As three-dimensional (3D) imaging and fluorescence techniques become standard in optical microscopy, novel approaches to 3D fluorescence microscopy are emerging. One such approach is based on the incoherent holography technique called optical scanning holography (OSH). The main advantage of OSH-based microscopy is that only a single two-dimensional (2D) scan is required to record 3D information, whereas most current 3D microscopes rely on sectioning techniques. To acquire a 3D representation of an object, current microscopes must physically scan the specimen in a series of 2D sections along the z-axis. In order to record holograms by OSH, the fluorescent specimen is scanned with an optically heterodyned laser field consisting of a Fresnel zone pattern. A unique acousto-optic modulator configuration is employed to generate a suitable heterodyne frequency for excitation of the fluorescent object. The optical response of a solution containing a high concentration of 15 um fluorescent latex beads to this type of excitation field has been recorded. In addition, holograms of the same beads have been recorded and reconstructed. To demonstrate the 3D imaging capability of the technique, the hologram includes beads with longitudinal separation of about 2 mm. A detailed comparison of 3D fluorescence microscopy by OSH and the confocal approach was conducted. Areas for comparison were 3D image acquisition time, resolution limits and photobleaching. The analysis shows that an optimized OSH-based fluorescence microscope can offer improved image acquisition time with equal lateral resolution, but with degraded longitudinal resolution when compared to confocal scanning optical microscopy (CSOM). For the photobleaching investigation, the parameter of concern is the fluence received by the specimen during excitation, which takes into account both the irradiance level and the time of illumination. Both peak and average fluence levels are addressed in the comparison. The analysis shows that during a 3D image acquisition, the OSH system will deliver lower peak fluence but higher average fluence levels to the specimen when compared to CSOM. / Ph. D.

Bilayer lipid membranes (BLMs) are formed from phospholipid molecules which self-assemble into a lipid bilayer with 4 to 9 nm thickness when submerged in an aqueous solution. This is due to their amphiphilic nature, meaning that one part of the molecule is hydrophilic, or attracted to water, and one part is hydrophobic, or repelled by water. They are the primary structural component of cell membranes in living organisms and therefore are useful for modeling the properties of cell membranes since they share many of the same chemical and physical properties. The objective of this dissertation is to investigate the maximum pressure that can be withstood by a BLM formed over a porous substrate, which will be referred to as the failure pressure. This work represents the first time that this quantity has been measured and reported. The failure pressure is investigated in several complementary ways, including mechanical, electrical, and optical measurements and modeling. The phospholipids that are tested include 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphatidylcholine (SOPC) and mixtures of SOPC and cholesterol (CHOL), which was chosen because cholesterol is known to increase the strength of SOPC BLMs. A new test methodology was developed to measure the failure pressure of BLMs. A custom test fixture was used to pressurize BLMs while measuring the applied pressure with a high degree of precision and repeatability. The BLMs were tested in an electrolyte solution over substrates that contained a single pore and also substrates that contained an array of many pores. SOPC BLMs were tested over single pore substrates with pore sizes ranging from 5 to 20 microns, and this resulted in failure pressures from 67 to 19 kPa, respectively. For single pore tests, the addition of 50 mol% cholesterol to SOPC resulted in a 56% higher failure pressure on average than SOPC alone. For multi pore substrates, SOPC BLMs were tested using pore sizes between 0.05 and 10 microns, which yielded bulk failure pressures of 380 to 1.5 kPa, respectively. For multi pore tests, SOPC/CHOL-50 mol% resulted in a 47% higher bulk failure pressure on average. A model was developed to predict the pressurization curve of BLMs and was applied to both the single and multi pore tests. It was found that the failure pressure of BLMs follows a distribution which was well modeled by a Weibull distribution with a positive skew. Parameters such as the Weibull shape parameter were determined by fitting the model to the experimental pressurization curves and it was found that the shape of the Weibull distribution was nearly the same for every pore size. Using the pressurization model it was estimated that the percentage of failed BLMs that were pressurized over a multi pore substrate ranged from 4% to 33%. The model also coupled the bulk failure pressure of BLMs formed over multiple pores to the failure pressure of a single BLM, showing that the bulk failure pressure of multiple BLMs is smaller than the failure pressure of a single BLM because it represents the failure of only the weakest BLMs in a group. Electrical impedance was measured before and after pressurization of the BLMs, and these measurements were modeled by assuming that the BLMs act as a resistor and a capacitor configured in parallel. In general, the impedance magnitude dropped by two to three orders of magnitude after BLM pressurization, which was a result BLMs failing and opening conductive pathways through the subsequently empty pores. It was found that normalized conductance values for SOPC BLMs were between G / A = 4 x 10^-12 and 2 x 10^-8 S/cm^2, and normalized capacitance values varied between C / A = 3 x 10^-14 and 1 x 10^-10 F/cm^2. In the literature these values ranged from G / A = 10^-1 to 10^-9 S/cm^2 and C / A = 10^-6 to 10^-8 F/cm^2, having a wide range of values due to the many variations of experimental test procedures. Visual images of BLMs were produced using fluorescence microscopy. Images were recorded before and after pressurization of SOPC BLMs formed over a multi pore substrate. As predicted by the pressurization model, it was found that some but not all BLMs fail after pressurization. It was also found that BLMs fail over the center of a pore, and leave remnants around the perimeter of the pore. / Ph. D.

A series of studies were conducted to examine the antioxidant status, drought and disease tolerance, and growth response to foliar application of soluble seaweed (Ascophyllum nodosum) extracts (SE) and humic acid (HA; 25% active HA or 2.9% active HA) in tall fescue (Festuca arundinacea Schreb), Kentucky bluegrass (Poa pratensis L.) and creeping bentgrass (Agrostis palusttis Huds.) grown under low (-0.5 MPa) and high (-0.03 MPa) soil moisture environments. Foliar application of humic acid (2.9 % active HA) at 23.7 and 47.4 l/ha improved leaf water status, shoot and root development in tall fescue, Kentucky bluegrass and creeping bentgrass grown under drought. Humic acid (2.9% active HA) at 15.5 l/ha or SE at 326 g/ha significantly reduced dollarspot incidence and improved turf quality in creeping bentgrass. Drought stress induced an increase of antioxidants alpha-tocopherol and ascorbic acid concentrations in the three turfgrass species. In the experiment with Kentucky bluegrass, drought stress increased beta-carotene concentration, but did not significantly influence superoxide dismutase (SOD) activity. Foliar application of humic acid (25% active HA) at 5 l/ha and/or SE at 326 g/ha consistently enhanced alpha-tocopherol and ascorbic acid concentrations, leaf water status, and growth in the three cool-season turfgrass species grown under low and high soil moisture environments. In the experiment with Kentucky bluegrass, application of HA at 5 l/ha plus SE at 326 g/ha also increased beta-carotene content and SOD activity under low and high soil moisture environments. There were close positive correlations between the antioxidant status and shoot or root growth in the three turfgrass species regardless of soil moisture levels. The antioxidant SOD activity, photosynthetic capacity in terms of Fvm690, and chlorophyll content in terms of Fm730/Fm690 exhibited a seasonal fluctuation in endophyte [Neotiphodium coenophialum (Morgan Jones and Gams) Glenn, Bacon, Price and Hanlin] -free and endophyte-infected tall fescue. Application of SE enhanced SOD activity, photosynthetic capacity, and chlorophyll content in tall fescue, especially at 4 weeks after SE treatment. The SOD activity, photosynthetic capacity and chlorophyll content were not significantly influenced by the endophyte infection. A close positive correlation between SOD and photosynthetic capacity during the summer was found in endophyte-free and endophyte-infected tall fescue. / Ph. D.

Titre de l'écran-titre (visionné le 23 novembre 2023) / Les bactéries lactiques sont indispensables pour la production de produits laitiers fermentés comme le fromage. Les espèces bactériennes Lactococcus lactis et Lactococcus cremoris sont les plus utilisées par l'industrie laitière. Cependant, la fermentation peut être ralentie par des bactériophages. Ils se multiplient via un cycle lytique débutant par leur adsorption à la surface cellulaire. Toutefois, l'adsorption ne mène pas toujours à l'infection dû à la présence de mécanismes de défense contre les phages chez certaines souches bactériennes. Ce phénomène est peu étudié puisque les essais classiques d'adsorption virale nécessitent beaucoup de ressources et les résultats obtenus peuvent être variables et ambigus. L'objectif général de ce projet consistait à explorer l'utilisation de la fluorescence pour optimiser les essais d'adsorption virale. Ainsi, cinq phages infectant des lactocoques et appartenant aux trois principaux groupes (Skunavirus, Ceduovirus et P335) isolés dans les usines laitières ont été sélectionnés afin d'adapter les protocoles à une diversité de phages. D'abord, le protocole classique d'adsorption virale a été utilisé pour cinq phages sur leur souche bactérienne hôte et sur une souche insensible, suspectée de bloquer l'adsorption. Un protocole de microscopie à fluorescence a ensuite été mis au point en utilisant le SYBR Gold pour marquer l'ADN viral et le rouge du Nil pour les membranes bactériennes. Cette méthode a permis une analyse qualitative efficace de l'adsorption des cinq phages et qui concordait avec les résultats obtenus par la méthode classique. Finalement, une méthode a été développée pour réaliser des essais d'adsorption en plaque 96 puits, afin de quantifier l'adsorption des phages sur plusieurs souches bactériennes en un seul essai. L'ajout de la fluorescence a aussi été tenté pour faire ces essais d'adsorption à un plus haut débit. Toutefois, la spécificité du fluorophore et la sensibilité du lecteur de plaque ne conviennent pas au contexte des tests d'adsorption. / Lactic acid bacteria are indispensable to produce fermented milk products, such as cheese. The bacterial species Lactococcus lactis and Lactococcus cremoris are the most used by the dairy industry. However, the fermentation process may be slowdown by the presence of lytic bacteriophages. These phages replicate through the lytic cycle that begins with their adsorption to the cell surface. However, phage adsorption does not always lead to a successful infection, due to the presence of phage defense mechanisms in some bacterial strains. This phenomenon is not often studied because classical phage adsorption assays need a lot of resources, and the results obtained are often variable and ambiguous. The general objective of this project was to explore the use of fluorescence to optimize viral adsorption assays. To do this, five lactococcal phages belonging to the three main groups (Skunavirus, Ceduovirus, and P335) isolated in cheese plants were selected to adapt the protocols to a diversity of phages. First, classical phage adsorption assays were performed with five of these phages on their host strain and on insensitive strains, suspected to block phage adsorption. A fluorescence microscopy protocol was then developed using SYBR Gold as a marker for viral DNA as well as Nile red for bacterial cell membranes. This method allowed a qualitative analysis of the adsorption of five phages on the different bacterial strains, and the results were in accordance with the ones obtained with the classical assays. Finally, a 96-well plate method was developed to quantify phage adsorption on multiple bacterial strains in a single assay. The addition of fluorescence has also been attempted to perform these phage adsorption assays at a higher throughput. However, the specificity of the fluorophore and the sensitivity of the plate reader were not suited to the context of phage adsorption tests.

Optical diagnostics have become indispensable tools for the study of turbulent flows and flames. However, optical diagnostics developed in the past have been primarily limited to measurements at a point, along a line, or across a two-dimensional (2D) plane; while turbulent flows and flames are inherently four-dimensional (three-dimensional in space and transient in time). As a result, diagnostic techniques which can provide 4D measurement have been long desired. The purpose of this dissertation is to investigate two of such 4D diagnostics both for the fundamental study of turbulent flow and combustion processes and also for the applied research of practical devices. These two diagnostics are respectively code named tomographic chemiluminescence (TC) and volumetric laser induced fluorescence (VLIF). For the TC technique, the emission of light as the result of combustion (i.e. chemiluminescence) is firstly recorded by multiple cameras placed at different orientations. A numerical algorithm is then applied on the data recorded to reconstruct the 4D flame structure. For the VLIF technique, a laser is used to excite a specific species in the flow or flame. The excited species then de-excite to emit light at a wavelength longer than the laser wavelength. The emitted light is then captured by optical sensors and again, the numerical algorithm is applied to reconstruct the flow or flame structure. This dissertation describes the numerical and experimental validation of these two techniques, and explores their capabilities and limitations. It is expected that the results obtained in this dissertation lay the groundwork for further development and expanded application of 4D diagnostics for the study of turbulent flows and combustion processes. / Ph. D.

Due to their scientific significance and practical applications, turbulent flows and flames have been under extensive and intensive research for a long time. Turbulent flows and flames of interests to practice inherently have three-dimensional (3D) spatial structures, and therefore diagnostic techniques that can instantaneously resolve their 3D spatial features have long been desired and probably are needed to ultimately answer some of the open research questions. The goal of this dissertation thus is to investigate such diagnostics and demonstrate their capability and limitations in a range of turbulent flows/flames. To accomplish this goal, this dissertation developed and evaluated the following three diagnostic methods: tomographic chemiluminescence (TC), volumetric laser induced fluorescence (VLIF), and super-resolution planar laser induced fluorescence (SR-PLIF). First, 3D flame topography of well-controlled laboratory flames was measured with TC method and validated by a simultaneous 2D Mie scattering measurement. The results showed that the flame topography obtained from TC and the Mie scattering agreed qualitatively, but quantitative difference on the order of millimeter was observed between these two methods. Such difference was caused by the limitations of the TC method. The first limitation involves TC's reliance on chemiluminescence of nascent radicals (mainly CH*) in reacting flows, causing ambiguity in the definition of flame front and limiting its applications to certain types of reactive flow only. The second limitation involves TC's inability to study an isolated region of interest because the chemiluminescence is emitted everywhere in the flame. Based on the above understanding of the TC technique, the second part of this dissertation studied a VLIF method to overcome the above limitations of the TC technique. Compared with the TC technique, the VLIF method can be used in either reacting or non-reacting flow and on any particular region of interest. In the VLIF technique, the fluorescence signal was generated by exciting a target species with a laser slab of certain thickness. The signal was recorded by cameras from different perspectives, and then a VLIF tomographic algorithm was applied to resolve the spatial distribution of the concentration of the target species. An innovative 3D VLIF algorithm was proposed and validated by well-designed experiment. This model enables analysis of VLIF performance in terms of signal level, size of the field of view in 3D, and accuracy. However, due to the limited number of views and the tomographic reconstruction itself, the spatial resolution of VLIF methods is limited. Hence, the third part of this dissertation investigated a SR-PLIF method to provide a strategy to improve the spatial resolution in two spatial directions, and also to extend the measurement range of scanning 3D imaging strategies. The SR-PLIF method used planar images captured simultaneously from two (or more) orientations to reconstruct a final image with resolution enhanced or blurring removed. Both the development of SR algorithm, and the experimental demonstration of the SR-PLIF method were reported. / Ph. D.

The Ca-ATPase is an integral membrane enzyme which translocates two calcium ions from the cytoplasm of the cell to the sarcoplasmic reticulum lumen utilizing ATP breakdown as its energy source, in order to promote muscle relaxation. The focus of this research is the cardiac isoform of the Ca-ATPase which undergoes allosteric regulation by the phosphoprotein phospholamban (PLN). The Ca-ATPase is thought to be a target for nitrative stress and is affected by several chronic diseases of the heart. In the heart, age-based nitration of the Ca-ATPase inhibits Ca²⁺ transport activity but the physical mechanism by which nitration inhibits Ca-ATPase activity is not understood. Conversely, nitroxyl (HNO), a new candidate for drug therapy for congestive heart failure (CHF), improves overall cardiovascular function by increasing Ca-ATPase activity in the heart. However, the physical mechanism for this activation is unknown. Therefore, we have used enzyme kinetics, fluorescence spectroscopy, and EPR spectroscopy studies to determine the effects of ONOO⁻ and HNO on the Ca-ATPase and the physical regulation of the Ca-ATPase by PLN. Treatment of Ca-ATPase with a nitrating agent, ONOO⁻, inhibited Ca-ATPase activity, and the [ONOO⁻]-dependent inhibition of the Ca-ATPase was more effective in the presence of PLN. ONOO⁻ did not affect the [Ca²]-dependence of Ca-ATPase activity either in the presence or absence of PLN. ONOO⁻ had no effect on Ca-ATPase rotational mobility or oligomeric interactions, as affected by PLN, but ONOO⁻ decreased the amplitude of the Ca²⁺-dependent E2 to E1•Ca2 conformational change, both in the absence and presence of PLN. Treatment with HNO had no affect on the [Ca²⁺]-dependence of Ca-ATPase activity in the absence of PLN; however in the presence of PLN, the [Ca²⁺]-dependent activity was shifted to lower Ca²⁺ levels and corresponded to the uncoupling of PLN from the Ca-ATPase. HNO decreased Ca-ATPase rotational mobility and increased the Ca-ATPase Ca²⁺-dependent conformational transition, consistent with uncoupling PLN from the Ca-ATPase. Taken together, these results suggest that ONOO⁻ inactivates a fraction of enzyme units to lower overall enzyme activity, whereas HNO uncouples PLN from the Ca-ATPase with increases in Ca-ATPase conformational flexibility and Ca-ATPase activity. / Ph. D.

Actin-based cell motility plays crucial roles throughout the lifetime of an organism. The dynamic rearrangement of the actin cytoskeleton triggers a plethora of cellular processes including cellular migration. Neural Wiskott Aldrich syndrome protein (N-WASP) is involved in transduction of signals from receptors on the cell surface to the actin cytoskeleton. N-WASP activated actin polymerization drives extension of invadopodia and podosomes into the basement layer. In addition to activating Arp2/3 complex, N-WASP binds actin filament barbed ends, and both N-WASP and barbed ends are tightly clustered in these invasive structures. We used nanofibers coated with N-WASP WWCA domains as model cell surfaces and single actin filament imaging to determine how clustered N-WASP affects Arp2/3-independent barbed end assembly. Individual barbed ends captured by WWCA domains of N-WASP grew at or below their diffusion limited assembly rate. At high filament densities, overlapping filaments formed buckles between their nanofiber tethers and myosin attachment points. These buckles grew 3.4-fold faster than the diffusion-limited rate of unattached barbed ends. N-WASP constructs with and without the native poly-proline (PP) region showed similar rate enhancements. Increasing polycationic Mg2+ or Spermine to enhance filament bundling increased the frequency of filament buckle formation, consistent with a requirement of accelerated assembly on barbed end bundling. Our preliminary data shows that tethered N-WASP construct containing one WH2 domain does not generate processive bundles or filament loops leading us to believe that tandem WH2 is required for processivity. We propose that this novel N-WASP assembly activity provides an Arp2/3-independent force that drives nascent filament bundles into the basement layer during cell invasion. Discovery of this bundle mediated unique pathway involved in invasion and metastasis will provide new targets for therapeutic development. / Ph. D.

A holographic recording method using an optical heterodyne 2-D scanning technique for 3-D imaging of fluorescent objects in turbid media is described and experimentally demonstrated. For the first time, 3-D imaging of fluorescentobjects in turbid media by a holographic method is achieved, and the diffused photon rejecting process through a heterodyne technique is analyzed. We also propose and realize a multiplexing and a digital decoding method for removing twin-image noise in optical scanning holography. The holographic method studied can be applied to 3-D biomedical imaging of fluorescent objects in turbid media as well as diffusely reflecting objects. / Master of Science

De par le monde, les virus, les bactéries pathogènes et les parasites ont causé plus de 21 millions de décès en 2010 [1, 2]. La détection dés agents pathogènes plus tôt dans le traitement antimicrobien permettrait de diminuer le nombre de victimes et réduirait le développement de résistance antibiotique [3]. Cependant, les techniques de biodétection nécessitent souvent quelques jours de délai dus à l'étape de culture microbienne. Pour éliminer cette étape, notre groupe de recherche développe une technique de biodétection optique très sensible basée sur les modes de galerie de microsphères fluorescentes. Bien que cette technique ait montré des résultats prometteurs, elle n'est toutefois pas prête à être intégrée en milieu clinique, puisque certaines étapes sont longues et expérimentalement non triviales. Dans nos travaux de recherche, les étapes de préparation des échantillons, de prise de mesures et de traitement de signal de la technique de biodétection WGMs en fluorescence ont. été réformées afin de simplifier et d'accélérer les manipulations. En premier lieu, les microsphères fluorescentes ont été étudiées afin de choisir un diamètre nominal et une plage spectrale offrant un maximum de WGMs selon la longueur d'onde d'excitation utilisée au laboratoire. Ensuite, l'intégration de la chambre d'écoulement d'un cytomètre en flux commercial et d'un système de déclenchement synchronisé au montage ont permis d'automatiser et d'accélérer considérablement l'étape de prise de mesures. De plus, l'optimisation de l'étape de traitement de signal a permis de générer des spectres théoriques très représentatifs des spectres de fluorescence de microsphères plus grandes que 6250 nm de rayon. Finalement, une nouvelle version de l'algorithme de mesure instantanée d'indice de réfraction apparent (IMARI 2.0) a été validée puis appliquée à la reconnaissance du rayon et de l'indice de réfraction apparent pour des microsphères de 6500 nm émettant de 580 nm à 650 nm.