<p>The interface between two dissimilar materials often presents a challenge for materials engineers. Mismatches of moduli, coefficients of thermal expansion, surface energies and chemical functionalities can create headaches for engineers seeking to control and understand interfacial bonding. In this work, I am interested in two specific interfacial problems: the adhesion of pressure sensitive adhesive tapes to various substrates and the interface in polymer reinforced composite materials between the reinforcement phase and the matrix.</p> <p>Pressure sensitive adhesive tapes (PSATs) are an important class of materials with applications ranging from medical adhesives to roadway markings. In this work, I present a novel 90° peel fixture to be used in the evaluation of road tapes on roadway surfaces in construction zones. This modular fixture was validated on control surfaces before demonstrating the capability to test pavement marking tapes from road surfaces. Within the context of medical adhesives, I am interested in the deformation of the skin around the PSAT during peeling. By developing a model to predict this deformation, adhesives can be tailored to mitigate skin damage. I present experimental evidence indicating the independence of peeling force to the elastic modulus of the substrate along with deformation measurements of skin analogs during the removal of a medical tape. A new model for predicting the deformation of soft substrates during peel is reported based on the contact mechanics of a rectangular prism indenting an elastic half space.</p> <p>Polymer matrix composites are another category of materials which are increasingly adopted to improve performance or efficiency by reducing the weight of components. These materials offer a high specific strength but often fail catastrophically rather than gradually. Using stress responsive fluorescent molecules called mechanophores, I present a methodology to quantify stresses within the polymeric matrix near the reinforcement phase. By correlating in situ fluorescence intensity measurements during a uniaxial tensile test to stresses predicted from a finite element analysis model, a calibration was developed. This calibration was then applied to increasingly complex composite geometries. Chemically bonding these mechanophores to the interface between two materials allows for the detection of interfacial failures through fluorescence microscopy. I present a technique to synthesize interfacial spirolactam mechanophores on industrially relevant epoxy and silica material systems. I demonstrate the ability of these systems to detect failures in the system through in situ confocal microscopy during deformation.</p>

The retinal pigment epithelium (RPE) is a transporting epithelium with polarized membrane domains. A unique characteristic of these cells is that their apical surface does not face a lumenal space, but is directly apposed to a layer of neurons (photoreceptors) and their associated extracellular matrix. Because the interaction occurring at this site is important for retinal attachment and particle phagocytosis, an attempt was made to identify epithelial molecules which potentially could mediate cell-cell or cell-matrix adhesion. In the present report, the subcellular localization of β1-integrins, the main receptors for extracellular matrix ligands, has been examined within Xenopus RPE. Several previously characterized antibodies were used in this analysis including: two rabbit polyclonal antibodies directed against purified chick muscle fibronectin receptor (pAbs No. 3818 and No. 2999), and a monoclonal antibody specific for Xenopus β1-integrin subunit (mAb 8C8). In Western blots of whole epithelial cell extracts, each of the antibodies intensely labeled a 115 kDa band, consistent with β1-integrin reactivity. One of the reagents (pAb No. 3818) also weakly stained unidentified bands of 50 and 100 kDa. Pre-clearing experiments demonstrated that pAb No. 3818 and mAb 8C8 both recognize the same detergent-soluble integrin: when cell extracts were depleted of β1-integrin by immunoprecipitation with mAb 8C8, the 115 kDa antigen recognized by pAb No. 3818 was not observed. Consistent with their similar immunochemical reactivities, each of the antibodies produced equivalent immunocytochemical staining of many eyecup tissues, including extraocular skeletal muscle cells, scleral and choroidal fibroblasts and vascular endothelium of the choroid and neural retina. In the native RPE, and isolated sheets of epithelium, however, qualitative differences in labeling between these antibodies were evident. Analysis by confocal microscopy showed that, while all three antibodies stained the basal surface of the epithelium, pAb No. 3818 also strongly labeled the apical microvillar surface. As the adjacent photoreceptors did not cross-react with this antibody in control experiments, the apical RPE staining could not be accounted for as contamination with retinal tissues during isolation. Furthermore, when the apical cell surface was selectively biotinylated in situ, and biotinylated proteins precipitated by streptavidin-agarose, β1-integrin was detected by immunoblotting with both mAb 8C8 and pAb No. 3818. This domain-specific material, however, represented only a fraction of the whole cell surface integrin: substantially greater amounts of tagged molecules could be detected when isolated epithelial sheets were biotinylated, most likely representing the basal protein. Based on these results, it can be concluded that β1-integrin is present in both basal and apical RPE plasma membranes. Molecules present in the apical, membrane may represent components of adhesion receptors responsible for retina-epithelium interactions.

Yes / A confocal microscope provides a sequence of images of the corneal layers and structures at different depths from which medical clinicians can extract clinical information on the state of health of the patient’s cornea. Hybrid model based on snake and particle swarm optimisation (S-PSO) is proposed in this paper to analyse the confocal endothelium images. The proposed system is able to pre-process (quality enhancement, noise reduction), detect the cells, measure the cell density and identify abnormalities in the analysed data sets. Three normal corneal data sets acquired using confocal microscope, and two abnormal endothelium images associated with diseases have been investigated in the proposed system. Promising results are achieved and the performance of this system are compared with the performance of two morphological based approaches. The developed system can be deployed as clinical tool to underpin the expertise of ophthalmologists in analysing confocal corneal images.

Yes / Corneal images can be acquired using confocal microscopes which provide detailed images of the different layers inside the cornea. Most corneal problems and diseases occur in one or more of the main corneal layers: the epithelium, stroma and endothelium. Consequently, for automatically extracting clinical information associated with corneal diseases, or evaluating the normal cornea, it is important also to be able to automatically recognise these layers easily. Artificial intelligence (AI) approaches can provide improved accuracy over the conventional processing techniques and save a useful amount of time over the manual analysis time required by clinical experts. Artificial neural networks (ANN) and adaptive neuro fuzzy inference systems (ANFIS), are powerful AI techniques, which have the capability to accurately classify the main layers of the cornea. The use of an ANFIS approach to analyse corneal layers is described for the first time in this paper, and statistical features have been also employed in the identification of the corneal abnormality. An ANN approach is then added to form a combined committee machine with improved performance which achieves an accuracy of 100% for some classes in the processed data sets. Three normal data sets of whole corneas, comprising a total of 356 images, and seven abnormal corneal images associated with diseases have been investigated in the proposed system. The resulting system is able to pre-process (quality enhancement, noise removal), classify (whole data sets, not just samples of the images as mentioned in the previous studies), and identify abnormalities in the analysed data sets. The system output is visually mapped and the main corneal layers are displayed. 3D volume visualisation for the processed corneal images as well as for each individual corneal cell is also achieved through this system. Corneal clinicians have verified and approved the clinical usefulness of the developed system especially in terms of underpinning the expertise of ophthalmologists and its applicability in patient care.

Yes / A confocal microscope provides a sequence of images of the various corneal layers and structures at different depths from which medical clinicians can extract clinical information on the state of health of the patient’s cornea. Preprocessing the confocal corneal images to make them suitable for analysis is very challenging due the nature of these images and the amount of the noise present in them. This paper presents an efficient preprocessing approach for confocal corneal images consisting of three main steps including enhancement, binarisation and refinement. Improved visualisation, cell counts and measurements of cell properties have been achieved through this system and an interactive graphical user interface has been developed.

Microporous carbonates host a significant portion of the remaining oil-in-place in the giant carbonate reservoirs of the Middle East. Improved understanding of petrophysical and multi-phase flow properties at the pore-scale is essential for the development of better oil recovery processes. These properties strongly depend on the 3D geometry and connectivity of the pore space. In this study, we harnessed the unique capabilities of fluorescence confocal laser scanning microscopy (CLSM) to capture both macroporosity and microporosity, down to 0.1 µm, to provide a more representative 3D representation of pore space compared to traditional methods. The experimental procedure developed was specifically designed to enable highresolution confocal 3D imaging of the pore space of carbonate systems. The protocol aims to render carbonates more "transparent" to CLSM by imaging etched epoxy pore casts of the sample and minimizing CLSM signal scattering. The resulting highquality 3D images of the multi-scale pore space allow more reliable petrophysical interpretation and prediction of transport properties. Additionally, we present a robust pore imaging approach that correlates 2D images produced by scanning electron microscopy (SEM) with the 3D models produced by CLSM that cover a range of scales, from millimeters in 3D to micrometers in 2D. For the first time, multi-color fluorescence confocal imaging was employed to characterize the geometric attributes of a porous medium. We foresee that the protocol developed in this study could be used as a standard protocol for obtaining high-quality 3D images of epoxy pore casts using confocal microscopy, and could contribute to improved characterization of micritic carbonate reservoirs and oil recovery methods. We also demonstrate the advantages of multi-scale and multi-color confocal images in realizing more accurate evaluations of petrophysical properties. Finally, we demonstrate that micro 3D printing (two-photon polymerization) can potentially be used to fabricate micromodels with sufficient resolution to capture the geometric attributes of micritic carbonates and that can replicate the inherent 3D interconnectivity between macro- and micro-pores.

No description available.

No description available.

<p>This report primarily focuses on effectively obtaining a Stimulated Emission Depletion fluorescence (STED) microscope, while using Fluorescence Correlation Spectroscopy (FCS) as a guide for the alignment of the system. STED is a super-resolution microscopy technique that has gained favour in the biological sciences due to its ability to successfully resolve sub-diffraction structures within live cells. Moreover the ease with which it can be combined with FCS has extended the applications of this technique to the study of the dynamics within a system as well. The central premise of this work focuses around building a STED-FCS system and developing an alignment tool for obtaining a symmetric STED doughnut. Since the point spread functions (PSF) seen in confocal microscopy can be generally approximated by a Gaussian function, we approximate the doughnut PSF with a difference of Gaussian functions. We calculated an autocorrelation function (ACF) corresponding to the simplified Gaussian form of the doughnut PSF and we found that this ACF contained three very similar diffusion times, all inversely proportional to the dye diffusion coefficient. In agreement with the fact that the doughnut PSF is spread out compared to the purely Gaussian PSF, the doughnut ACF amplitude is lower and its average diffusion time large. Lastly we calculated the quality factor, which is the product of the amplitude of the correlation function with the average intensity, Q=G(0)*I, for the purposes of alignment of the system. When translating the confocal pinhole along an axis of the doughnut we were able to identify the centre of the doughnut due to the presence of a minimum in Q which can be very handy for alignment of the doughnut with respect to the pinhole. This operation is essential when aligning the excitation and STED beam. For future work, a road map for alignment of the two beams in the focal plane is also presented utilizing the cross correlation function between the two beams.</p> / Master of Science (MSc)

Protein-protein interactions are important for biological processes. Therefore, many small molecules target a specific protein or interaction in the cell to have biological consequence. While we can measure some protein-protein interactions in a test tube, many proteins cannot be purified making it difficult to properly test that a drug is “on target”. An alternative is to measure these interactions in live cells. We express the proteins of interest fused to fluorophores allowing the use of fluorescence techniques. Förster Resonance Energy Transfer (FRET) provides a molecular level ruler to measure the distance, within a few nanometers, between two proteins. FRET indicates binding. The gold standard for measuring FRET in live cells is by quantifying changes in fluorescence lifetime using Fluorescence lifetime imaging microscopy (FLIM). The change in fluorescence lifetime is inversely proportional to the ratio of bound to non-bound proteins. Tradition FLIM-FRET microscopy is too slow for screening applications. Our aim was to develop a highly multiplexed confocal system for rapid FLIM-FRET acquisition. We present the development of multiple prototypes for confocal multiplexing. In this work, our final design includes 32×32 multiplexed excitation points which scan the sample using refractive window scanners. We coupled this excitation scheme to a 64×32 time-gated single-photon avalanche photodiode (SPAD) sparse array detector. This multiplexed setup allows the use of the sparse array with high frame rate and sub-nanosecond time-gating to achieve high throughput FLIM acquisition. Using our multiplexed FLIM prototype we measured Bcl-2 family protein-protein interactions in live cells (310×310 μm FOV) with two-channel confocal FLIM in 1.5 s. Protein binding affinities were estimated by measuring the changes in FRET as a function of acceptor to donor ratio. The resulting speed of this system meets requirements for implementation in screening applications. / Thesis / Candidate in Philosophy / Inside a cell, proteins are the “workers” and they interact with each other, doing that work. Many of these interactions are important for the cell to live. Pharmaceutical companies may design drugs that can interfere with a specific interaction in order to cause an effect in the cell. Scientists are interested in measuring these interactions and we can do this by “taking a picture” of the interaction using a specialized microscope. One of the major issues with these microscopes is that it takes scientists a long time to collect pictures of these interactions. This means only a few drugs can be tested in a day. To speed up the drug discovery and testing we want to design faster microscopes that can test hundreds of drugs in a day. In my thesis I contributed to building a state-of-the-art super fast microscope. We made progress in steps, and by the third attempt we successfully measured interactions in cells in seconds! Our new microscope is ~400x faster than current technologies. We hope that this research will be useful to speed up drug discovery in the future.