Fluorescence microscopy is an important tool for biological research. However, the resolution of a standard fluorescence microscope is limited by diffraction, which makes it difficult to observe small details of a specimen’s structure. We have developed two fluorescence microscopy methods that achieve resolution beyond the classical diffraction limit. The first method represents an extension of localisation microscopy. We used nonnegative matrix factorisation (NMF) to model a noisy dataset of highly overlapping fluorophores with intermittent intensities. We can recover images of individual sources from the optimised model, despite their high mutual overlap in the original dataset. This allows us to consider blinking quantum dots as bright and stable fluorophores for localisation microscopy. Moreover, NMF allows recovery of sources each having a unique shape. Such a situation can arise, for example, when the sources are located in different focal planes, and NMF can potentially be used for three dimensional superresolution imaging. We discuss the practical aspects of applying NMF to real datasets, and show super-resolution images of biological samples labelled with quantum dots. It should be noted that this technique can be performed on any wide-field epifluorescence microscope equipped with a camera, which makes this super-resolution method very accessible to a wide scientific community. The second optical microscopy method we discuss in this thesis is a member of the growing family of structured illumination techniques. Our main goal is to apply structured illumination to thick fluorescent samples generating a large out-of-focus background. The out-of-focus fluorescence background degrades the illumination pattern, and the reconstructed images suffer from the influence of noise. We present a combination of structured illumination microscopy and line scanning. This technique reduces the out-of-focus fluorescence background, which improves the quality of the illumination pattern and therefore facilitates reconstruction. We present super-resolution, optically sectioned images of a thick fluorescent sample, revealing details of the specimen’s inner structure. In addition, in this thesis we also discuss a theoretical resolution limit for noisy and pixelated data. We correct a previously published expression for the so-called fundamental resolution measure (FREM) and derive FREM for two fluorophores with intermittent intensity. We show that the intensity intermittency of the sources (observed for quantum dots, for example) can increase the “resolution” defined in terms of FREM.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Lactic acid bacteria (LAB) are common inhabitants of the human gastro-intestinal tract (GIT). Some LAB, especially lactobacilli, are well known for their application in fermented foods and probiotic properties. These microorganisms exert many beneficial effects on human health, such as digestion and assimilation of food and preventing pathogens colonising the GIT. Furthermore, some selected probiotic strains are believed to perform a critical role in the treatment of gastro-intestinal disorders, lactose intolerance and in the stimulation of the immune system. Despite the ever increasing consumer interest in probiotic LAB, the mechanisms by which they exert their beneficial effects and the activities of probiotics in the GIT often remain poorly understood. Understanding survival mechanisms of LAB in the GIT, especially the interaction between LAB and pathogens, would be facilitated by the direct in vivo monitoring of these processes. Using the mCherry fluorescence gene, we successfully constructed Lactobacillus plantarum 423 and Enterococcus mundtii ST4SA reporter strains. With this study we showed that fluorescence imaging can be used to detect Lb. plantarum 423 and Ent. mundtii ST4SA in the GIT of mice. The two species colonized the cecum and colon for at least 24 h after one oral administration. To our knowledge, this is the first report on fluorescence imaging of LAB expressing mCherry in a mouse model. Using a bioluminescence marker system, we evaluated the impact of Lb. plantarum 423 and Ent. mundtii ST4SA on orally acquired Listeria monocytogenes infection and the ability of the probiotics to compete with the pathogen in the GIT of mice. Challenging Lb. plantarum 423 and Ent. mundtii ST4SA that were already established in the GIT of mice with L. monocytogenes EGDe had no effect on the survival and persistence of the probiotic strains. We demonstrated that the colonization of mice with Lb. plantarum 423 and Ent. mundtii ST4SA, or a combination of the strains, protected the animals against colonization of the GIT by L. monocytogenes EGDe. Enterococcus mundtii proved more effective than Lb. plantarum 423 in reducing the number of L. monocytogenes EGDe in the mouse model. / AFRIKAANSE OPSOMMING: Melksuurbakterieë (MSB) kom algemeen in die mens se spysverteringkanaal (SVK) voor. Verskeie MSB, veral lactobacilli, is bekend vir hul gebruik in gefermenteerde voedsel en as probiotika. Die bakterieë het baie eienskappe wat die mens se gesondheid kan bevoordeel, insluitend vertering en assimilasie van voedsel en voorkoming van kolonisering van die SVK deur patogeniese bakterieë. Sekere probiotiese rasse speel ook ʼn belangrike rol in die behandeling van SVK versteurings, laktose intoleransie en die stimulering van die immuun stelsel. Alhoewel die belangstelling in probiotiese bakterieë toeneem, is daar min inligting bekend oor die meganismes wat MSB gebruik om hulle voordelige eienskappe in die SVK uit te voer. Die oorlewing van MSB in die SVK, veral die interaksies tussen MSB en patogene, kan met behulp van ʼn in vivo moniteringsisteem bestudeer word. Deur die mCherry fluorisensie geen in Lactobacillus plantarum 423 en Enterococcus mundtii ST4SA te kloneer, het ons daarin geslaag om ʼn effektiewe verklikker sisteem te ontwikkel en kon die voorkoms en migrasie van die twee spesies in die SVK van muise bestudeer word. Lactobacillus plantarum 423 en Ent. mundtii ST4SA het veral die blindederm en kolon gekoloniseer. Beide rasse het na ʼn enkele mondelingse toediening vir ten minste 24 h in die SVK oorleef. Sover ons kennis strek, is hierdie die eerste verslag van fluoriserende MSB wat met behulp van die mCherry geenproduk in die SVK bestudeer is. Deur gebruik te maak van ʼn bioliggewende verklikker sisteem, het ons die vermoë van Lb. plantarum 423 en Ent. mundtii ST4SA om met Listeria monocytogenes in die SVK te kompeteer, bestudeer. Listeria monocytogenes het geen invloed gehad op die kolonisering van Lb. plantarum 423 en Ent. mundtii ST4SA nie. Deur die muise vooraf met Lb. plantarum 423 en Ent. mundtii ST4SA te koloniseer (in kombinasie of met net een van die twee rasse), kon ons daarin slaag om kolonisering van die SVK met L. monocytogenes te voorkom. In die muis model wat in hierdie studie gebruik is, was Ent. mundtii ST4SA meer effektief as Lb. plantarum 423 in die verlaging van Listeria selgetalle.

This thesis begins with a brief review about the role and importance of the small molecules containing fluorine atoms in medicine and imaging. Then, the first part of the thesis will discuss the synthesis, purification and characterization of pentamethine cyanine dyes. The structure identification of the final dyes is done by using 1H NMR, 13C NMR, 19F NMR, and mass spectrometry. The studies performed after full characterization were the determination of optical and physicochemical properties. After these properties were performed, the fluorophores were evaluated to be good candidates for in vivo testing.

Indocyanine green (ICG) is a fluorescent dye used as an indicator in medicine and surgery. The maximum absorption wavelength of ICG is at 785 nm, while the maximum emission is around 820 nm. ICG is nontoxic and is rapidly excreted into the bile. Near infrared (NIR) fluorescence imaging or spectroscopy offer new settings for seeing the blood vessels, and also in oncological applications for finding sentinel lymph nodes (SLN) to investigate if the cancer has spread from the tumor to the lymphatic system. Given the aforementioned applications, the aim of this thesis was to develop a hardware control and a user interface in LabVIEW, and to evaluate the software, as well as the instrumentation using phantom measurements.The system consisted of a spectrometer, a laser (785 ± 5 nm) for ICG excitation, optical filters, and a fiber optical probe containing five fibers for light excitation, and one for light collection. The basic LabVIEW program designed for the spectrometer was used, and additional features were added such as the recording functions, online measurements, opening of the recorded files, saving comments, and a loop was created for the laser control. Optical phantoms were prepared to model tissue for measurements using 20 % intralipid that gave μs = 298 mm−¹ at the excitation wavelength. Agar 1% w/v and ICG were added to the phantoms using different fluorophore concentrations of 2 μg/mL, 10 μg/mL, 20 μg/mL, 25 μg/mL, and 40 μg/mL. The objective was to perform controlled measurements of steady state ICG fluorescence, the dynamics of photobleaching at different concentrations, and to find the optimal ICG concentration for obtaining the maximum fluorescence intensity. The light to excite ICG fluorescence emission was provided by using a laser output power of 10.4 mW and 200 ms of integration time in the spectrometer for optimal measurements.Measurements using the different gel phantoms showed maximum fluorescence ICG concentration to be between 16 μg/mL and 20 μg/mL. Moreover, photobleaching measurements showed to be ICG concentration-dependent, where those concentrations higher than the optimal one incrementally photobleached with time after being exposed to light. Higher concentrations presented an incremental photobleaching where they first reached a maximum peak and then the intensity decayed with time. Additionally, laser reflection at 782 nm showed that the reflection increased with time ranging from 130% – 460% as the ICG photobleached to 50% of its initial value. Normalization of ICG by the laser reflection signal was investigated to compensate for the intensity variations due to the measurement parameters including the distance from the light source to the target, and the angle of inclination of the probe. The lowest ICG concentration detectable by the system was 0.05 μg/mL.In conclusion, a LabVIEW hardware control and user interface was developed for controlling the spectrometer and the laser. Several measurements were made using the different phantoms, where the optimal concentration of ICG was estimated. It was shown that ICG fluorescence intensity and photobleaching behavior were dependent on the concentration. The results gave suggestions for future experimental design. / NIRF

Coatings are often used as a means of protecting aluminum alloy and steel structures in industry. The assessment of corrosion under these coatings can be challenging. Corrosion sensing coatings can exhibit properties that allow undercoating corrosion to be identified before it can be seen with the naked eye. This would be very advantageous and could potentially result in tremendous savings in time and money when structures undergo routine maintenance. Our work involved the study of corrosion sensing coatings with incorporated fluorescent indicators that can be used to sense the undercoating corrosion on metal substrates. The fluorescent indicator in the coated-aluminum system was a negative indicator, i.e. the indicator in the coating was initially fluorescent and subsequently non-fluorescent due to the reduced pH at the anodic sites of corrosion. The fluorescent indicator in coated-steel system was positive, in the sense that the coating changed from non-fluorescent to fluorescent over the cathodic areas due to increased pH. The corrosion sensing coating was composed of commercial epoxy-polyamide and the indicator: 7-amino-4-methylcoumarin (7-AMC) for the coated-aluminum alloy system and 7-diethylamino-4-methylcoumarin (7-DMC) for the coated-steel system. The feasibility of using 7-AMC for sensing early undercoating corrosion was demonstrated by using fluorescent observations, Electrochemical Impedance Spectroscopy (EIS), Scanning Electron Microscope (SEM) and Energy Dispersive Spectroscopy (EDS) tests. EIS results estimated that with continuous immersion the undercoating corrosion occurred within 24 hours after immersion in the salt solution. When corrosion occurred, the corrosion was invisible under natural light. However, small spots appeared in the fluorescent image, changing from initially fluorescent to non-fluorescent where the anodic sites were identified by SEM and EDS. In other words, the fluorescent indicator could sense the early undercoating corrosion, although blistering can be a competing mechanism associated with corrosion under some conditions. The sensitivity of the 7-AMC corrosion detection system was tested by applying anodic current to the metal and measuring the charge at which fluorescence quenching was detected. The critical charge for a detectable pit under the coating was approximately 2x10⁻⁵ C, which implied a critical radius of a single corrosion spot or set of spots of approximately 10 [mu]m. The fluorescent properties of 7-AMC, its effect on the protectiveness, its sensitivity to pH and its concentration in the coating are explored as well. Fourier transform spectroscopy (FTIR) was used to characterize the structure of the coating with and without 7-AMC. The results suggested that there is no structure change occurring after adding 7-AMC into the coating. Fluorescence behavior, electrochemical behavior, microscopic evidence, and visual observations of coated steel specimens with 7-DMC are compared based on exposure to saltwater conditions. Some of the challenges associated with the use of these types of coatings will be presented. This includes the interference from the increased production of ferrous and ferric ions. All of this information is aimed at the development of corrosion sensing coatings that can reveal undercoating corrosion before it is visible to the naked eye. / text

Compressed and supercritical fluids, such as pressurized CO2, ethane, orpropane, provide a versatile and environmentally acceptable alternative to conventionalliquid organic solvents in bioprocessing applications – specifically in the areas ofproduct extraction, protein purification, microbial sterilization, and enzymatic and wholecellbiocatalysis. While their advantages have been well demonstrated, the effects ofcompressed and supercritical fluids on whole cells are largely unknown.Metabolic and structural perturbations of whole cells by compressed andsupercritical fluid solvents were examined. These perturbations exist as cell metabolismand membrane structure are influenced by pressure and the presence of a solventphase. Continuous cultures of Clostridium thermocellum (a model ethanol-producingthermophilic bacterium) were conducted under elevated hydrostatic and hyperbaricpressure to elucidate pressure- and solvent-effects on metabolism and growth.Fluorescence anisotropy was employed to study liposome fluidization due to thepresence of compressed and supercritical fluids and their partitioning/accumulation inthe phospholipid bilayer.Under elevated hydrostatic pressure (7.0 and 13.9 MPa; 333 K), significantchanges in product selectivity (towards ethanol) and growth were observed in C.thermocellum in conjunction with reduced maximum theoretical growth yields andincreased maintenance requirements. Similarly, metabolism and growth were greatlyinfluenced under hyperbaric pressure (1.8 and 7.0 MPa N2, ethane, and propane; 333K); however, severe inhibition was observed in the presence of supercritical ethane andliquid propane. These changes were attributed to mass-action effects on metabolicpathways, alterations in membrane fluidity, and the dominant role of phase toxicityassociated with compressed and supercritical fluids.Fluorescence anisotropy revealed fluidization and melting point depression ofdipalmitoylphosphatidylcholine liposomes in the presence of CO2, ethane, and propane(1.8 to 20.7 MPa; 295 to 333 K). The accumulation of these fluids within the bilayerupon pressurization and the ordering effects of pressure influenced liposome fluidity, themelting temperature, and the gel-fluid phase transition region. These resultsdemonstrate the disordering effects of compressed and supercritical fluids on biologicalmembranes and the ability to manipulate liposomes.

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Semiconductor quantum dots (QDs), particles several nanometers in diameter, exhibit a range of interesting properties that arise as a result of quantum confinement. Among these characteristics is photoluminescence, and unlike traditional fluorophores, the fluorescence emission of QDs is characterized by broad absorption and narrow emission that is a function of the particle diameter. This allows high spatial resolution to be achieved using spectral discrimination of closely spaced QDs.We propose applying QD fluorescence as a tool to sense the local environment of the QD to achieve wide-field sensing at high-resolution. Many factors influence QD fluorescence from the growth parameters and choice of ligand to the local environment of the QD post-fabrication. Nano-materials in the local QD environment influence the spectral or temporal characteristics of the QD fluorescence and detecting these changes enables identification of the location and motion of these nanoparticles with resolution on the order of a few nanometers.We have fabricated aqueous colloidal cadmium telluride QDs, experimenting with the choice of thiol-based ligand to influence the chemistry in post-processing and application. A wide range of tools have been used to characterize the spectral and physical properties of the QDs. We have successfully immobilized QDs on a variety of substrates including glass coverslips, silicon and indium tin oxide coated glass. Immobilization is achieved with even and consistent distributions of QDs on the substrate by using self-assembly of the colloidal particles onto substrates functionalized with N1-(3-Trimethoxysilylpropyl)diethylenetriamine (DETA) silane.Using fluorescence microscopy we have successfully demonstrated the detection of interactions between QDs and other nano-materials including green fluorescent protein and gold seed particles, demonstrating that QDs may, in principle, be used in a wide field microscopy technique to sense nano-materials with high resolution.

This study examined the interaction of model cationic, neutral and anionic endocrine disrupting compounds, pharmaceuticals and personal care products (EDC/PPCPs) with bulk and fractionated freshwater and waste water dissolved organic matter (DOM). The chemical composition of the freshwater DOM (Suwannee River, GA, SROM) proved to be rich in plant-derived hydrophobic aromatics, while the wastewater DOM (WWOM) contained a greater proportion of microbial biomolecular products, presumably resulting mainly from human waste. Studies focused on the fluorescence quenching of excitation-emission matrices (EEMs) of WWOM indicated that interaction with bis-phenol A (BPA), carbamazepine (CBZ), and ibuprofen (IBU) occurred preferentially with soluble protein-like and fulvic acid-like constituents. However, upon introduction to bulk SROM, BPA and CBZ were observed to quench humic acid-like regions of the EEMs, while negatively charged ibuprofen preferentially quenched the protein-like and fulvic acid components irrespective of DOM source and/or fraction. Despite this evidence of EDC/PPCP interactions with both DOM types, the strength of bonds formed was generally not sufficient to preclude full recovery and quantification of all three contaminants by liquid chromatography tandem mass spectrometry (LC-MSMS). An important exception, however, was for the hydrophilic acid fraction (HiA) of both DOM types, whose apparent bonding to cationic CBZ and anionic IBU significantly diminished LC-MSMS recovery. Thus, water sources rich in HiA character might produce a concentration underestimation of ionized EDC/PPCPs even with the use of sophisticated instruments such as LC-MSMS.The results of this research are consistent with the evolving ""supramolecular"" theory of natural organic matter, which postulates that organic matter itself is comprised of fragments of partially degraded biomolecules that are aggregated into ""supramolecular"" structures of apparent higher molar mass via relatively weak electrostatic, hydrophobic, and van der Waals interaction. Our findings suggest that EDC/PPCP contaminants, which comprise many of the same functional groups as waste water and freshwater DOM, may be incorporated into such DOM supramolecular structures, likely via the same types of intermolecular bonding, when they are present in natural waters under environmentally-relevant conditions.

We investigated the elastic scattering and fluorescence from laser dye solutions inside 5000, 1100, and 96.5 micron inner-diameter hollow-core capillaries. Incident 4416 A laser illumination of Coumarin 7 dye dissolved in ethanol caused fluorescence from approximately 4600 to 6000 A. This was studied over an angular range from 0° to 360°. A light scattering nephelometer coupled with a spectrometer gave intensity measurements as functions of wavelength (at fixed detection angles) and angle (at fixed wavelengths), while the illumination source, dye-filled capillary, and detector remained stationary. We saw capillary size and detection-angle dependence of the fluorescence and elastic scattering. Results show that angular variations of the elastic scattering and emitted fluorescence can be used to determine an optimum detection angle from the capillary with respect to the incident illumination direction. This work is important in the design and execution of Capillary Zone Electrophoresis (CZE) experiments.