In this Thesis, we have developed a microtechnology-based method for culturing and visualizing high numbers of individual cells and cell-cell interactions over extended periods of time. The foundation of the device is a silicon-glass multiwell microplate (also referred as microchip) directly compatible with fluorescence microscopy. The initial microchip design involved thousands of square wells of sizes up to 80 µm, for screening large numbers of cell-cell interactions at the single cell level. Biocompatibility and confinement tests proved the feasibility of the idea, and further investigation showed the conservation of immune cellular processes within the wells. Although the system is very reliable for screening, limitations related to synchronization of the interaction events, and the inability to maintain conjugations for long time periods, led to the development of a novel ultrasonic manipulation multiwell microdevice. The main components of the ultrasonic device is a 100-well silicon-glass microchip and an ultrasonic transducer. The transducer is used for ultrasonic actuation on the chip with a frequency causing half-wave resonances in each of the wells (2.0-2.5 MHz for wells with sizes 300-350 µm). Therefore, cells in suspension are directed by acoustic radiation forces towards a pressure node formed in the center of each well. This method allows simultaneous aggregation of cells in all wells and sustains cells confined within a small area for long time periods (even up to several days). The biological target of investigation in this Thesis is the natural killer (NK) cells and their functional properties. NK cells belong to the lymphatic group and they are important factors for host defense and immune regulation. They are characterized by the ability to interact with virus infected cells and cancer cells upon contact, and under suitable conditions they can induce target cell death. We have utilized the ultrasonic microdevice to induce NK-target cell interactions at the single cell level. Our results confirm a heterogeneity within IL-2 activated NK cell populations, with some cells being inactive, while others are capable to kill quickly and in a consecutive manner. Furthermore, we have integrated the ultrasonic microdevice in a temperature regulation system that allows to actuate with high-voltage ultrasound, but still sustain the cell physiological temperature. Using this system we have been able to induce formation of up to 100 solid tumors (HepG2 cells) in parallel without using surface modification or hydrogels. Finally, we used the tumors as targets for investigating NK cells ability to infiltrate and kill solid tumors.  To summarize, a method is presented for investigating individual NK cell behavior against target cells and solid tumors. Although we have utilized our technique to investigate NK cells, there is no limitation of the target of investigation. In the future, the device could be used for any type of cells where interactions at the single cell level can reveal critical information, but also to form solid tumors of primary cancer cells for toxicology studies. / <p>QC 20150113</p>

The theme of this dissertation is to advance safety hazard mitigation by detecting and characterizing hidden targets of concern. Ground-penetrating radar (GPR) is used to detect and characterize hidden targets that pose safety hazards at Earth's surface, within shallow soil, and within rock. The resulting images detect unexploded ordnance (UXO) and detect fractures that pose collapse hazards in a mine. Detecting and characterizing fractures and voids within rock prior to excavation can enable mitigation of mine collapse hazards. GPR data were acquired on the wall of a pillar in an underground mine. Strong radar reflections in the field data correlate with fractures and a cave exposed on the pillar walls. Pillar wall roughness was included in migration, a wavefield imaging algorithm, to quantitatively locate fractures and voids and map their spatial relationships within the rock. Quantifying the radar reflection amplitudes enabled mapping the distance between fracture walls. Detecting and characterizing UXO and landmines from a safe distance can enable de-mining. Migration was used to improve GPR imaging for unmanned aerial vehicle (UAV) data acquisitions. Existing algorithms were adapted for UAV flight irregularities and surface topography, and a new algorithm was developed that does not depend on the unknown soil wavespeed. Errors associated with wavespeed and raypath assumptions were quantified. The algorithms were tested with real and synthetic datasets. The improved and new algorithms are more successful than previous algorithms. To detect linear targets at all orientations, fully polarized GPR data are needed. Polarity combinations were investigated to optimize the detection of surface and subsurface small targets and linear targets. Scattering caused by topographic roughness is the primary shallow subsurface noise. For subsurface targets, detection is optimized by migration plus a polarity combination that captures all scattered energy. Strong reflection and scattering from the air-ground boundary can hide surface targets. Detection is optimized by removing the strong isotropic surface scattering, imaging targets by their anisotropic scattering. / Doctor of Philosophy / The theme of this dissertation is to advance safety hazard mitigation by detecting and characterizing hidden targets of concern. Ground-penetrating radar (GPR) is used to detect and characterize hidden targets that pose safety hazards at Earth's surface, within shallow soil, and within rock. The resulting images detect unexploded ordnance (UXO)/landmines and detect fractures that pose collapse hazards in a mine. Detecting and characterizing fractures and voids within rock prior to mining can enable mitigation of mine collapse hazards. GPR data were acquired on the wall of a pillar in an underground mine. Strong radar reflections in the field data correlate with fractures and a cave exposed on the pillar walls. Pillar wall roughness was included in migration, a wavefield imaging algorithm, to quantitatively locate fractures and voids and map their spatial relationships within the rock. Quantifying the radar reflection amplitudes enabled mapping the distance between fracture walls. Detecting and characterizing UXO, landmines from a safe distance can enable de-mining. Migration was used to improve GPR imaging for an unmanned aerial vehicle (drone) data acquisition. Existing algorithms were adapted for drone flight irregularities and surface topography, and a new algorithm was developed that does not depend on the unknown soil properties. Errors associated with the algorithms' assumptions were quantified. The algorithms were tested with real and computer-generated datasets. The improved and new algorithms are more successful than previous algorithms. To detect all targets regardless of their orientation, GPR data need to be acquired with antenna pointing in multiple directions (different polarities). Polarity combinations were investigated to optimize the detection of surface and subsurface small targets and linear targets. Scattering caused by topographic roughness is the primary shallow subsurface noise. For subsurface targets, detection is optimized by migration plus a polarity combination that captures all scattered energy. Strong radar reflection from the air-ground boundary can hide surface targets. Detection is optimized by removing the strong ground surface from the data, and imaging targets by differences in their radar scattering.

Magnetic resonance imaging (MRI) is the preferred imaging modality for visualization of intracranial soft tissues. Surgical planning, and increasingly surgical navigation, use high resolution 3-D patient-specific structural maps of the brain. However, the process of MRI is a multi-parameter tomographic technique where high resolution imagery competes against high contrast and reasonable acquisition times. Resolution enhancement techniques based on super-resolution are particularly well suited in solving the problems of resolution when high contrast with reasonable times for MRI acquisitions are needed. Super-resolution is the concept of reconstructing a high resolution image from a set of low-resolution images taken at dierent viewpoints or foci. The MRI encoding techniques that produce high resolution imagery are often sub-optimal for the desired contrast needed for visualization of some structures in the brain. A novel super-resolution reconstruction framework for MRI is proposed in this thesis. Its purpose is to produce images of both high resolution and high contrast desirable for image-guided minimally invasive brain surgery. The input data are multiple 2-D multi-slice Inversion Recovery MRI scans acquired at orientations with regular angular spacing rotated around a common axis. Inspired by the computed tomography domain, the reconstruction is a 3-D volume of isotropic high resolution, where the inversion process resembles a projection reconstruction problem. Iterative algorithms for reconstruction are based on the projection onto convex sets formalism. Results demonstrate resolution enhancement in simulated phantom studies, and in ex- and in-vivo human brain scans, carried out on clinical scanners. In addition, a novel motion correction method is applied to volume registration using an iterative technique in which super-resolution reconstruction is estimated in a given iteration following motion correction in the preceding iteration. A comparison study of our method with previously published methods in super-resolution shows favorable characteristics of the proposed approach.

The nematode Caenorhabditis elegans has served as one of the primary model organisms in neuroscience. As C. elegans research became more specific, so have the biological tools for manipulating C. elegans improved and matured. Additionally, in some avenues of research, technologies have been developed to manipulate the animals in very efficient and quantitative ways. However, the field of dynamic studies has remained without significant technological support. Dynamic studies focus on processes occurring over time and span a range of time-scales of i) minutes to hours requiring continuous imaging for accurate observation, ii) hours to days requiring periodic imaging of the same animal, and iii) days to weeks requiring daily monitoring. Because of a lack of suitable tools and technologies to perform these studies, researchers have to either apply standard biological methods with limited ability to observe processes dynamically or simply cannot perform such studies with the desired set of experimental conditions. To address this problem, a comprehensive microfluidic toolkit for dynamic studies has been created. The first element is a novel method for reversible and repeatable immobilization at benign conditions in tandem with a microfluidic system for isolated culture of C. elegans with integrated temperature control. The second element is a system for efficient handling of C. elegans embryos in a high-throughput and scalable fashion for chemical and thermal embryonic stimulation with subsequent study of development. The third component is a system capable of selective immobilization of animals’ bodies, while simultaneously facilitating feeding and normal physiological function for live imaging. The last component is capable of culturing animals over their life-span with efficient animal handling, environmental control (temperature and dietary conditions), and high data content experimentation. As a whole, the work in this thesis enables dynamic studies over the whole range of time scales applicable to C. elegans research. These types of studies were previously very difficult or near impossible to perform practically. Now, instead of building population composites to understand the dynamics of a process, risking affecting physiology via the experiment itself, or dealing with extremely labor intensive physical handling of animals, a toolkit for efficient handling of C. elegans facilitating dynamic and direct observation of individual animals is available. The biological applications range from dynamically studying lipid droplet morphology or studying synaptic vesicle transport, through observing the dynamics of synaptic re-arrangement during development or the effect of cancer drugs on development, to performing high-content life-span experiments able to ascertain the relationship between aging and behavior. Additionally, many of the principles in these designs can be expanded to accommodate research on other model organisms, such as other nematode species, zebra fish embryos, or cells and embryoid bodies.

Light allows to non-invasively study the complex and dynamic biological phenomenon undergoing within cells and tissues in their native state. The development of super-resolution microscopes in recent years has helped to overcome the fundamental limitation imposed by Abbe’s diffraction limit, thereby revolutionizing the field of molecular and cellular biology. With the advancement of various super-resolution techniques (like STED, PALM, and 4Pi) it is now possible to visualize the nanometeric cellular structures and their dynamics in real time. The limitations of existing fluorescence microscopy techniques are: poor axial resolution when compared to their lateral counterpart, and their inability to produce high resolution images of dynamic samples. This thesis covers two broadly connected areas of fluorescence imaging techniques while addressing these limitations. First, the PSF engineering and spatial filtering technique for axial super-resolution microscopy and second, the integration of light sheet illumination PSF with microfluidic cytometry for imaging cells on-the-go. The first chapter gives an explicit description on the fundamentals of fluorescence imaging. This introductory chapter includes a variety of optical microscopes, PSF engineering, the resolution limit imposed by the wave nature of light, the photochemistry of the fluorescent dyes, and their proper selection for fluorescence experiments. In addition to the state-of-art imaging techniques, namely Laser Scanning Confocal Microscopy and Light Sheet Microscopy, this chapter also gives a brief explanation on the evolution of imaging cytometry techniques. Their high speed analytic capability (i.e sorting and counting) makes this technique an important tool in health care diagnosis and other various biomedical applications. The chapter ends with a discussion on the operating principle of the flow cytometers and their limitations. The second chapter in this thesis describes the spatial filtering technique for engineering the PSF to eliminate the side-lobes in the system PSF of the 4Pi Confocal Microscopes. Employing an amplitude mask with binary light transmission windows (also called binary filters), the incident light is structured to minimize the secondary lobes. These lobes are responsible for exciting the off-focal planes in the specimen, hence provide incorrect map of the fluorophore distribution in the object. The elimination of the side-lobes is essential for the artifact-free axial super-resolution microscopy. This second chapter describes the spatial filtering technique in details (its mathematical formulation, application in fluorescence microscopy for generation of desired PSF including Bessellike beam). Specifically, spatial filtering technique is employed in 4Pi type-C Confocal Microscope. The spatial mask used results in the reduction of the side-lobes in 1PE case while they are nearly eliminated in 2PE variant of the proposed technique. The side-lobes are reduced by 46% and 76% for 1PE and 2PE when compared to the existing 4Pi type-C Confocal Microscope system. Moreover, OTF of the proposed system confirms the presence of higher frequencies in the Fourier domain indicating high resolution imaging capability. Apart from the resolution in lateral and axial dimension, achieving high resolution while imaging dynamic samples is another challenge that is limiting the field of fluorescence microscopy to flourish. The third and fourth chapters are entirely dedicated towards the work that was carried out to develop imaging techniques on a microfluidic platform for imaging dynamic samples. The fusion of microscopy and flow cytometry has given rise to the celebrated field of imaging flow cytometry. In recent years, the focus has shifted towards miniaturized cytometry devices. Apart from the reduced cost of the sample reagents and the assays, portability and easy handling make the microfluidic devices more relevant to developing countries. The commercially available cytometers are bulky and quite costly. In addition to these practical concerns, they are complex in operation and limited in performance. Most of the existing cytometers use different inlets for sheath and sample flow to achieve the hydrodynamic focusing of the sample assays in a narrow and confined region. The laser beam in the illumination arm interrogates with the flowing samples at this region and the response is captured by the detection optics. The same principle is extensively used in most of the microfluidic based flow cytometers reported till date. Apart from the hydrodynamic force other effects like electro-osmotic, acoustic, and dielectrophoresis have also been exploited to achieve flow focusing in the microfluidic channel. Despite omitting the necessity of external syringe pump as required in pressure driven based cytometers, they all rely upon point-source based excitation scheme and thereby can not interrogate the cells flowing through the entire microfluidic channel. The third chapter describes the integration of light sheet illumination PSF with microfluidic flow cytometry for simultaneous counting and imaging cells on-the-go. The chapter starts with the description on photolithography procedure for preparing SU8 master and PDMS casting procedure adopted to prepare dedicated microfluidic chips for the developed imaging system. The research work reported here demonstrates the proof-ofprinciple of light sheet based imaging flow cytometer. A light sheet fills the entire microfluidic channel and thus omits the necessity of flow focusing and point-scanning based technology. Another advantage lies in the orthogonal detection geometry that totally cuts-off the incident light, thereby substantially reducing the background in the acquired images. Compared to the existing state-of-the-art techniques, the proposed technique shows marked improvement. Using fluorescently coated Saccharomyces cerevisiae cells, cell counting with throughput as high as 2090 cells/min was recorded. Overall the proposed system is cost-effective and simple in channel geometry. Apart from achieving efficient counting in operational regime of low flow rate, high contrast images of the dynamic samples are also acquired using the proposed cytometry technique. Further, visualization of intra-cellular organelles is achieved during flow in light sheet based high-throughput cytometry system. The fourth chapter demonstrates the proof of concept of light-sheet-based microfluidic cytometer in conjugation with 2π/3 detection system for high-throughput interrogation and high resolution imaging. This system interrogates the flow channel using a sheet of light rather than the existing point-scanning based techniques. This ensures single-shot scanning of specimens flowing through the microfluidic flow channel at variable flow rates. In addition to high throughput counting at low flow rate, visualization of the intra-cellular organelle (mitochondrial network in human cancerous cells) during flow is achieved with sub-cellular resolution. Using mitochondrial network tagged HeLa cells, a maximum count of 2400 cells/min at the optimized flow rate of 700 nl/min was recorded. The 2π/3 detection system ensures efficient photon collection and minimal background caused by scattered illumination light. The other advantage of this kind of detection system which includes 8f detection optics, is the capability to produce variable magnification using the same high NA objective. This thesis opens up in vivo imaging of sub-cellular structures and simultaneous cell counting in a miniaturized flow cytometry system. The developed imaging cytometry technique may find immediate applications in the diverse field of healthcare diagnostics, lab-on-chip technology, and fluorescence microscopy. The concluding chapter summarizes the results with a brief discussion on the future aspects of this field (e.g., live-cell imaging of infectious RBC in microfluidic device and 3D optical sectioning of flowing cells). The field of imaging flow cytometry has immense applications in the overlapping areas of physics and biology. The hydrodynamic forces which are used to achieve flow focusing of the sample assays can have an adverse effect in the cell morphology, thereby altering the cellular functions. Light sheet based cytometry system lifts off the requirement of flow focusing and ensures a single shot scanning of entire samples flowing through the microfluidic channel. The similar concept can be used to study the developmental biology of an entire organism, such as C. elegans. This enables the direct observation of developmental and physiological changes in the entire body. Such an organism can be kept alive for a longer duration in microfluidic chambers, and the neural development and mating behaviors can be extensively studied.

Ce mémoire porte sur la synthèse et la caractérisation de composés hexaferrites dans le système Ca-Fe-O. Ce travail a permis d’isoler quatre composés sous forme polycristalline ayant pour composition (Ca4Fe5O13)1-x(Fe9O12)1+x (x= 0,334 ; 0,301 et 0,128) et (Ca4Fe5O13)(Fe4O4). Leur structure cristalline a été déterminée à partir de données de diffraction électronique acquises en mode tomographie par précession des électrons et validée à l’aide de l’imagerie haute résolution (HREM et HAADF). Les différents modèles structuraux ont également été confirmés par diffraction des rayons X et des neutrons sur poudre. L’analyse fine des défauts d’intercroissance en imagerie HAADF a révélée des écarts significatifs de composition par rapport à la composition idéale (Ca4Fe5O13)(Fe9O12) à l’origine des trois polymorphes observés. En complément des études menées sur des cristaux de taille micrométrique dans les années 80, l’obtention d’échantillons polycristallins a rendu possible l’étude des propriétés physiques de ces composés. Malgré la complexité de ces structures et la présence de défauts étendus, la spectrométrie Mössbauer a mis en exergue un degré d’oxydation unique pour les atomes de fer (+3) et de confirmer les nombreuses transitions magnétiques initialement détectées par les mesures d’aimantation, ainsi que leur évolution en fonction de l’écart à la stœchiométrie x. Les composés ont également été caractérisés par des mesures de résistivité électrique et de coefficient Seebeck. / This thesis reports on the synthesis and the characterization of hexaferrite compounds in the Ca-Fe-O system. This work has allowed to isolate four polycrystalline compounds presenting the chemical formula (Ca4Fe5O13)1-x(Fe9O12)1+x (x= 0.334; 0.301 and 0.128) and (Ca4Fe5O13)(Fe4O4). Their crystalline structure has been determined using the precession electron diffraction tomography and has been validated through high resolution imaging microscopy (HREM/HAADF). X-ray diffraction and neutron diffraction studies on polycrystalline samples have confirmed the different structural models. Fine analysis of intergrowth defects in HAADF imaging revealed significant deviations in composition with respect to the ideal composition (Ca4Fe5O13)(Fe9O12) at the origin of the three observed polymorphs. In addition to the studies on micron-sized crystals in the 80s, obtaining polycrystralline samples allowed the measurement of their physical properties. Despite the complexity of these structures and the presence of extensive defects, the Mössbauer spectroscopy has highlighted a unique oxidation degree of iron (+3) and confirmed as well the various magnetic transitions initially detected by magnetization measurements, as well as their evolution versus the x deviation value. Electrical resistivity and Seebeck coefficient measurements were performed on the samples.

No description available.

During the last decade, ultrasonic manipulation has matured into an important tool with a wide range of applications, from fundamental cell biological research to clinical and industrial implementations. The contactless nature of ultrasound makes it possible to manipulate living cells in a gentle way, e.g., for positioning, sorting, and aggregation. However, when manipulating cells using ultrasound, especially using high acoustic amplitudes, a great deal of heat can be generated. This constitutes a challenge, since the viability of cells is dependent on a stable physiological temperature around 37°C.      In this Thesis we present applications of ultrasonic manipulation of fluids, particles, and cells in temperature-controlled micrometer-sized devices fabricated using well established etching techniques, directly compatible with high-resolution fluorescence microscopy. Furthermore, we present ultrasonic manipulation in larger up to centimeter-sized devices optimized for fluid mixing and cell lysis. In the present work, two new ultrasonic manipulation platforms have been developed implementing temperature control. These platforms are much improved with increased performance and usability compared to previous platforms. Also, two new ultrasonic platforms utilizing low-frequency ultrasound for solubilization and cell lysis of microliter-volumed and milliliter-volumed samples have been designed and implemented.      We have applied ultrasound to synchronize the interaction between large numbers of immune, natural killer cells, and cancer cells to study the cytotoxic response, on a single cell level. A heterogeneity was found among the natural killer cell population, i.e., some cells displayed high cytotoxic response while others were dormant. Furthermore, we have used temperature-controlled ultrasound to form up to 100, in parallel, solid cancer HepG2 tumors in a glass-silicon multi-well microplate. Next, we investigated the immune cells cytotoxic response against the solid tumors. We found a correlation between the number of immune cells compared to the size of the tumor and the cytotoxic outcome, i.e., if the tumor could be defeated.             Finally, the effect of high acoustic pressure amplitudes in the MPa-range on cell viability has been studied in a newly developed platform optimized for long-term stable temperature control, independent on the applied ultrasound power. Lastly, we present two applications of ultrasonic fluid mixing and lysis of cells. One platform is optimized for small microliter-sized volumes in plastic disposable chips and another is optimized for large milliliter-sized volumes in plastic test tubes. The latter platform has been implemented for clinical sputum sample solubilization and cell lysis for genomic DNA extraction for subsequent pathogen detection / Ultraljudsmanipulering har under de senaste tio åren mognat och utvecklats till ett verktyg med ett brett användningsområde. Idag kan man finna applikationer inom allt från cellbiologisk grundforskning till industri samt sjukvård. Ultraljudsmanipuleringens kontaktlösa natur gör det till en varsam metod för att manipulera celler, till exempel inom positionering, sortering och aggregering. När ultraljud med hög amplitud används kan värmeutvecklingen, som är oundviklig, bli ett problem. För att kunna säkerställa hög cellviabilitet krävs temperaturkontroll som kan hålla en fysiologisk, stabil temperatur på 37°C.      I denna avhandling presenterar vi tillämpningar av temperaturkontrollerad ultraljudsmanipulering i mikrometerstora anordningar fabricerade med väletablerade etsningstekniker.  Dessa anordningar är optimerade för att vara fullt kompatibla med högupplöst fluorescensmikroskopi.  Vi demonstrerar även ultraljudsmanipulering i centimeterstora anordningar optimerade för omrörning och blandning av vätskor samt lysering av celler. Två nya plattformar för ultraljudsmanipulering med inbyggd temperaturkontroll har utvecklats. Dessa två plattformar erbjuder ökad prestanda, flexibilitet samt även användarvänlighet. Utöver dessa plattformar har ytterligare två anordningar för lågfrekvent ultraljudssolubilisering och cellysering av mikroliter- och milliliterstora prover konstruerats.      I denna avhandling har vi tillämpat ultraljud för att synkronisera interaktionen mellan populationer utav immunceller (natural killer-celler) och cancerceller för att på cellnivå studera det cytotoxiska gensvaret. Vi fann en heterogenitet hos immuncellspopulationen. Det manifesterade sig i en fördelning av immuncellerna, från celler med stort cytotoxiskt gensvar till inaktiva immunceller. Vi har dessutom använt temperaturkontrollerad ultrasljudsmanipulering för att skapa solida cancertumörer utav HepG2-cancerceller, upp till 100 stycken parallellt, i en multihåls-mikrotiterplatta bestående av glas och kisel. Med hjälp av dessa tumörer har vi studerat det cytotoxiska gensvaret från immuncellerna. Vi fann att förhållandet mellan antalet immunceller och storleken på tumören bestämde utfallet, det vill säga om tumören kunde bekämpas.      Vi presenterar dessutom effekten utav högamplitudsultraljudsexponering av cancerceller i en plattform speciellt designad för höga tryckamplituder med implementerad ultraljudseffektsoberoende temperaturkontroll. Slutligen presenterar vi två tillämpningar av ultraljud för vätskeblandning och cellysering. Den första tillämpningen är anpassad för små volymer i plastchip för engångsbruk och den andra är optimerad för större volymer i plastprovrör. Den senare tillämpningen är speciellt framtagen för ultraljudssolubilisering och cellysering utav kliniska sputumprover för att möjliggöra DNA-extrahering för detektion av smittämnen. / <p>QC 20150522</p>

La localisation de source Distribuée Cohérente (DC) présente un défi du traitement d'antenne. Les contributions de cette thèse s’articulent principalement autour de trois aspects. Premièrement, un estimateur conjoint de l'angle, la distance, la dispersion et la forme de la source appelée JADSSE est proposé pour le cas champ proche. L’estimation d’un paramètre de forme de distribution de la dispersion permet d’éviter des erreurs de modèles sur l’a priori de la forme de la distribution. Deuxièmement, on généralise l'estimateur Decoupled DSPE en champ proche. Cette approche permet de découpler l'estimation de la Direction D’Arrivée (DDA) et de la distance de l'estimation de la dispersion. Afin de permettre l’estimation de la dispersion sans connaître a priori les formes de distribution, on propose le DADSSE qui consiste à estimer successivement la DDA, la distance et ensuite la dispersion et la forme de la distribution de la source. Troisièmement, on généralise le modèle DC avec une dispersion spatiale bidimensionnelle de la source ainsi que l’estimateur JADSSE. Deux approches sont proposées pour l’estimation de la puissance prenant en compte le modèle d’étalement des sources. Les méthodes proposées sont testées sur les données expérimentales de la soufflerie de Renault. Les résultats mettent en évidence des sources aéro-acoustiques proches et de faibles puissances. L’ensemble de ces travaux permet de fournir un outil pour une meilleure cartographie et caractérisation des sources aéro-acoustiques grâce à l’estimation de la position, l'étalement, la puissance et la forme. / Localization of Coherently Distributed (CD) source presents a challenge in the array signal processing. Our work motivates the localization of aero-acoustic source based on its spatial extension. This challenge is practically ignored in the literature of acoustic imaging field where many applications consist in mapping noisy source to reduce its contribution. The thesis presents the three following contributions. First, we propose a Joint Angle, Distance, Spread and Shape Estimator called JADSSE. The estimation of the so-called spread shape distribution parameter proposed by JADSSE avoids the modeling error due to the required a priori knowledge on the source shape when using classical estimators. Second, we expand the Decoupled DSPE to the near field. This method decouples the Direction of Arrival (DoA) and the range estimation from the spread estimation. Meanwhile, this method prevents the spread estimation for unknown shape distribution. Therefore, we propose the DADSSE to successively estimate the DOA, the range and then the spread and the shape distribution of the source. Third, we generalize the CD model and the JADSSE to consider the bi-dimensional spread of the source. Next, we propose two source power estimation approaches accounting the spatial spread of the source. The proposed methods are tested using a set of experimental data of the Renault wind tunnel application. Results show the presence of new aero-acoustic sources especially the overlapped ones with weak powers. We provide a tool to better map and characterize the aero-acoustic source by estimating the position, spread, power and shape.

Transmission Electron Microscopy (TEM) on light element materials and soft matters is problematic due to electron irradiation damage and low contrast. In this doctoral thesis techniques were developed to address some of those issues and successfully characterize these materials at high resolution. These techniques were demonstrated on graphene flakes, DNA/magnetic beads and a number of water containing biomaterials. The details of these studies are given below. A TEM based method was presented for thickness characterization of graphene flakes. For the thickness characterization, the dynamical theory of electron diffraction is used to obtain an analytical expression for the intensity of the transmitted electron beam as a function of thickness. From JEMS simulations (experiments) the absorption constant λ in a low symmetry orientation was found to be ~ 208 nm (225 ± 9 nm). When compared to standard techniques for thickness determination of graphene/graphite, the method has the advantage of being relatively simple, fast and requiring only the acquisition of bright-field (BF) images. Using the proposed method, it is possible to measure the thickness change due to one monolayer of graphene if the flake has uniform thickness over a larger area. A real-space TEM study on magnetic bead-DNA coil interaction was conducted and a statistical analysis of the number of beads attached to the DNA-coils was performed. The average number of beads per DNA coil was calculated around 6 and slightly above 2 for samples with 40 nm and 130 nm beads, respectively. These results are in good agreement with magnetic measurements. In addition, the TEM analysis supported an earlier hypothesis that 40 nm beads are preferably attached interior of the DNA-coils while 130 nm beads closer to the exterior of the coils. A focused ion-beam in-situ lift-out technique for hydrated biological specimens was developed for cryo-TEM. The technique was demonstrated on frozen Aspergillus niger spores which were frozen with liquid nitrogen to preserve their cellular structures. A thin lamella was prepared, lifted out and welded to a TEM grid. Once the lamella was thinned to electron transparency, the grid was cryogenically transferred to the TEM using a cryo-transfer bath. The structure of the cells was revealed by BF imaging. Also, a series of energy filtered images was acquired and C, N and Mn elemental maps were produced. Furthermore, 3 Å lattice fringes of the underlying Al support were successfully resolved by high resolution imaging, confirming that the technique has the potential to extract structural information down to the atomic scale. The experimental protocol is ready now to be employed on a large variety of samples e.g. soft/hard matter interfaces.