M.Sc., Faculty of Science, University of the Witwatersrand, 2011 / The objective of the study was to investigate the preparation of glass beads for base metal analysis of mining samples prior to x-ray fluorescence analysis. The research method used included the investigation of different fluxes, oxidising, non-wetting agents, fusion temperature and time. The experiments were carried out using different fusion instruments: Electrofluxer and Katanax followed by both EDXRF and WDXRF analysis. The x-ray spectrometers were calibrated with standards prepared from pure oxides and the results compared to values determined by alternative techniques. Different statistical methods were used to validate the experiments including factorial designs. Not all the elements and oxides were recovered successfully, however, perfect glass beads were prepared. The two areas of concern were addressed successfully: firstly the loss of copper was overcome by using an alternative heating mechanism of the Katanax and sodium iodide as the non-wetting agent. Secondly, the sulphur was successfully oxidised and retained in the glass beads.

Atualmente os maiores gastos da citricultura concentram-se na sanidade do pomar. Os custos fitossanitários, juntamente com fertilizantes, ultrapassam 60% do custo da produção. O controle de doenças como a Morte Súbita dos Citros (MSC), Declínio e o Greening, também conhecida como Huanglongbing, são feitos de forma visuais os que conferem ao método um elevado grau de subjetividade e imprecisão. O presente trabalho teve por finalidade desenvolver uma metodologia rápida e segura de diagnóstico para as doenças acima citadas utilizando espectroscopia de fluorescência e fluorescência induzida por laser (FIL). Foram utilizados neste estudo dois porta-enxertos, Cravo e Cleópatra, e quatro variedades de copa, Pêra, Natal, Valência e Hamilim. As doenças estudadas também foram classificadas em diferentes níveis de severidade. O índice 1 foi utilizado para baixa severidade da MSC, e 2 para o estágio mais avançado da doença. Para o Greening fez-se uma distinção entre folhas sintomáticas e assintomáticas. A doença Declínio dos Citros também fez parte deste estudo em função dos sintomas serem muito parecidos aos da fase inicial de MSC, sendo este um problema sério para as avaliações de contaminação no campo. Inicialmente, foram realizados estudos a respeito da emissão de fluorescência dos pigmentos fotossintéticos provenientes dos extratos de folhas doentes e sadias através de um fluorímetro de bancada. Para as análises foliares utilizou-se um sistema portátil de espectroscopia de fluorescência induzida por laser desenvolvido no LOLEIA. O estudo dos extratos revelaram que as maiores variações ocorrem nas concentrações de clorofila a e b e que os pigmentos acessórios como ? e ?- caretonos não fluorescem no comprimento de onda estudado. De uma maneira geral, é possível dizer que plantas doentes sofrem uma diminuição das clorofilas a e b. Os resultados das folhas foram analisados por um método estatístico de Análises de Componentes Principais (PCA) para facilitar a observação das alterações espectrais. Através da metodologia desenvolvida foi possível a identificação e a distinção das doenças com nível de acerto superior a 93%. Contudo, estes resultados contribuíram para o desenvolvimento de uma metodologia para diagnóstico das doenças de citros de uma forma rápida, precisa e economicamente viável. / Currently the largest portion of money applied to citriculture focus on health of the orchard. The phytosanitary costs, along with fertilizers, exceed 60% of the production cost. The control of diseases such as Sudden Death of Citrus (SDC), Decline and Greening, also known as Huanglongbing, is done by visual inspection, which confers to the method a high level of subjectivity and imprecision. This work was intended to develop a quick and safe methodology of diagnosis for the diseases mentioned above using fluorescence spectroscopy and laser-induced fluorescence (LIF). It was used in this study two rootstocks, Cravo and Cleopatra, and four varieties of crown: Pera, Natal, Valencia and Hamlin. The diseases studied were also classified into different levels of severity. The index 1 was used for low severity SDC, and 2 for a more advanced stage of this disease. For Greening has been made a distinction between symptomatic and asymptomatic leaves. The Decline of Citrus disease was also part of this study because its symptoms are very similar to those in the initial phase of SDC, and this is a serious problem for the evaluation of contamination in the field. Initially, studies have been made on the fluorescence of photosynthetic pigments from the extracts of ill and healthy leaves using a non-portable fluorimeter. For the leaf analyses we used a portable system of laser-induced fluorescence spectroscopy developed in LOLEIA. The study of extracts revealed that the largest cariations occur in the concentrations of chlorophyll a and b, and that acessory pigments like ? and ?-carotones do not fluoresce in the wavelength used. In general, it is possible to say that ill plants have a decrease of chlorophylls a e b. The leaf results were analyzed through the statistical method Principal Component Analysis (PCA) in order to facilitate the observation of spectral changes. Through the developed methodology it was possible to identify and distinguish the diseases with efficiency greater than 93%. However, these results contributed for the development of a methodology for diagnosis of citrus diseases in a quick, accurate and economically viable way.

by Siu-kai Tse. / Bibliography: leaves 100-103 / Thesis (M.Ph.)--Chinese University of Hong Kong, 1984

Methods for polyamine derivatization with a panel of extrinsic fluorophores followed by HPLC with fluorescence and UV absorption detection have been developed. Four fluorophores were examined using polyamines and aminoglycosides. o-Phthalaldehyde (OPA) and fluorescamine are selective fluorophores that only react with primary amines; 9- fluorenylmethyl chloroformate (FMOC Cl) and dansyl chloride react with both primary and secondary amines. Reaction and HPLC conditions were optimized with each of the above fluorophores using a series of model mono- and diamines and then applied to natural and semi-synthetic polyamines. The amines that have been investigated are natural di- and polyamines: putrescine, cadaverine, spermidine, spermine, thermospermine, aminoglycosides: kanamycin, paramomycin, neomycin, and synthetic polyamine conjugates e.g. N⁴,N⁹-dioleoylspermine, N¹-cholesteryl spermine carbamate. The resultant derivatives were confirmed by off-line high resolution electrospray ionization mass spectrometry (HR ESI MS). The results show that the synthesis of polyamine derivatives in quantitative yield depends on the time of reaction, the temperature and the ratio of fluorophore reagent. Linearity of derivatization was calculated and regression coefficients ranged from 0.968 to 0.999 with good reproducibility. HR ESI MS analysis of the reaction products demonstrated complete derivatization of both primary and secondary amino groups with dansyl and FMOC fluorescence derivatives and of primary amine groups for OPA and fluorescamine derivatives. Under the ionization conditions used the dansyl derivatives showed, in addition to monovalent ions [M+H]⁺, divalent cations [M+2H]²⁺ because this chromophore contains a basic amine that can be easily protonated. FMOC derivatives gave prominent [M+Na]⁺ ions. The OPA derivatization reaction is rapid, but the products have poor stability. The derivatization with fluorescamine gave multiple products with glucosamine due to the presence of a chiral centre in the fluorophore. The relative quantum yields of the polyaminefluorophore derivatives were examined to determine the effect of intramolecular fluorescence quenching. Dansylation is the fluorescent derivatization method of choice.

Reactive oxygen species (ROS) and reactive nitrogen species (RNS), saccharide (i.e. monosaccharide, disaccharide and polysaccharide), are continuously generated, transformed and consumed in the living systems. As a consequence of their significant value towards human health in aerobic life, it is very important and has drawn much attention in the chemical and biological sensing of the species. It is our long-standing interest in the recognition of monosaccharide (e.g. glucose) through exploration of various boronate-based fluorescence probes, thus, based on the previous work, we started on the design, synthesis and evaluation of novel fluorescent chemosensors for breakthrough discoveries in the detection of saccharide and ROS selectively and specifically, which are made up of different receptors and diverse singaling fluorophores, e.g. anthracene, coumarin, fluorescein, naphthalimine. Firstly, “integrated” and “insulated” boronate-based fluorescent probes (2-naphthylboronic acid and N-Methyl-o-(aminomethyl)phenylboronic acid) have been evaluated for the detection of hydrogen peroxide in the presence of saccharides (i.e. D-fructose). In the presence of D-fructose the initial fluorescence intensity of the “insulated” system is much higher and produces a blue visible fluorescence. Based on the experimental observation above in the boronate-based systems (i.e. B-N bond protection), a new water-soluble boronate-based fluorescent probe was designed and evaluated for the detection of peroxynitrite (much stronger oxidant) in the presence of D-fructose. The enhanced fluorescence of probe when bound with D-fructose was switched off in the presence of peroxynitrite. While, other reactive oxygen/nitrogen species led to only slight fluorescence decreases due to protection by the internal N-B interaction. The interaction of probe with D-fructose not only strengthens the fluorescence signal, but also protects the boronic acid to oxidation by other ROS/RNS. Therefore, under conditions generating various ROS/RNS, the boronate-based sugar complex preferentially reacts with peroxynitrite (ONOO−). The sensor displays good “on-off” response towards peroxynitrite both in RAW 264.7 cells and HeLa cells. A new ICT (internal charge transfer) sensing system was developed for the detection of hydrogen peroxide and peroxynitrite. The probe displayed an enhanced fluorescence change when bound with D-fructose due to the prolonged N-B distance. The fluorescence intensity of the probe dropped down both in the detection of H2O2 and ONOO− which was attributed to the oxidation of arylboronic acid even though in the presence of D-fructose. Using the self-assembly of aromatic boronic acids with Alizarin Red S (ARS), we developed a new chemo/biosensor for the selective detection of peroxynitrite. Phenylboronic acid, benzoboroxole and 2-(N, N-dimethylaminomethyl) phenylboronic acid were employed to bind with ARS to form the complex probes. In particular the ARS-NBA system with a high binding affinity can preferably react with peroxynitrite over hydrogen peroxide and hypochlorite due to the protection of the boron via the solvent-insertion B-N interaction. Our simple system produces a visible naked-eye colorimetric change and on-off fluorescence response towards peroxynitrite. By coupling a chemical reaction that leads to an indicator displacement, we have developed a new sensing strategy, referred to herein as RIA (Reaction-based Indicator displacement Assay). Next, we developed a novel class of simple materials for sensing monosaccharides by the functionalization of graphene oxide (GO) with boronate-based fluorescence probes. The composite materials were characterized by atomic force microscopy, Raman spectroscopy, and UV-vis/fluorescence spectroscopy. The strong fluorescence of the fluorescence probes is quenched in the presence of GO through fluorescence resonance energy transfer (FRET). The BA@GO composite sensors formed provide a useful platform for fluorogenic detection of monosaccharides based on the strong affinity between the boronic acid receptor and monosaccharides. The BA@GO composite sensor displayed a “turn-on” fluorescence response with a good linear relationship towards fructose over a range of other saccharides. Next, new water-soluble copper (II) complex fluorescence probes were developed and evaluated for the detection of nitric oxide and nitroxyl in a physiological condition. A significant fluorescence “off-on” response displayed by using the copper (II) complex for the detection of NO and HNO (Na2N2O3 as a donor). Under pathological conditions generating various ROS/RNS, the copper (II) complex fluorescent probe preferentially reacts with NO/HNO over other reactive oxygen species. The dual-analyte recognitions of the simple, sensitive probe were further applied in living cell for the exogenous NO/HNO. In the following work, we synthesised a phosphorous-based compound for the detection of HNO which derived from Angeli’s salt in a biological condition. Significantly, it displayed a high sensitivity and selectivity toward HNO over other various ROS species, especially NO since they have a similar chemical property. The underlying mechanism was attributed to the cleavage of C-O bond induced by Staudinger Ligation.

The aluminum industry is in need of high-speed in-situ elemental identification technology for molten metals. wTe Corporation of Bedford, MA was granted funds to research this technology for aluminum through the Advanced Technology Program (ATP) of the National Institute of Standards and Technology (NIST). As a secondary metal scrap processor, wTe Corporation is interested in increasing the value of scrap and better identification technology is one way of doing this. With better identification technology, foundries and smelters will be more confident in the melt chemistry and more apt to use secondary aluminum which decreases the energy required for processing by approximately 85%. wTe Corporation is exploring two avenues for in-situ molten metal identification: Laser Induced Breakdown Spectroscopy (LIBS) and X-Ray Fluorescence (XRF). The objectives of this work were to contribute to developing XRF technology for in-situ identification of molten metal by establishing a method of data instrumentation and analysis for XRF to determine aluminum melt composition, investigate the major alloying elements in the Al380 alloy series and determine the relationship between intensity and concentration, and to determine the effect of temperature on XRF Spectra. The XRF instrumentation development and the technical challenges associated with high temperature measurements are presented. The relationship between intensity and concentration is presented for three alloys within the 380 alloy series, and lastly it is observed that there are significant differences between liquid and solid spectra and that a calibration curve for liquid data is necessary. Several hypotheses are given as to why this difference between liquid and solid state spectra may occur.

The self-assembly of amyloidogenic proteins to form cytotoxic species that give rise to brain deterioration underlies numerous neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases. Increasing evidence indicates that it is the rare, low-molecular-weight species (oligomers) rather than the more abundant high-molecular-weight fibrils of certain proteins that are the most cytotoxic in several neurodegenerative diseases. However, these species have proven difficult to study using traditional methods due to their transient nature and the heterogeneity of aggregation mixtures. In this thesis, I describe my work to develop advanced methods where I combine single-molecule and ensemble fluorescence techniques with microfluidic strategies to enable the study of protein aggregation, spanning small, transient oligomers to large, insoluble aggregates. In Chapter 1 I give an overview of the biological context and relevance of this work, including the background of neurodegenerative disease, amyloidogenic aggregation and key proteins involved. I then briefly review fluorescence microscopy techniques and the field of microfluidics. In Chapter 2 I describe how complex microfluidics can be integrated with single-molecule confocal techniques to provide a highly sensitive method to continuously probe protein aggregation in vitro. I show, for the first time, that the dilution of aggregating mixtures may be automated, by up to five orders of magnitude, down to the picomolar concentrations suitable for single-molecule measurements. By incorporating this microfluidic dilution device I greatly improve the temporal resolution of the technique and facilitate the observation of more transient species through the ability to rapidly dilute and take fluorescence measurements of samples. In Chapter 3 I overcome the need for in situ labels to monitor amyloidogenic aggregation using single-molecule confocal microscopy. I describe my work to adapt the single-molecule confocal technique to achieve the ultrasensitive detection of individual aggregate species under flow without covalently-attached labels. I have demonstrated the ability of this new method to monitor the aggregation of label-free amyloidogenic proteins using extrinsic labels ex-aggregation, opening the way for biological samples to be probed in a high-throughput manner. In Chapter 4 I describe my work to combine the high precision of confocal microscopy with a microfluidic device developed to directly characterise the sizes and interactions of biomolecules in the continuous phase. By monitoring the spatial and temporal mass transport on the micron scale, the diffusion coefficient, and thus hydrodynamic radius, of species may be determined. The technique delivers much greater sensitivity for size quantification, allowing scarce and other challenging samples to be characterised, and provides significant steps towards accurate sizing for single-molecule aggregation experiments under flow. In Chapter 5 I describe my work to determine the microscopic driving force for the spatial propagation of amyloid-beta. The epifluorescence instrument I built has enabled the proliferation of aggregate species to be monitored over a macro distance on a timescale of minutes. This has greatly improved the scope of the experimental data attained, which will be used in conjunction with Monte Carlo simulations to deliver a model for the propagation of amyloid-beta in vitro. Together this thesis represents my work developing the above novel fluorescence techniques to improve their temporal and size resolution, sensitivity and adaptability to study highly complex and fundamental protein aggregation linked to neurodegenerative disease.

Quantitative three dimensional maps of cellular structure, activity and function provide the key to answering many prevalent questions in modern biological research. Fluorescence microscopy has emerged as an indispensable tool in generating such maps, but common techniques are limited by fundamental physical constraints which render them incapable of simultaneously achieving high spatial and temporal resolution. This thesis will describe the development of novel microscopy techniques and complementary computational tools capable of addressing some of the aforementioned limitations of fluorescence microscopy and further outline their application in providing novel biological insights. The first section details the design of a light sheet microscope capable of high-throughput imaging of cleared, macroscopic samples with cellular resolution. In light sheet microscopy, the combination of spatially confined illumination with widefield detection enables multi-megapixel acquisition in a single camera exposure. The corresponding increase in acquisition speed enables systems level biological studies to be performed. The ability of this microscope to perform rapid, high-resolution imaging of intact samples is demonstrated by its application in a project which established a niche and hierarchy for stem cells in the adult nervous system. Light sheet microscopy achieves fast volumetric imaging rates, but the two dimensional nature of each measurement results in an inevitable lag between acquisition of the initial and final planes. The second section of this thesis describes the development and optimization of a light field microscope which captures volumetric information in a snapshot. Light field microscopy is a computational technique and images are reconstructed from raw data. Both the fidelity of computed volumes and the efficiency of the algorithms are strongly dependent on the quality of the rectification. A highly accurate, automated procedure is presented in this section. Light field reconstruction techniques are investigated and compared and the results are used to inform the re-design of the microscope. The new optical configuration is demonstrated to minimize the long-object problem. In the final section of the thesis, the spatial resolution limits of light field microscopy are explored using a combination of simulations and experiments. It is shown that light field microscopy is capable of localizing point sources over a large depth of field with high axial and lateral precision. Notably, this work paves the way towards frame rate limited super resolution localization microscopy with a depth of field larger than the thickness of a typical mammalian cell.

The ability to resolve multiple fluorescent emissions from different biological targets in video rate applications, such as endoscopy and intraoperative imaging, has traditionally been limited by the use of filter-based imaging systems. Hyper and multispectral imaging facilitate the detection of both spatial and spectral information in a single data acquisition, however, instrumentation for spatiospectral data acquisition is typically complex, bulky and expensive. This thesis seeks to overcome these limitations by using recently commercialised compact and robust hyper/multispectral cameras based on spectrally resolved detector arrays. Following sensor calibrations, which devoted particular attention to the angular sensitivity of the sensors, we integrated spectrally resolved detector arrays into a wide-field and an endoscopic imaging platform. This allowed multiplexed reflectance and fluorescence imaging with spectrally resolved detector array technology in vitro, in tissue mimicking phantoms, in an ex vivo oesophageal model and in vivo in a mouse model. A hyperspectral linescan sensor was first integrated in a wide-field near-infrared reflectance based imaging set-up to assess the suitability of spectrally resolved detector arrays for in vivo imaging of exogenous fluorescent contrast agents. Using this fluorescence hyperspectral imaging system, we could accurately resolve the presence and concentration of seven fluorescent dyes in solution. We also demonstrated high spectral unmixing precision, signal linearity with dye concentration, at depth in tissue mimicking phantoms, and delineation of four fluorescent dyes in vivo. After the successful demonstration of multiplexed fluorescence imaging in a wide-field set-up, we proceeded to combine near-infrared multiplexed fluorescence imaging with visible light spectral reflectance imaging in an endoscopic set-up. A multispectral endoscopic imaging system, capable of simultaneous reflectance and fluorescence imaging, was developed around two snapshot spectrally resolved detector arrays. In the process of system integration and characterisation, methods to characterise and predict the imaging performance of spectral endoscopes were developed. With the endoscope we demonstrated simultaneous imaging and spectral unmixing of chemically oxy/deoxygenated blood and three fluorescent dyes in a tissue mimicking phantom, and of two fluorescent dyes in an ex vivo oesophageal porcine model. With further developments, this technology has the potential to become applicable in medical imaging for detection of diseases such as gastrointestinal cancers.

Les interactions plasma surface (PSI) sont considérées comme l'un des défis scientifiques majeurs de la fusion nucléaire magnétique contrôlée. L'interaction entre les isotopes d'hydrogène et les matériaux de l’enceinte à plasma tels que le tungstène revêt une importance particulière. Le coefficient de perte en surface des isotopes atomiques (γ) est un point clé dans les études PSI. Il peut donner des informations sur le recyclage de l'hydrogène atomique à la paroi et constitue ainsi un paramètre clé dans les modélisations des interactions plasma surface. Le but de ce projet est de déterminer les coefficients de perte de surface de l'hydrogène atomique et du deutérium sur échantillons de tungstène (W) et de nitrure de tungstène (WN) en utilisant une technique de fluorescence induite par plasma (PIF) et une technique de fluorescence induite par laser à deux photons (TALIF). Ce projet s'effectue dans le réacteur CAMITER qui est un réacteur plasma radiofréquence à basse pression au laboratoire PIIM de l'Université Aix-Marseille. / Plasma surface interaction (PSI) is considered to be one of the key scientific challenges in nuclear fusion. The interaction between hydrogen isotopes and plasma-facing materials such as tungsten is of particular importance. The atomic hydrogen isotope surface loss coefficient (γ) is a key point in PSI studies. It can give information on hydrogen isotope inventory and is an important input for modeling and theoretical work. The aim of this project is to determine atomic hydrogen and deuterium surface loss coefficients on tungsten (W) sample by using two-photon-absorption laser induced fluorescence (TALIF) and pulsed induced fluorescence (PIF) technique. This project is carried out in CAMITER reactor which is a low-pressure radio-frequency ICP reactor at PIIM laboratory in Aix-Marseille University.