L'étude des cellules souches est l'un des champs de recherches les plus importants dans le domaine biomédical. La vision par ordinateur et le traitement d'images ont été fortement mis en avant dans ce domaine pour le développement de solutions automatiques de culture et d'observation de cellules. Ce travail de thèse propose une nouvelle méthodologie pour l'observation et la modélisation de la prolifération de cellule souche neuronale sous microscope à contraste de phase. À chaque observation réalisée par le microscope durant la prolifération, notre système extrait un modèle en trois dimensions de la structure de cellules observées. Cela est réalisé par une suite de processus d'analyse, synthèse et sélection. Premièrement, une analyse de la séquence d'images de contraste de phase permet la segmentation de la neurosphère et des cellules la constituant. À partir de ces informations, combinées avec des connaissances a priori sur les cellules et le protocole de culture, plusieurs modèles 3-D possibles sont générés. Ces modèles sont finalement évalués et sélectionnés par rapport à l¿image d¿observation, grâce à une méthode de recalage 3-D vers 2-D. A travers cette approche, nous présentons un outil automatique de visualisation et d'observation de la prolifération de cellule souche neuronale sous microscope à contraste de phase.

La miniaturisation des pièces est une étape importante pour la progression de la microtechnologie dans plusieurs domaines (connectique, médical, optique, microsystèmes mécaniques). Pour cela, le moulage par microinjection, semble être la solution clé pour la production à grande échelle de micro-composants de polymères. Pour les polymères semi-cristallins, la cristallisation, sous fort taux de cisaillement et sous des vitesses de refroidissement élevées (about 100 K/s), induit des morphologies et des propriétés spécifiques. Elle prend donc une importance considérable dans le processus de microinjection par rapport au moulage par injection classique où les épaisseurs injectées sont généralement supérieures à 1 mm. Ces microstructures ont une grande influence sur les propriétés mécaniques du produit final. La prédiction de ces propriétés à partir de la description de la microstructure est un défi technique et scientifique. Durant cette thèse, deux polymères semi-cristallins ont été microinjectés, le polyéthylène haute densité et le polyamide 12. Les analyses obtenues par la microscopie otiques montrent que les morphologies cristallines varient entre les micro- et les macro-pièces. Tandis que la morphologie de 'peau-cœur' est présente dans les macropièces, les micropièces présentent une morphologie plutôt particulière. Les analyses combinées de diffusion et de diffraction des rayons X (SAXS et WAXS) avec un microfaisceau synchrotron, nous ont permis de déterminer la microstructure induite par le processus de microinjection dans toute l'épaisseur des pièces. Nous avons constaté que la morphologie et les orientations cristallines induites sont très dépendantes des conditions d'injection ou de microinjection. Une diminution de l'épaisseur, de la vitesse et de la température du moule, augmente l'orientation cristalline en limitant la relaxation des chaînes de polymères.

Dans le cadre du projet Laser MégaJoule (LMJ), des chaînes de mesure, appelées diagnostics, sont nécessaires à la qualification de l'atteinte de l'ignition. Parmi ces diagnostics, des imageurs X devront observer le développement d'instabilités hydrodynamiques à la surface du microballon. L'imagerie de ces instabilités sera faite dans le domaine X par radiographie ou en utilisant l'émission propre de la cible. Aucun imageur X conçu aujourd'hui pour le LMJ ne permet de réaliser une telle image. L'imageur X développé dans cette thèse devra donc réaliser une image à haute résolution et à haute énergie tout en respectant les contraintes de fonctionnement lié à une installation telle que le LMJ. Nous avons tout d'abord étudié et amélioré un diagnostic existant : EHRXI. Nous avons optimisé la bande d'énergie X utile à l'imagerie par ce diagnostic en l'étendant jusqu'à 12 keV. Nous avons obtenu des résolutions inférieures à 5 μm dans un champ de 1 mm de diamètre. Ce diagnostic a été déployé avec succès sur les installations laser ELFIE 100 TW et OMEGA. Avec le retour d'expérience obtenu avec EHRXI nous avons conçu un prototype de diagnostic pour le LMJ : Merssix. Ce microscope aura une résolution inférieure à 5 μm dans un champ de 500 μm de diamètre pour des énergies s'étendant jusqu'à 22 keV. Merssix a été pensé et adapté aux conditions expérimentales du LMJ. Sa conception met notamment en jeu une sélection spectrale pour permettre une utilisation en radiographie en présence d'un environnement X complexe.

Inconel 718 is a high nickel content superalloy possessing high strength at elevated temperatures and resistance to oxidation and corrosion. The non-traditional manufacturing process of wire-electrical discharge machining (EDM) possesses many advantages over traditional machining during the manufacture of Inconel 718 parts. However, certain detrimental effects are also present. The top layer of the machined surface is melted and resolidified to form what is known as the recast layer. This layer demonstrates microstructural differences from the bulk workpiece, resulting in altered material properties. An experimental investigation was conducted to determine the main machining parameters which contribute to recast layer formation in wire-EDM of Inconel 718. It was found that average recast layer thickness increased with energy per spark, peak discharge current, current pulse duration, and open-voltage time and decreased with sparking frequency and table feed rate. Over the range of parameters tested, the recast layer was observed to be between 5 and 10 μm in average thickness, although highly variable in nature. Surface roughness of the cut parts showed an increase with energy per spark. Electron Probe Microanalysis (EPMA) revealed the recast layer to be alloyed with elements from the wire electrode. X-ray diffraction testing showed the residual tensile stresses evident near the cut surface to decrease with energy per spark. Additionally, nano-indentation hardness testing indicated that the recast layer is reduced in hardness and elastic modulus compared to the bulk material. Vibratory tumbling was found to be a moderately effective post-processing tool for recast layer removal when using pre-formed ceramic abrasive media or fine grained aluminum oxide.

Development of a three dimensional (3D) HYBROT model with targeted in vivo like intact cellular circuitry in thick brain slices for multi-site stimulation and recording will provide a useful in vitro model to study neuronal dynamics at network level. In order to make this in vitro model feasible, we need to develop several associated technologies. These technologies include development of a thick organotypic brain slice culturing method, a three dimensional (3D) micro-fluidic multielectrode Neural Interface system (µNIS) and the associated electronic interfaces for stimulation and recording of/from tissue, development of targeted stimulation patterns for closed-loop interaction with a robotic body, and a deep-tissue non-invasive imaging system. To make progress towards this goal, I undertook two projects: (i) to develop a method to culture thick organotypic brain slices, and (ii) construct a multiphoton imaging system that allows long-term and deep-tissue imaging of two dimensional and three dimensional cultures. Organotypic brain slices preserve cytoarchitecture of the brain. Therefore, they make more a realistic reduced model for various network level investigations. However, current culturing methods are not successful for culturing thick brain slices due to limited supply of nutrients and oxygen to inner layers of the culture. We developed a forced-convection based perfusion method to culture viable 700µm thick brain slices. Multiphoton microscopy is ideal for imaging living 2D or 3D cultures at submicron resolution. We successfully fabricated a custom-designed high efficiency multiphoton microscope that has the desired flexibility to perform experiments using multiple technologies simultaneously. This microscope was used successfully for 3D and time-lapse imaging. Together these projects have contributed towards the progress of development of a 3D HYBROT. ----- 3D Hybrot: A hybrid system of a brain slice culture embodied with a robotic body.

Au cours de ce travail, nous avons procédé à une analyse extensive des dislocations d'interface et de la relaxation des contraintes dans les couches épitaxiales de GaSb sur GaAs (ou GaP) par microscopie électronique en transmission. Sur le substrat de GaAs, nous avons étudié le rôle de l'épaisseur de couches intermédiaires AlSb et le traitement de surface du substrat sur la relaxation des contraintes et la densité de dislocations émergeantes de la couche GaSb. Pareillement, nous avons étudié les effets des paramètres de croissance, tels que, le traitement de surface du substrat, la vitesse et la température de croissance sur la relaxation des contraintes des premières monocouches de GaSb sur la GaP. Avec ces paramètres de croissance optimisés, nous avons pu réaliser une couche de GaSb tampon (600 nm) et des hétérostructures AlSb/InAs avec une mobilité température ambiante de 30000 cm2V-1s-1 et 25500 cm2V-1s-1 sur la GaAs et GaP, respectivement. De plus nous avons mis en évidence, une dépendance du type de dislocation d'interface au mode de croissance: une croissance 2D de GaSb favorise la génération de dislocations de Lomer; alors que des dislocations 60o et des paires de 60o sont principalement générées en mode de croissance 3D. Nous avons aussi déterminé de façon quantitative le mécanisme général de formation des dislocations d'interface: l'interaction d'une dislocation 60o qui se forme en surface et glisse sous interaction avec celle qui se trouve déjà dans l'interface, mais aussi la tension de surface, permettent de déterminer la direction de son vecteur de Burger et donc la configuration de la dislocation résultante à l'interface. Les structures des dislocations et leur stabilité ont été étudiées par HAADF avec résolution atomique et modélisation par dynamique moléculaire. L'étude quantitative des vecteurs de Burger par analyse fine des images a confirmé le mécanisme de formation des dislocations d'interface en accord avec notre modèle.

Research Doctorate - Doctor of Philosophy (PhD) / Helium has the highest ionization energy of any species and is as a consequence difficult to detect by conventional means. On the other hand, it is the ideal surface probe, having no net charge or spin, a low mass and a short de Broglie wavelength. Therefore, there exists a strong incentive to develop a microscopy technique based on helium atom scattering. The purpose of this thesis is to investigate in detail how an efficient helium detector might be developed using the phenomenon of field ionization, an ionization method that relies on quantum mechanical tunneling rather than the more conventional electron impact ionization techniques. In particular, the work focusses on the potential use of a novel nanomaterial, carbon nanotubes, as the source of the high electric fields required for field ionization detection. In Chapter 1 we review the history of field ionization research and the properties and synthesis methods for carbon nanotubes. Chapter 2 describes the experimental apparatus and procedures used for the present research, and Chapter 3 introduces the theoretical framework and background for field ionization. In Chapter 4, the prototypical field ionization system is considered from a detector viewpoint. The work demonstrates that existing theory is not sufficiently quantitative for describing a field ionization detector and therefore a semi-empirical theory is advanced for that purpose. Chapter 5 considers the problem of nanotube field enhancement in detail using computational methods, leading to a complete description of the maximum field enhancement of a nanotube array based on the four fundamental array parameters. Efforts to synthesize carbon nanotubes in the Newcastle plasma-enhanced chemical vapor deposition system are described in Chapter 6. Several procedures are developed for reproducible growth of nanotube films and the chemical vapor deposition system is characterized with single parameter studies. Chapter 7 presents the results of electron field emission and helium field ionization experiments carried out using the grown nanotube films. We demonstrate for the first time the field ionization of helium using a planar film of carbon nanotubes. Finally, we conclude the investigation of field ionization detection in Chapter 8 with a discussion on how such a detection method integrates into a helium microscope and in particular we detail the design and initial calculations for the planned Newcastle helium microscope.

The ongoing merger of the digital and optical components of the modern microscope is creating opportunities for new measurement techniques, along with new challenges for optical modelling. This thesis investigates several such opportunities and challenges which are particularly relevant to biomedical imaging. Fourier optics is used throughout the thesis as the underlying conceptual model, with a particular emphasis on three--dimensional Fourier optics. A new challenge for optical modelling provided by digital microscopy is the relaxation of traditional symmetry constraints on optical design. An extension of optical transfer function theory to deal with arbitrary lens pupil functions is presented in this thesis. This is used to chart the 3D vectorial structure of the spatial frequency spectrum of the intensity in the focal region of a high aperture lens when illuminated by linearly polarised beam. Wavefront coding has been used successfully in paraxial imaging systems to extend the depth of field. This is achieved by controlling the pupil phase with a cubic phase mask, and thereby balancing optical behaviour with digital processing. In this thesis I present a high aperture vectorial model for focusing with a cubic phase mask, and compare it with results calculated using the paraxial approximation. The effect of a refractive index change is also explored. High aperture measurements of the point spread function are reported, along with experimental confirmation of high aperture extended depth of field imaging of a biological specimen. Differential interference contrast is a popular method for imaging phase changes in otherwise transparent biological specimens. In this thesis I report on a new isotropic algorithm for retrieving the phase from differential interference contrast images of the phase gradient, using phase shifting, two directions of shear, and non--iterative Fourier phase integration incorporating a modified spiral phase transform. This method does not assume that the specimen has a constant amplitude. A simulation is presented which demonstrates good agreement between the retrieved phase and the phase of the simulated object, with excellent immunity to imaging noise.

The biological, technical and computational aspects of this project collectively focused on using electron tomography (ET) for the high-resolution (10-20 nm) 3D reconstruction of entire insulin-secreting beta cells within islets of Langerhans isolated from mouse pancreata. Islets were cultured overnight to represent either steady-state (non-stimulated) or elevated glucose (stimulated) conditions, prior to fast-freezing, freeze-substitution, plastic embedment and cutting into 250-400 nm thick sections for tomographic imaging using intermediate voltage electron microscopy (EM). 3D images (tomograms) of each section were used to evaluate the performance of the new technical and computational approaches developed, and make biological comparisons of intercellular structure-function. Analysis focused on key compartments/organelles of the insulin-secretory pathway - Golgi apparatus, mitochondria, insulin secretory granules and multi-granular bodies. To allow the application of ET to entire mammalian cells, several technical limitations were addressed. Since segmenting (delimiting compartments of interest) tomograms manually, represented the major ërate-limiting stepí of ET, an interactive approach for 3D segmentation using novel interpolation algorithms (crude smooth, pointwise smooth and spherical interpolation) to iteratively predict the shape of 3D surfaces between user-drawn contours was developed. The performance of these tools in segmenting a range of compartment types was examined, and found to significantly enhance the speed and accuracy of manual segmentation. To better compensate for the physical collapse of plastic sections in the EM, a novel method was developed for estimating section collapse by analyzing approximately spherical organelles. Using this method on mature insulin granules in high-resolution datasets, coupled with measurements from the whole cell reconstructions, section collapse was found to be substantially less (~25%) than the value (40%) previously used to re-scale 3D models. Other new approaches developed to further improve the accuracy and quality of tomograms, included interactive tools for fiducial tracking, and the use of larger gold particles, a ëreduced second axisí to account for the missing wedge problem, and deformation grids to account for anisotropic deformation. As well as affording more efficient and precise mapping of cell ultrastructure in 3D for subsequent quantitative analysis, these developments provided new insights for future automated (hybrid) segmentation pipelines and new computational approaches for improving quality and isotropic accuracy of volumetric image data. The Interpolator and DrawingTools for segmentation, AnalysisTools for estimating section collapse and BeadHelper for tracking fiducial particles, written as plug-ins for the IMOD software package distributed by the University of Colorado, are now being used by the wider ET community with significant positive feedback. Using the novel approaches developed, four insulin-secreting beta cells - two from the periphery of an islet frozen 1 hr after stimulation with 11 mM glucose, and two from the periphery of another islet under steady-state 5.6 mM glucose conditions - were reconstructed in their entirety in 3D. Quantitative data on the key compartments/organelles provided new information regarding global changes in cellular organization, and enabled robust comparisons of each pair of functionally equivalent cells at unprecedented spatial resolution. Relative differences in the number, dimensions, architecture and distribution of organelles per cubic micron of cellular volume (including mitochondrial branching) reflected differences in the cellsí individual capacity/readiness to respond to secretagogue stimulation. In the two stimulated cells this was reflected by inverse relationships between the number/size of mature granules versus immature granules, the number/size of mitochondria, and the volume of the trans-Golgi network relative to the entire Golgi ribbon. Complementary stereological analysis of whole islets indicated which cells were the most representative under stimulated versus non-stimulated conditions, and revealed a marked natural heterogeneity between cells both within and between individual islets. Overall, this project led to significant improvements in efficiency and accuracy for segmenting cellular compartments/organelles, and in image quality and accuracy for tomogram computation and reconstruction through use of the newly developed techniques. The improved 3D reconstruction and analysis of pancreatic beta cells in toto in native tissue provided a powerful approach for quantitatively mapping the organelles involved in insulin synthesis/secretion at unprecedented detail, and afforded a level of insight into the complex 3D organization of mammalian cells not previously achieved by any other analytical technique or imaging method.

Τα νανοϋλικά, τα υλικά δηλαδή που οι διαστάσεις τους είναι στην νανοκλίμακα, έχουν διαφορετικές ιδιότητες από τα αντίστοιχα υλικά σε μεγαλύτερη κλίμακα. Για αυτό το λόγο, τα νανοϋλικά παρουσιάζουν ιδιαίτερο ενδιαφέρον για περαιτέρω μελέτη και έρευνα. Σε αυτά τα υλικά ανήκει και το οξείδιο του ψευδαργύρου (ZnO). Το οξείδιο του ψευδαργύρου είναι ένας σύνθετος ημιαγωγός τύπου II-IV με άμεσο ενεργειακό χάσμα (Eg=3.37 eV) σε θερμοκρασία δωματίου και με μεγάλη ενέργεια σύνδεσης εξιτονίου (60 meV). Σκοπός της παρούσας διπλωματικής εργασίας είναι η σύνθεση, ο χαρακτηρισμός και η μελέτη ιδιοτήτων νανοσύνθετων υλικών οξειδίου του ψευδαργύρου. Προς αυτόν τον σκοπό, θα παρασκευάσουμε νανοδομές ZnO, με τη μέθοδο του ατμού-υγρού-στερεού (VLS), σε τριζωνικό φούρνο. Στην πορεία της εργασίας, στο πρώτο κεφάλαιο θα δοθούν οι ορισμοί της νανοτεχνολογίας και των νανουλικών. Στο δεύτερο κεφάλαιο, θα αναλυθεί η δομή και οι ιδιότητες του οξειδίου του ψευδαργύρου (ZnO), βάσει των κυρίων μεθόδων ανάπτυξης των νανοδομών του ZnO. Στο τρίτο κεφάλαιο, θα γίνει ανάλυση των μεθόδων χαρακτηρισμού των νανοδομών του ZnO,που θα ακολουθήσουμε στην παρούσα εργασία. Στο επόμενο κεφάλαιο (4ο), θα παρουσιαστεί η μέθοδος παρασκευής των νανοδομών του ZnO και στο αμέσως επόμενο, παρουσιάζονται τα αποτελέσματα του χαρακτηρισμού των νανοδομών ZnO, με τις μεθόδους PL, SEM και XRD. Στο τελευταίο κεφάλαιο αναφέρονται τα συμπεράσματα που προέκυψαν από την παρούσα διπλωματική εργασία. / Nanomaterials, materials on the scale of a few nanometers, have different properties in comparison with larger-scale materials. For this reason, nanomaterials are of particular interest for further study and research. Zinc Oxide (ZnO) belongs to these materials. ZnO is a complex II-VI semiconductor with a direct band-gap energy (Eg = 3.37 eV) at room temperature and a large exciton binding energy (60 meV). The aim of the present diploma thesis, is the preparation, the characterization and the study of the properties of ZnO nanoparticles. In order to accomplish that, we are going to produce nanostructures of ZnO, using the Vapor-Solid-Solid method, in a three-zone furnace. In the first chapter, we give the definitions of nanomaterials and various methods of producing them. In the chapter that follows, we are analyzing the properties of ZnO, the main methods of producing nanostructures of ZnO as well as the ways of exploiting these specific nanostructures. Thereafter, in chapter three, the three methods of characterizing the samples with the nanostructures, are analyzed. In the next chapter, the experimental procedure is presented, whereas on the fifth chapter the results are presented, using the methods of SEM, PL and XRD. In the final chapter we make about conclusion about this diploma thesis