PURPOSE: To determine, under flow conditions, whether reduced connections between Schlemm’s canal (SC) inner wall (IW) and juxtacanalicular tissue (JCT) cells play a role in giant vacuole (GV) formation; and whether decreased amount of cell margin overlap between adjacent IW cells promotes paracellular pore formation using serial block-face scanning electron microscopy (SBF-SEM). METHODS: Normal human eyes were immersion-fixed (0 mmHg, N=2) or perfusion-fixed (15 mmHg, N=1). Frontal and radial sections of SC were processed for SBF-SEM. IW and JCT cells, GVs, and pores were 3D-reconstructed. In each IW cell, total number of connections with underlying JCT cells/matrix was determined. Total cell margin length (TCML) and zero-overlap length (ZL) of each IW cell were measured to calculate percent zero-overlap length (PZL=ZL/TCML). All data were compared between the eyes fixed at 0 and 15 mmHg. RESULTS: Total number of IW/JCT connections in individual IWs significantly decreased in the eye fixed at 15 mmHg (33±5, N=5 cells) compared to those fixed at 0 mmHg (189±12, N=4 cells, p<0.001). The summed GV volume in individual cells significantly increased in the eye fixed at 15mmHg (218.03±19.65 μm3) compared to those fixed at 0 mmHg (82.33±27.22 μm3, p=0.0043). PZL increased 26.68% (p=0.001) in the eye fixed at 15mmHg vs. those fixed at 0mmHg, and all paracellular pores were found only in regions where the overlap length was 0 μm. CONCLUSIONS: Cellular connections between IW/JCT and IW/IW cells play a role in GV and pore formation in normal human eyes under flow conditions. Our results provide a baseline for future comparison with primary open angle glaucoma eyes.

Aluminium sheet production is a multi-stage process in which altering processing conditions can drastically alter the size and type of second phase particles found in the final product. The properties of these second phase particles also affects deformation and annealing processes, meaning that any attempt to create a through process model would require the ability to predict both how the particles would develop in the material, and how these particles then affect the alloy moving forward. This project first focuses on gaining insight into how the particles in a model aluminium alloy change during homogenisation heat treatment and hot rolling. This has been accomplished by utilising serial block face scanning electron microscopy (SBF-SEM), a technique which allows the capture of 3D data sets at sub micron resolutions. This has allowed the populations of primary (constituent) and secondary (dispersoid) particles to be analysed at different stages of sheet production, and thus allowing the effects of homogenisation and hot rolling on particle populations to be quantified. To discover how the particles would go on to affect further processing, digital image correlation has been used to examine the localised strain in the alloy near to a selection of particle configurations. This highlighted the heterogeneity in slip behaviour within the alloy and illustrated that plumes of rotation develop near to non deformable regions. Rotation plumes have previously been modelled using a crystal plasticity model, and so further work is also presented expanding upon this model to simulate a variety of particle configurations. This has shown that in the case of single particles, local deformation is dependent on both the aspect ratio of the particle and how it is aligned to the active slip system. With the incorporation of a second particle, the interparticle spacing must also be considered.

Electrical trees are micrometre-size tubular channels of degradation in high voltage polymeric insulation, a precursor to failure of electrical power plant. Hence, electrical trees critically affect the reliability of power systems and the performance of new insulation designs. Imaging laboratory-grown electrical trees has been an important tool for studying how trees develop. Commonly, electrical trees prepared in transparent or translucent polymers are imaged using traditional optical methods. Consequently, most of the analysis has been based on two-dimensional (2D) images of trees, thus, valuable information may be lost. However, electrical trees are complex interconnected structures that require a tree-dimensional (3D) approach for more complete analysis. This thesis investigates a method for imaging and analysis of electrical trees to characterise their 3D structure and provide a platform for further modelling. Laboratory created electrical trees were imaged using X-ray Computed Tomography (XCT) and Serial Block-Face Scanning Electron Microscopy (SBFSEM), 3D imaging techniques that provide sub-micrometre spatial resolution. Virtual replicas of the trees, which are the 3D geometrical models representing the real electrical trees, were generated and new indices to characterise the 3D structure of electrical trees were developed. These parameters were indicative of differences in tree growth and thus, they can be used to investigate patterns and classify the structure of electrical trees. The progression of the tree was analysed using cross-sections of the tree that are orthogonal to the growth: the number of tree channels and area covered by them were measured. The fractal dimension of the tree was calculated from the 3D model and from the 2D projections, the latter being lower for all the tree-type structures studied. Parameters from the skeleton of the tree such as number of nodes, segment length, tortuosity and branch angle were measured. Most of the mean segment lengths ranged 6-13 µm, which is in accordance to the 10µm proposed by various tree-growth models. The capabilities of XCT and SBFSEM imaging techniques were evaluated in their application to electrical trees. Bush and branch trees, including early-growth electrical trees (of length 20-40 µm), were analysed and compared using the comprehensive tool of visualisation and characterisation developed. A two-stage tree-growth experiment was conducted to analyse the progression and development of tree branches using XCT: tree channels after the second stage of growth were wider than after the first, while the fractal dimension remained the same. The capabilities of XCT and SBFSEM were tested for imaging electrical trees in optically-opaque materials such as micro and nano-filled epoxy compounds. The general structure of trees in epoxy filled up to 20 wt% micro-silica was observed using both techniques. The use of a virtual replica as the 3D geometrical model for the simulation of the electric field distribution using Finite Element Analysis (FEA) was preliminary explored. A combination of the imaging techniques is proposed for a more complete structural analysis of trees. It is believed that a great impact towards understanding electrical treeing will be achieved using the 3D technical platform developed in this thesis.

BACKGROUND: Netarsudil, a Rho kinase and norepinephrine transport (NET) inhibitor, is a new FDA approved drug used for decreasing raised intraocular pressure (IOP) in ocular hypertensive and primary open-angle glaucoma (POAG) patients. Previous studies reported that netarsudil increased outflow facility and lowered IOP by increasing active outflow areas around the circumference of the eye and dilating the episcleral veins (ESV; Kiel and Kopczynski, 2015; Ren et al., 2016). However, the mechanisms by which netarsudil increases outflow facility have not yet been fully elucidated. Moreover, the effects of netarsudil on the inner wall (IW) endothelium I-pores and B-pores of the Schlemm’s canal (SC) have also not been investigated yet. AIM: The goal was to determine if netarsudil-treatment increased the effective filtration areas (EFA) by increasing pore density in both high- and non-flow type areas, compared to untreated control eyes. METHODS: In this study, the effects of netarsudil on the pore densities on IW of SC were investigated by serial block-face scanning electron microscopy (SBF-SEM). Two pairs of eyes were perfused with green fluorescent tracers in order to determine the outflow pattern prior to treatment. Then, one eye of each pair was perfused with netarsudil, while the fellow eye of each pair was perfused with vehicle solution. All eyes were then perfused with red fluorescent tracers in order to determine the outflow pattern once they were treated with netarsudil. Both pairs of eyes were perfused and fixed at 15 mmHg. Global imaging was performed for all eyes to visualize high- and non- flow areas in the trabecular meshwork (TM) and ESV’s. A SBF-SEM was used to image eight wedges of tissue including the IW of SC and TM (high- and non-flow areas from four eyes) for a total of 16,378 images. The study analyzed the percentage of pore-types (GV-associated I-pores, Non-GV associated I-pores, B-pores), the median pore spans, the GV-associated I-pore locations, and the pore densities (per IW nuclei and IW area) between the equivalent control and netarsudil-treated flow areas. RESULTS: In global images, an increase in high-flow areas were observed in netarsudil-treated eyes due to recruitment from low-flow and non-flow areas. A greater percentage of GV-associated I-pores, B-pores, and total pores were found in high-flow in contrast to non-flow areas in both control and netarsudil-treated eyes (all P ≤ 0.05). However, the percentage of GV-associated I-pores in non-flow areas were significantly greater in treated compared to control eyes (P ≤ 0.05). Qualitative observations from two pairs of eyes showed a trend of greater I-pore, B-pore, and total pore density/per IW nucleus and density/per IW surface area in high-flow in contrast to non-flow areas for both treated and control eyes. No difference in I-pore, B-pore, and total pore density/per IW nucleus and density /per IW surface area were observed in equivalent flow-type areas when comparing control and netarsudil-treated eyes. In addition, there was a significant greater percentage of I-pores located on the side of GVs than the top of GVs in all cases (P ≤ 0.05). CONCLUSIONS: Netarsudil increased high-flow areas. A greater pore density was found in high-flow in contrast to non-flow areas. Netarsudil also significantly increased the proportion of GV-associated I-pores in non-flow areas when compared to control eyes. Our results suggests that one mechanism of netarsudil increasing outflow facility is acting through recruiting the high-flow areas around the circumference of the eye, which is associated with higher pore density and increasing the proportion of GV-associated I-pores in non-flow areas.

No description available.

No description available.

La microscopie non linéaire a transformé le domaine de la neurobiologie depuis les années 1990, en permettant d'acquérir des images tridimensionnelles de tissus épais avec une résolution subcellulaire. Cependant, les profondeurs d'imagerie accessibles sont limitées à quelques centaines de micromètres dans des tissus diffusants tels que le tissu cérébral. Au cours des dernières années, plusieurs stratégies ont été développées pour dépasser cette limitation de profondeur et accéder à de plus grands volumes de tissu. Ces avancées récentes ont jusqu'à présent été limitées en terme de modes de contrastes accessibles, et ont souvent été réduites à des approches monochromes. Ce travail de thèse vise à développer des techniques d'imagerie non linéaires de grands volumes et de grande profondeur dotées de diverses possibilités de contrastes, indispensables pour l'étude de tissus complexes tels que le tissu cérébral. Dans un premier chapitre, nous présentons les difficultés associées à l'imagerie de grand volume de tissu cérébral, avec une emphase particulière sur les puissantes stratégies de marquages génétiques dont l'usage à jusqu'à présent été limité à des faibles étendues. Ensuite, nous introduisons la microscopie Chrom-SMP (chromatic serial multiphoton), une méthode développée au cours de cette thèse et consistant à combiner l’excitation deux-photon multicouleurs par mélange de fréquences avec une technique d'histologie automatisée (i.e découpe sériée) pour accéder à plusieurs contrastes non linéaires à travers de grands volumes de tissus ex vivo, allant de plusieurs mm3 à des cerveaux entiers, avec une résolution micrométrique et un coalignement intrinsèque des canaux spectraux. Dans un troisième chapitre, nous explorons le potentiel de cette nouvelle approche pour la neurobiologie. En particulier, nous démontrons l'histologie multicouleur de plusieurs mm3 de tissu "Brainbow" avec une résolution constante dans l’ensemble du volume imagé. Nous illustrons le potentiel de notre approche à travers l'analyse de la morphologie, des interactions et du lignage des astrocytes du cortex cérébral de souris. Nous explorons également l’apport du Chrom-SMP pour le suivi multiplexé de projections neuronales marquées par des traceurs de couleurs distinctes sur de grandes distances. Enfin, nous présentons dans un quatrième chapitre le développement de la microscopie à trois photons multimodale, approche permettant d’augmenter la profondeur d’imagerie sur tissus vivants. / Multiphoton microscopy has transformed neurobiology since the 1990s by enabling 3D imaging of thick tissues at subcellular resolution. However the depths provided by multiphoton microscopy are limited to a few hundreds of micrometers inside scattering tissues such as the brain. In the recent years, several strategies have emerged to overcome this depth limitation and to access larger volumes of tissue. Although these novel approaches are transforming brain imaging, they currently lack efficient multicolor and multicontrast modalities. This work aims at developing large-scale and deep-tissue multiphoton imaging modalities with augmented contrast capabilities. In a first chapter, we present the challenges of high-content large-volume brain imaging, with a particular emphasis on powerful multicolor labeling strategies which have so far been restricted to limited scales. We then introduce chromatic serial multiphoton (Chrom-SMP) microscopy, a method which combines automated histology with multicolor two-photon excitation through wavelength-mixing to access multiple nonlinear contrasts across large volumes, from several mm3 to whole brains, with submicron resolution and intrinsic channel registration. In a third chapter, we explore the potential of this novel approach to open novel experimental paradigms in neurobiological studies. In particular, we demonstrate multicolor volumetric histology of several mm3 of Brainbow-labeled tissues with preserved diffraction-limited resolution and illustrate the strengths of this method through color-based tridimensional analysis of astrocyte morphology, interactions and lineage in the mouse cerebral cortex. We further illustrate the potential of the method through multiplexed whole-brain mapping of axonal projections labeled with distinct tracers. Finally, we develop multimodal three-photon microscopy as a method to access larger depths in live settings.