Transmission electron microscopy (TEM) is an important tool for the characterization of materials as it can provide clear understanding of the relationship between structure, property and composition of nanomaterials. For this, the in-situ TEM analysis is performed and requires specially manufactured sample holders. In particular, those designed to carry out electrical biasing can be used to understand not just the I-V characteristics but also the failure mechanism, structure-property relationship, Joule heating dynamics, electromigration, field emission properties, etc. at the nanoscale. The platforms holding the sample in most modern in-situ TEM holders rely on an insulating ceramic membrane which needs to be (almost) transparent to the imaging electron beam. Electrodes are defined through lithography and patterned on this membrane. Unfortunately, the presence of this membranes introduces several limitations such as electrostatic charging, reduction of image contrast and poor mechanical stability. To circumvent this issue it is necessary to fabricate a novel type of sample platform which does not rely on the presence of a membrane. In this work, novel membraneless sample-holding platforms were designed and manufactured using advanced microfabrication methods and tools. Besides fitting into an array of analytical tools, the novel platforms (or “chips”) can be subjected to thermal and/or chemical processing without compromising their function or structure. To test these, the electrical response of one-, two- and zero-dimensional nanoparticles were studied. Firstly, we investigated current-induced modifications in silver nanowires and expandable graphite flakes and studied various phenomenon involved. Along with these, corresponding ex-situ studies were also performed. Next, graphene oxide was explored as an alternative support platform for in-situ TEM. We successfully achieved temperature as high as 2000o C by Joule heating of graphene oxide. Furthermore, this graphene oxide platform was used as a heater and chemical processing substrate for investigating thermal stability and synthesis of inorganic nanoparticles, respectively.

Nanofluidics is an emerging field with many science and engineering applications. The physics of material transport through nanochannels are of interest in filtration, sensing, device miniaturization, and biomimetics. To address such ambitions with nanofluidic tools will require advancements in our understanding and control over nanofluidic systems. This work contributes to electrokinetic phenomena, characterization techniques, and applications in nanofluidics. Ion transport data through nanopipettes are used to validate a finite element model for nonlinear electrokinetic flows. With the model, we conclude that asymmetric surfaces induce fluid vortices and provide insight into supporting mathematical techniques. We then establish nanobubble-plugged nanopipettes as promising ionic devices due to the electrokinetic effects of three-phase interfaces. Using cryogenic transmission electron microscopy, ion current measurements, and extensive physical modeling, we conclude that nanobubble plugs are metastable, slow-growing, and induce strong current rectification and enhancement. All these insights let us study microbial surfaces using electrokinetic phenomena detected by a scanned nanopipette. Over immobilized Pseudomonas aerugonsa cells and Δphz-type biofilms, we detect topographic and surface charge properties due to voltage-dependent signals through a scanned nanopipette probe. Our efforts establish a fast hopping probe scanning ion conductance microscopy technique for long-range surface charge detection. Finally, we use an integrated carbon nanotube channel to demonstrate how solid-state charge can drive electrokinetic flows through Coulomb drag coupling.

Les plasmons de surface (SP) sont des ondes électromagnétiques se propageant à l'interface entre deux milieux, typiquement un métal et un diélectrique. Les plasmons de surface ont la capacité de confiner le champ électromagnétique dans de très petite région de l’espace, typiquement quelques nanomètres, c’est à dire bien en dessous de la limite de diffraction de la lumière. Une conséquence de ce confinement sub-longueur d’onde de la lumière est que leur observation nécessite une résolution spatiale nanométrique - ce qui exclut l’utilisation de techniques optiques standard. Néanmoins, le microscope électronique en transmission à balayage (STEM) est un outil particulièrement adapté à l'étude des plasmons de surface car il emploie des électrons rapides ayant une longueur d’onde typique comprise entre 1 et 10 picomètres. Ainsi, durant la dernière décennie, les spectroscopies électroniques appliquées à la nano-optique se sont fortement développées, parmi elle comptent : la spectroscopie de perte d'énergie électronique (EELS), la spectroscopie cathodoluminescence (CL) ou l'interférométrie de Hanbury Brown et Twiss (HBT) appliquée à la CL. Dans cette thèse, j’ai exploré différents problèmes ouverts de la plasmonique et de la nano-optique dans le cadre particulier de la microscopie électronique. Dans le chapitre 3, je présente un formalisme prenant en compte à la fois la nature quantique et relativiste des expériences d’EELS en faisant appel notamment à des éléments de théorie quantique des champs. Dans le chapitre 4, nous démontrons que la réalisation d’une expérience d’EELS avec de tels faisceaux permet de mesurer des propriétés jusqu’alors inatteignable à l’échelle du nanomètre telle que la phase des plasmons, leurs chiralité optique voire même leur longueur de cohérence. Dans le chapitre 5, je présente plusieurs résultats théoriques et expérimentaux concernant des expériences de couplage. En particulier, j’étudie le phénomène contre-intuitif d’auto-hybridation qui est une conséquence de la nature non-hermitienne du problème aux valeurs propres associé aux résonances de plasmon et établit une analogie avec les systèmes quantiques ouverts. Enfin, au chapitre 6, je discute des récentes mesures de phonon réalisées dans un STEM grâce au développement de monochromateur électroniques. / Surface plasmons (SP) are electromagnetic waves propagating at the interface between two media typically a metal and a dielectric. SPs can confine electromagnetic fields in very short volumes (typically one to few nanometers), well below the light diffraction limit. This property has a tremendous number of applications ranging from fundamental physics (e.g. quantum optics) to applications (e.g. cancer therapy). However, the price to pay is that SPs suffer from huge ohmic losses in the metal which leads to very short lifetimes (typically few femtoseconds). Theoretically, this presence of dissipation dramatically hardens the theoretical description of SPs. Another consequence of the sub-wavelength confinement of light associated with SPs is that their observation requires a nanometric resolution - which excludes the use of standard optical techniques. Yet, the scanning transmission electron microscope (STEM) is a particularly suitable tool to study SPs as it employs fast electrons with typical wavelength from 1 to 10 picometers. Thus, the last decade has seen the tremendous development of electron-based spectroscopies applied to nano-optics such as electron energy loss spectroscopy (EELS), cathodoluminescence spectroscopy (CL) or STEM- Hanbury Brown and Twiss interferometry (HBT). In this thesis, I explored different open problems of plasmonics and nano-optics under the scope of electron microscopy and spectroscopies. In chapter 3, I develop a formalism taking into account both the quantum and relativistic nature of EELS experiments using elements of quantum field theory. In chapter 4, I apply the latter formalism to the case of EELS measurements of SPs using electrons with shaped phase. In chapter 5, I give several theoretical and experimental results on coupling experiments involving SPs. Particularly, I demonstrate a counterintuitive type of coupling, the so-called self- hybridization which is a consequence of the non-Hermitian nature of the LSP eigenproblem and draw analogy with open quantum system. Finally, in chapter 6, I discuss the recent result on vibrational EELS in monochromated STEM.

Hliníková slitina AA8079 připravena plynulým odléváním mezi válce je běžně používána pro výrobu tenké potravinářské fólie. Vzhledem k použité metodě odlévání a složení slitiny je struktura litého materiálu značně nehomogenní a obsahuje intermetalické fáze, které se shlukují v eutektických koloniích. Litý materiál tak vyžaduje tepelné zpracování před dalšími kroky výroby. Mikrostruktura materiálu po homogenizačních žíháních na různých teplotách je rozdílná, což ovlivňuje další zpracování. Po homogenizaci je materiál válcován což způsobuje snížení tažnosti. Rekrystalizační žíhání je vyžadováno po zválcování na střední tloušťku před finálním válcování, aby byla tažnost obnovena. Chování materiálu během rekrystalizačního žíhání je ovlivněno přítomnými fázemi, jejich velikostí a rozdělením. Byl studován vliv mikrostruktury po homogenizaci na rozdílných teplotách na rekrystalizaci, byla vyhodnocena kinetika rekrystalizace a byly identifikovány dva hlavní mechanismy ovlivňující rekrystalizaci - částicemi stimulovaná nukleace a Zenerův tlak.

Dental amalgams have been used for centuries as major restorative materials for decaying teeth. Amalgams are prepared by mixing alloy particles which contain Ag, Sn, and Cu as the major constituent elements with liquid Hg. The study of microstructure is essential in understanding the setting reactions and improving the properties of amalgams. Until the work reported in this dissertation, optical microscopy (OM), scanning electron microscopy (SEM), and x-ray diffractometry (XRD) were used commonly to analyze amalgam microstructures. No previous systematic transmission electron microscopy (TEM) study has been performed due to sample preparation difficulties and composite structure of dental amalgams. The goal of this research was to carry out detailed microstructural and compositional studies of dental amalgams. This was accomplished using the enhanced spatial resolution of the TEM and its associated microanalytical techniques, namely, scanning transmission electron microscopy (STEM), x-ray energy dispersive spectroscopy (XEDS) and micro-microdiffraction (μμD). A new method was developed for thinning amalgam samples to electron transparency using the "wedge technique." Velvalloy, a low-Cu amalgam, and Tytin, a high-Cu amalgam, were the two amalgams characterized. Velvalloy is composed of a Ag₂Hg₃ (γ₁)/HgSn₇₋₉ (γ₂) matrix surrounding unreacted Ag₃Sn (γ) particles. In addition, hitherto uncharacterized reaction layers between Ag₃Sn(γ)/Ag₂Hg₃ (γ₂) and Ag₂Hg₃ (γ₁)/HgSn₇₋₉ (γ₂) were observed and analyzed. An Ag-Hg-Sn (β₁) phase was clearly identified for the first time. In Tytin, the matrix consists of Ag₂Hg₃ (γ₁) grains. Fine precipitates of Cu₆Sn₅ (η') are embedded inside the γ₁ and at the grain boundaries. These precipitates are responsible for the improved creep resistance of Tytin compared to Velvalloy. The additional Cu has completely eliminated the γ₂ phase which is the weakest component of amalgams. Ag-Hg-Sn (β₁) and large grains of Cu₆Sn₅ (η') are found adjacent to the unreacted alloy particles. Tytin alloy particles contain Cu₃Sn (ε) precipitates in a matrix of Ag₃Sn (γ) and Ag₄Sn (β). SEM was used to correlate the TEM findings in the context of the general microstructure. The results are in good agreement with those published in the literature. The microstructural details reported here, many of which were not previously available, will help provide insight into the deformation mechanisms of dental amalgams.

Philosophiae Doctor - PhD / The blood-brain barrier (BBB) is a restrictive interface located between the blood circulation and the central nervous system (CNS), regulating the homeostatic environment of the neuronal milieu, by controlling the permeability of the cerebrovasculature. Currently, we cannot fully comprehend the regulatory features and the complexity of BBB morphology to allow for intervention clinically. The thesis consists of four publications. The methodology paper proposes a novel experimental design to visualize the morphological architecture of immortalized mouse brain endothelial cell lines (bEnd3/bEnd5). The brain endothelial cells (BECs) were grown on cellulose matrices and fixed in 2.5 % glutaraldehyde in preparation for visualization of the paracellular (PC) spaces between adjacent BECs, employing high-resolution electron microscopy (HREM), with vested interest in the morphological profile of the developing BEC.

While much is known about the attachment of the chlamydiae to the host cell and intracellular events during the developmental cycle, little is known about the mechanism(s) by which elementary bodies exit the cell. In this report, we use the guinea-pig conjunctival model of Chlamydia caviae infection to present in vivo ultrastructural evidence supporting two mechanisms for release of chlamydiae from the mucosal epithelia. Four days after infection, histopathologic observation shows an intense infiltration of polymorphonuclear leukocytes (PMN) in the conjunctival epithelium. Using transmission electron microscopy, a gradient-directed PMN response to chlamydiae-infected epithelial cells was observed. As PMN infiltration intensifies, epithelial hemidesmosome/integrin/focal adhesion adherence with the basal lamina is disconnected and PMNs literally lift off and release infected superficial epithelia from the mucosa. Many of these infected cells appear to be healthy with intact microvilli, nuclei, and mitochondria. While lysis of some infected cells occurs with release of chlamydiae into the extracellular surface milieu, the majority of infected cells are pushed off the epithelium. We propose that PMNs play an active role in detaching infected cells from the epithelium and that these infected cells eventually die releasing organisms but, in the process, move to new tissue sites via fluid dynamics.

La grande majorité des bactériophages connus ont un virion équipé d'une queue permettant la reconnaissance de l'hôte, la perforation de l'enveloppe bactérienne et l'éjection du matériel génétique viral directement dans le cytoplasme de la bactérie. La famille des Siphoviridae représente 60% des phages caudés et est caractérisée par une queue longue et non-contractile. Le tube de la queue est formé par un empilement de protéine majeur de tube (TTP) polymérisé autour de la protéine vernier (TMP). L'extrémité distale de la queue est équipé d'un complexe de protéine dans lequel se trouve les protéines de liaison au récepteur (RBP). La séquence d'évènement permettant l'éjection de l'ADN et l'infection est encore mal décrite.Au cours de cette thèse, la structure de pb6, la TTP du phage T5 qui s'assemble de façon non-canonique en trimères, a été résolue par cristallographie à une résolution de 2,2 Å. L'analyse de cette structure confirme cependant une homologie structurale de pb6 avec les autres TTPs et avec des protéines bactériennes du système de sécrétion de type VI et des pyocines R. Une étude RMN comparant pb6 dans ses états de monomère et de tube polymérisé est en cours et permettra à terme une description très fine de cet assemblage.De plus, les structures du complexe distal de queue et du tube de la queue (tube de pb6) ont été résolue des résolutions intermédiaires avant et après interaction avec le récepteur bactérien. Ces structures obtenues par cryo-microscopie électronique révèle une absence de changements structuraux au niveau du tube, en contradiction avec le modèle jusque là proposé que la TTP transmettait l'information de fixation du récepteur à la capside.Bien qu'à un stade préliminaire, les reconstructions du complexe distal sont très informatives sur les rôles de protéines pb2 et pb4.L'ensemble de ses données ainsi que des expériences biochimique et la comparaison avec d'autres systèmes bien décrits dans la littérature permet de proposer un nouveau modèle pour les premières étapes de l'infection des Siphoviridae. Ce modèle a également un intérêt pour l'étude du mécanisme d'autres familles de virus (Myoviridae). Les différences, similarité et parenté d'éléments de la queue du phage T5 avec d'autres systèmes de perforation de membrane sont discutés. / The vast majority (96%) of bacteriophages possess a tail that allows host cell recognition, cell wall perforation and safe viral DNA channelling from the capsid to the cytoplasm of the bacterium. Siphoviridae is a familly representing 60% of all tailed phages characterized by a long flexible tail. The tail tube is formed by stacks of hexamers of the tail tube protein (TTP) polymerised around the tape measure protein (TMP). At the distal end of the tail, the tail tip complex harbours the receptor binding proteins (RBP). For these phages, little is known on the mechanism that triggers DNA ejection after binding to the host.We report the crystal structure at 2.2 Å resolution of pb6, an unusual trimeric TTP, of siphophage T5. Structure analysis however confirms the homology of pb6 with all TTPs, related tube proteins of bacterial puncturing devices (type VI secretion system and R-pyocin) and procapsid proteases. We fit this structure into the cryo-electron microscopy map of the tail tube determined at 6 Å resolution. Comparing the structure of the tail tube before and after interaction with the host receptor, we show that unlike previously proposed, the host binding information is not propagated to the capsid by the tail tube, as the two structures, at that resolution, are identical. An ambitious NMR comparative study of the TTP in its monomeric and tube form is underway to further describe this assembly.Moreover, the structures of the tail tip complex prior and after interaction with the bacterial receptor were solved at intermediate resolution. These structures reveal interesting conformationnal changes triggered by the RBP binding to the bacterial receptor. Those rearrangements are the first to occur after phage irreversible binding to its host and they induce the TMP ejection, the capsid opening, the enveloppe perforation and ultimately DNA channeling to the host cytoplasm.Together with biochemical data and comparison with other known system in the litterature we are able to propose a model for Siphoviridae very first steps of infection. These findings might be of interest for the mechanism of other viral familly (notably Myoviridae) and similarity with other membrane perforating systems is discussed.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第19180号 / 人健博第28号 / 新制||人健||3(附属図書館) / 32172 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 高桑 徹也, 教授 市橋 則明, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM