laser induced chlorophyll fluorescence (LICF) are emerging as useful tools in plant physiology and agriculture since these methods allow an early detection of plant stress and transformation of plant tissue, before visual symptoms appear. Chlorophyll fluorescence is governed by photosynthetic efficiency and it depends on the plant species and physiological state. In addition, the laser induced fluorescence of chlorophyll molecules in the red and far red spectral range is also used to study basic processes and phenomena in photo-excited molecules. In the work reported here experimental setups used for laser induced chlorophyll fluorescence imaging and spectroscopy techniques were developed to investigate chlorophyll fluorescence under constant illumination and also to detect green-fluorescent protein (GFP) by looking at the chlorophyll fluorescence spectrum and image. He-Ne (wavelength 632 nm), tunable argon ion (wavelength 455 nm), and excimer (wavelength 308 nm) lasers were used as excitation sources. An Ocean Optics spectrometer was used to record the spectrum of the chlorophyll fluorescence and the variation of the chlorophyll fluorescence spectrum with time. The chlorophyll fluorescence spectrum of tobacco leaves expressing GFP was compared to that of control leaves. A charge-coupled device (CCD) camera was used to image the fluorescence from GFP expressing and control tobacco leaves to investigate the effect of GFP genes on chlorophyll fluorescence in relation to the state of the plant material. The spectral analysis technique and image processing procedures were elaborated in order to obtain better information on chlorophyll fluorescence. The results of this work show that the experimental setups and analytical procedures that were devised and used are suitable for laser induced chlorophyll fluorescence analysis. Fluorescence bleaching could be obtained from the time variation of the fluorescence spectrum, and plant expressing GFP can be distinguished from control plants by differences in the laser induced chlorophyll fluorescence.

This thesis presents the preparation of hyaluronan-based biomaterials with imaging capability and their application as scaffolds in tissue engineering. First, we have synthesized HA derivatives functionalized with different chemoselective groups. Then, functional ligands with capacities for hydrophobic drug loading, imaging, and metal ion coordination were chemically conjugated to HA by chemoselective reactions with these groups. An injectable in situ forming HA hydrogel was prepared by hydrazone cross-linking between hybrid iron-oxide nanogel and HA-aldehyde (paper-I). The degradation of this hydrogel could be monitored by MRI and UV-vis spectroscopy since it contained iron oxide as a contrast agent and pyrene as a fluorescent probe. Additionally, this hydrogel has a potential for a delivery of hydrophobic drugs due to its pyrene hydrophobic domains. The degradation study showed that degradability of the hydrogel was correlated with its structure. Based on the obtained results, disulfide cross-linked and fluorescently labeled hydrogels with different HA concentration were established as a model to study the relationship between the structure of the hydrogel and its degradability (paper-II). We demonstrated that disulfide cross-linked HA hydrogel could be tracked non-invasively by fluorescence imaging. It was proved that the in vivo degradation behavior of the hydrogel is predictable basing on its in vitro degradation study. In paper-III, we developed a disulfide cross-linked HA hydrogel for three-dimensional (3D) cell culture. In order to improve cell viability and adhesion to the matrix, HA derivatives were cross-linked in the presence of fibrinogen undergoing polymerization upon the action of thrombin. It led to the formation of an interpenetrating double network (IPN) of HA and fibrin. The results of 3D cell culture experiments revealed that the IPN hydrogel provides the cells with a more stable environment for proliferation. The results of the cellular studies were also supported by in vitro degradation of IPN monitored by fluorescence measurements of the degraded products. In paper-IV, the effect of biomineralization on hydrogel degradation was evaluated in a non-invasive manner in vitro. For this purpose, two types of fluorescently labeled hydrogels with the different ability for biomineralization were prepared. Fluorescence spectroscopy was applied to monitor degradation of the hydrogels in vitro under two different conditions in longitudinal studies. Under the supply of Ca2+ ions, the BP-modified hydrogel showed the tendency to bio-mineralization and reduction of the rate of degradation. Altogether, the performed studies showed the importance of imaging of hydrogel biomaterials in the design of optimized scaffolds for tissue engineering.

<p>Altered metabolism is a hallmark of almost all cancers. A tumor’s metabolic phenotype can drastically change its ability to proliferate and to survive stressors such as hypoxia or therapy. Metabolism can be used as a diagnostic marker, by differentiating neoplastic and normal tissue, and as a prognostic marker, by providing information about tumor metastatic potential. Metabolism can further be used to guide treatment selection and monitoring, as cancer treatments can influence metabolism directly by targeting a specific metabolic dysfunction or indirectly by altering upstream signaling pathways. Repeated measurement of metabolic changes during the course of treatment can therefore indicate a tumor’s response or resistance. Recently, well-supported theories indicate that the ability to modulate metabolic phenotype underpins some cancer cells’ ability to remain dormant for decades and recur with an aggressive phenotype. It follows that accurate identification and repeated monitoring of a tumor’s metabolic phenotype can bolster understanding and prediction of a tumor’s behavior from diagnosis, through treatment, and (sadly) sometimes back again.</p><p>The two primary axes of metabolism are glycolysis and mitochondrial metabolism (OXPHOS), and alteration of either can promote unwanted outcomes in cancer. In particular, increased glucose uptake independent of oxygenation is a well-known mark of aggressive cancers that are more likely to metastasize and evade certain therapies. Lately, mitochondria are also gaining recognition as key contributors in tumor metabolism, and mitochondrial metabolism has been shown to promote metastasis in a variety of cell types. Most tumor types rely on a combination of both aerobic glycolysis and mitochondrial metabolism, but the two axes’ relative contributions to ATP production can vary wildly. Knowledge of both glycolytic and mitochondrial endpoints is required for actionable, systems-level understanding of tumor metabolic preference. </p><p>Several technologies exist that can measure endpoints informing on glycolytic and/or mitochondrial metabolism. However, these technologies suffer from a combination of prohibitive cost, low resolution, and lack of repeatability due to destructive sample treatments.</p><p>There is a critical need to bridge the gap in pre-clinical studies between single-endpoint whole body imaging and destructive ex vivo assays that provide multiple metabolic properties, neither of which can provide adequate spatiotemporal information for repeated tumor monitoring. Optical technologies are well-suited to non-destructive, high resolution imaging of tumor metabolism. A carefully chosen set of endpoints can be measured across a variety of length scales and resolutions to obtain a complete picture of a tumor’s metabolic state. First, the fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) can be used to report on glucose uptake. The fluorophore tetramethylrhodamine, ethyl ester (TMRE) reports on mitochondrial membrane potential, which provides information regarding capacity for oxidative phosphorylation. Vascular oxygenation (SO2) and morphological features, which are critical for interpretation of 2-NBDG and TMRE uptake, can be obtained using only endogenous contrast from hemoglobin. </p><p>Three specific aims were proposed toward the ultimate goal of developing an optical imaging toolbox that utilizes exogenous fluorescence and endogenous absorption contrast to characterize cancer metabolic phenotype in vivo. </p><p>In Aim 1, we optimized the fluorescent glucose analog 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG) to report on glycolytic demand in vivo. Our primary goal was to demonstrate that correcting 2-NBDG uptake (NBDG60) by the rate of delivery (RD) showed improved contrast between distinct tumor phenotypes. We showed that the ratio 2-NBDG60/RD served as a delivery-corrected measure of glucose uptake in the dorsal skin flap window chamber models containing normal tissues and tumors. Delivery correction was able to minimize the effects of a large change in the injected 2-NBDG dose. Further, the endpoint showed a significant inverse correlation with blood glucose levels. Since glucose has been shown to competitively inhibit 2-NBDG transport into cells, this finding indicating that we were indeed reporting on glucose uptake. Importantly, the ratio was able to distinguish specific uptake of 2-NBDG from accumulation of a fluorescent control, 2-NBDLG, which is identical to 2-NBDG in molecular weight and fluorescent spectrum, but is unable to undergo active transport into the cell. </p><p>The ratio 2-NBDG60/RD was then leveraged to compare different tumor phenotypes and to characterize the dependence of glucose uptake on vascular oxygenation within these tumors. Our results showed that 2-NBDG60/RD was an effective endpoint for comparing in vivo glucose uptake of metastatic 4T1 and nonmetastatic 4T07 murine mammary adenocarcinomas. Further, the addition of vascular information revealed metabolic heterogeneity within the tumors. The primary conclusion of Aim 1 was that delivery-corrected 2-NBDG uptake (2-NBDG60/RD) is an appropriate indicator of glucose demand in both normal and tumor tissues.</p><p>In Aim 2, we optimized fluorescent tetramethyl rhodamine, ethyl ester (TMRE) for measurement of mitochondrial membrane potential (MMP). We then leveraged the relationships between MMP, glucose uptake, and vascular endpoints to characterize the in vivo metabolic landscapes of three distinct and extensively studied murine breast cancer lines: metastatic 4T1 and non-metastatic 67NR and 4T07. </p><p>Using two-photon microscopy, we confirmed that TMRE localizes to mitochondrial-sized features in the window chamber when delivered via tail vein. The kinetics of TMRE uptake were robust across both normal and tumor tissues, with a stable temporal window for measurement from 40-75 minutes after injection. We saw that TMRE uptake decreased as expected in response to hypoxia in non-tumor tissue, and in response to chemical inhibition with a mitochondrial uncoupler in both non-tumor and 4T1 tissue. MMP was increased in all tumor types relative to non-tumor (p<0.05), giving further confirmation that TMRE was reporting on mitochondrial activity.</p><p>We leveraged the relationships between the now-optimized endpoints of MMP (Aim 2), glucose uptake (Aim 1) and vascular endpoints (Aims 1 and 2) to characterize the in vivo metabolic landscapes of three distinct and extensively studied murine breast cancer lines: metastatic 4T1 and non-metastatic 67NR and 4T07. Imaging the combination of endpoints revealed a classic “Warburg effect” coupled with hyperpolarized mitochondria in 4T1; 4T1 maintained vastly increased glucose uptake and comparable MMP relative to 4T07 or 67NR across all SO2. We also showed that imaging trends were concordant with independent metabolomics analysis, though the lack of spatial and vascular data from metabolomics obscured a more detailed comparison of the technologies.</p><p>We observed that vascular features in tumor peritumoral areas (PA) were equally or more aberrant than vessels in the tumor regions that they neighbored. This prompted consideration of the metabolic phenotype of the PA. Regional metabolic cooperation between the tumor region and the PA was seen only in 4T1, where MMP was greater in 4T1 tumors and glucose uptake was greater in 4T1 PAs. </p><p>Because of their regional metabolic coupling as well as their demonstrated capacity for glycolysis and mitochondrial activity, we hypothesized that the 4T1 tumors would have an increased ability to maintain robust MMP during hypoxia. 67NR and 4T07 tumors showed expected shifts toward decreased MMP and increased glucose uptake during hypoxia, similar to the trends we observed in normal tissue. Surprisingly, 4T1 tumors appeared to increase mitochondrial metabolism during hypoxia, since MMP increased and SO2 dramatically decreased. Overall, this aim demonstrated two key findings: 1. TMRE is a suitable marker of mitochondrial membrane potential in vivo in normal tissue and tumors, and 2. imaging of multiple metabolic and vascular endpoints is crucial for the appropriate interpretation of a metabolic behavior. </p><p>Finally, in Aim 3 we evaluated the feasibility of combined 2-NBDG and TMRE imaging. The primary objective was to enable simultaneous imaging of the two fluorophores by minimizing sources of “cross-talk”: chemical reaction, optical overlap, and confounding biological effects. A secondary objective was to transition our imaging method to a new platform, a reflectance-mode, high-resolution fluorescence imaging system built in our lab, which would expand the use of our technique beyond the dorsal window chamber model. We first used liquid chromatography- mass spectrometry to confirm that the chemical properties of the two fluorophores were compatible for simultaneous use, and indeed saw that the mixing of equimolar 2-NBDG and TMRE did not form any new chemical species. </p><p>We also performed a phantom study on the hyperspectral imaging system, used for all animal imaging in Aim 1 and Aim 2, to estimate the range of 2-NBDG and TMRE concentrations that are seen at the tissue level in normal and tumor window chambers. We created a new phantom set that spanned the range of estimated in vivo concentrations, and imaged them with the reflectance-mode fluorescence imaging system. The phantom experiments gave us two important findings. First, we saw that fluorescence intensity increased linearly with fluorophore concentration, allowing for accurate quantification of concentration changes between samples. Most importantly, we found that we could exploit the optical properties of the fluorophores and our system’s spectral detection capability to excite the two fluorophores independently. Specifically, we could excite 2-NBDG with a 488nm laser without detectable emission from TMRE, and could excite TMRE with a 555nm laser without detectable emission from 2-NBDG. With this characterization, the optical properties of the two fluorophores were considered compatible for simultaneous imaging. </p><p>Next, we sought to determine whether biological or delivery interactions would affect uptake of the two fluorophores. Surprisingly, both in vitro and in vivo imaging suggested that simultaneous dosing of the 2-NBDG and TMRE caused significant changes in uptake of both probes. Since we previously found that TMRE equilibrates rapidly at the tissue site, we hypothesized that staggering the injections to allow delivery of TMRE to tissue before injecting 2-NBDG would restore the full uptake of both fluorophores. Two sequential injection protocols were used: in the first group, TMRE was injected first followed by injection of 2-NBDG after only 1-5 minutes, and in the second group, TMRE was injected first followed by injection of 2-NBDG after 10-15 minutes. Both sequential injection strategies were sufficient to restore the final fluorescence of both fluorophores to that seen in the separate TMRE or 2-NBDG imaging cohorts; however, the shorter time delay caused changes to the initial delivery kinetics of 2-NBDG. We concluded that sequential imaging of TMRE followed by 2-NBDG with a 10-15 minute delay was therefore the optimal imaging strategy to enable simultaneous quantification of glucose uptake and mitochondrial membrane potential in vivo. </p><p>Applying the sequential imaging protocol to 4T1 tumors demonstrated a highly glycolytic phenotype compared to the normal animals, as we had seen in Aim 2. However, mitochondrial membrane potential was comparable for the normal and tumor groups. The next study will test an extended delay between the injections to allow more time for TMRE delivery to tumors prior to 2-NBDG injection. Overall, the key finding of Aim 3 was that a carefully chosen delivery strategy for 2-NBDG and TMRE enabled simultaneous imaging of the two endpoints, since chemical and optical cross-talk were negligible.</p><p>The work presented here indicates that an optical toolbox of 2-NBDG, TMRE, and vascular endpoints is well poised to reveal interesting and distinct metabolic phenomena in normal tissue and tumors. Future work will focus on the integration of optical spectroscopy with the microscopy toolbox presented here, to enable long-term studies of the unknown metabolic changes underlying a tumor’s response to therapy, its escape into dormancy, and ultimately, its recurrence.</p> / Dissertation

Prions are infectious agents that cause fatal neurodegenerative diseases in the brain. The wide-accepted protein-only hypothesis states that the misfolded form of prion protein (PrP) is the sole constituent of prions, and the self-propagating process of PrP is considered to play a central role in prion pathogenesis. Prions are believed to propagate when a PrP assembly enters a cell and replicates to produce two or more fibrils, leading to an exponential increase in PrP aggregate number with time. However, the molecular basis of this process has not yet been established in detail. This prion-like replication is also suggested to be the mechanism in the development of other notorious neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. In this thesis, I use single-aggregate imaging to study fibril fragmentation and elongation of individual murine PrP aggregates from seeded aggregation in vitro. From fluorescence imaging of individual PrP aggregates on the coverslip surface, elongation and fragmentation of the PrP assemblies have been directly observed. PrP elongation occurs via a structural conversion from a proteinase K (PK)-sensitive to PK-resistant conformer. Fibril fragmentation was found to be length-dependent and resulted in the formation of PK-sensitive fragments. To gain more insights into the mechanism of the spread of PrP, the quantified kinetic profiles allows the determination of the rate constants for these processes through the use of kinetic modelling. This enables the estimation of a simple framework for aggregate propagation through the brain, assuming that doubling of the aggregate number is rate-limiting. In contrast, the same method was applied to measurement for α-Synuclein (αS) aggregation, which has been suggested to be prion-like and is associated with Parkinson's disease. While αS aggregated by the same mechanism, it showed significantly slower elongation and fragmentation rate constants than PrP, leading to much slower replication rate. Furthermore, the measurements in αS aggregation has been extended to the cellular environment, I use super-resolution imaging to study the amplification of endogenous αS aggregation in cells and the transcellular spread of αS. Endogenous αS showed a clear amplification in number of aggregates with time after seed transduction, and the newly-formed αS aggregates are likely to spread through cell-to-cell transmission. The proteasome was demonstrated to possess a novel disaggregase function for αS fibrils and thus produce more seeds for further replication. It partially explains that αS aggregation in cells was found to replicate at a substantially faster rate than that in vitro. Determining the nature of the oligomers formed during aggregation has been experimentally difficult due to the lack of suitable methods capable of detecting and characterising the low level of oligomers. To address this problem, I have studied the early formation of PrP oligomers formed during aggregation in vitro using various single-molecule methods. The early aggregation of PrP is observed to form a thioflavin T (ThT)-inactive and two ThT-active species of oligomers, which differ in size and temporal evolution. The ThT-active oligomers undergo a structural conversion from a PK-sensitive to PK-resistant conformer, while a fraction of which grow into mature fibrils. These results also enable the establishment of a kinetic framework for elucidating temporal evolution of PrP aggregation and the relationship between oligomers and fibrils. Overall, my research identifies fibril elongation with fragmentation are the key molecular processes leading to PrP and αS aggregate replication, an important concept in prion biology, and provides a simple framework to estimate the rate of prion and prion-like spreading in animals. The results also show that a diverse range of oligomers is formed and co-exist during PrP aggregation which differ both in their structure and properties and provides mechanistic insights into a prion aggregation. The work provides a new quantitative approach to describe the prion-like property in neurodegenerative diseases from a kinetic perspective that can be verified in extending studies in other proteins or in cells.

The objective of this research project was to establish a better understanding of the mechanism(s) and route(s) by which selenium (Se) may enter an aquatic ecosystem that has been receiving treated metal mine effluent from an upstream uranium milling operation. Synchrotron based X-ray absorption spectroscopy (XAS) and X-ray fluorescence (XRF) imaging, which require little sample pre-treatment, were employed to study the speciation and distribution of Se in complex sediment and benthic invertebrates samples collected from the field. Laboratory based inductively coupled plasma mass spectrometry (ICP-MS) provided quantitative Se concentrations. Samples were taken from Fox Lake and Unknown Lakes, downstream of the mill, and Yeoung Lake as a control. The variation in Se speciation as a function of depth in intact sediment cores may provide insight into the species of Se available to the sediment dwelling benthic invertebrate communities. Therefore, a custom sample holder was designed to facilitate analysis of intact sediment cores at cryogenic temperatures. Additionally, laboratory reared chironomids were water-exposed to various Se species, to compare their Se speciation and localization to chironomids collected in the field. The successful demonstration of the custom sample holder and viable use of synchrotron XAS and XRF in studying sediment and chironomid samples have revealed that biologically relevant Se forms were present in sediment at depths accessible by the benthic invertebrate community. These Se forms included selenomethionine-like and selenite species, and to a lesser degree elemental Se; an increased proportion of reduced Se species was observed as depth increased. Other elements measured concurrently with Se included As, Zn, Cu, Ni, Fe, and Mn, providing an estimation of the redox boundary found both in Fox and Unknown Lake, as well as suggesting the presence of iron species that could aid in the reduction of Se. Field and laboratory reared chironomids showed similar Se species, and XRF imaging revealed the localization of Se in 4 distinct regions: head capsule, brain, salivary glands, and gut lining. Overall, the project has provided important insights into the interactions of Se with this aquatic ecosystem, which may have future applications in cold water systems with elevated Se concentrations.

The main objective of this research was to develop a two-stage optical imaging platform to improve detection of cancer in Barrett’s esophagus (BE). BE caused by chronic reflux and patients with BE are at a higher risk for developing esophageal adenocarcinoma (EAC). However, neoplasia in BE is often unidentifiable under standard endoscopy, and studies have shown nearly half of early cancers can go unidentified by this method. Widefield imaging (resolves ~100 microns) allows efficient surveillance of large BE segments. Two widefield imaging techniques were identified to improve contrast between benign and abnormal lesions during an ex vivo 15 patient feasibility study. Cross-polarized imaging (CPI) reduced specular reflection and improved vascular contrast. Vital-dye fluorescence imaging (VFI) using topically-applied proflavine improved visualization of glandular pattern. Moreover, relevant pathologic features visible during VFI were seen in corresponding histology slides as well as high resolution images of the same sites. Based on these results, a cap-based Multispectral Digital Endoscope (MDE) was designed and built. The MDE can image in three different imaging modes: white light imaging, CPI, and VFI. Modifications to a Pentax EPK-i video processor and a Pentax endoscope were made to incorporate these imaging modes into one system. A 21 patient in vivo pilot study with 65 pathologically correlated sites demonstrated the feasibility of using this system in vivo; image criteria were developed to classify neoplasia with a sensitivity and specificity of 100% and 76% respectively. High resolution imaging (resolves ~2-5 micron) may verify the disease presence in suspicious areas identified using widefield techniques. 2-NBDG, a fluorescent metabolic marker, was used as to identify neoplastic biopsies. In a study with 21 patients yielding 38 pathologically correlated biopsies and 158 image sites, 2-NBDG imaging allowed classification of cancerous biopsies with a sensitivity of 96% and specificity of 90%. The unique contributions of these results is the development of a multimodal cap-based endoscopic system to identify suspicious areas in BE, and using a metabolic marker to verify the presence of disease. This application extends beyond esophageal cancer detection and may be explored for cancer detection in other organ sites characterized by columnar epithelium.