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The use of aplanatic solid immersion lenses (ASILs) made of high refractive index optical glasses provides a route to wide-field high-resolution optical microscopy. Structured illumination microscopy (SIM) can double the spatial bandwidth of a microscope to achieve high-resolution imaging. This research aims to investigate the combination of the ASILs and SIM in fluorescence microscopy, which we call structured illumination solid immersion fluorescence microscopy (SISIM), to pursue a microscopic system with very large numerical aperture and high lateral resolution. The first stage of the research shows the development of solid immersion fluorescence microscopy (SIF) employing an ASIL allows us to obtain a fluorescence microscope with effective numerical aperture of 1.85. The aberration issues, especially chromatic aberration, that need to be circumvented are analysed by both optical simulation and experimental verification. The near-field imaging property is also discussed and demonstrated. Then the SIM using a diffraction grating to generate structured illumination pattern via two-beam interference is developed. Finally, the SISIM system is constructed by combining the structured illumination with the SIF, and an effective numerical aperture of 3 has been obtained. Future developments of the SISIM system to make it achieve higher resolution and suit routine use are proposed. SISIM is a promising high-resolution microscopic technique with extensive potential applications in cell biology.

In this thesis for the first AFM has been employed for the high resolution imaging of a protein assembly. The DnaB-DnaG Helicase-Primase interaction in Bacillus is the key reaction that causes the switch from primase mode to polymerisation mode. This assembly was imaged using the AFM to a sub-molecular resolution revealing structural detail of the interaction. It is shown that the binding of the primase causes the structure of the helicase to switch from a hexamer to a trimer of dimers with one primase molecule bound to each dimer; also the existance of sub-populations with one and two primases bound suggests a sequential mode of binding. Recently crystallography data has been published that confirms the structural observations generated by AFM here. This is the first time that AFM and crystallography data have been used concurrently to solve the molecular structure of a protein assembly and it shows the potential application of AFM for sub-molecular resolution imaging of other protein assemblies. The role of DnaD in the Bacillus primosome is well established, however, its exact function was unknown. In this thesis AFM was applied to help solve this biomolecular problem, it revealed that DnaD has a pivotal role in early primosome assembly, opening up the DNA allowing other components of the cascade to bind. DnaD was shown to cause supercoiled DNA to adopt an open circular formation; this reaction was shown to be both reversible and universally applicable to all sequences of DNA. Comparisons are made between the role of DnaD and the roles of the histone-like proteins H-NS and HU. These experiments show that AFM can be applied to the imaging of proteins and their interactions with DNA and used to solve biomolecular problems that other techniques cannot solve. The design and implementation of a novel cryogenic AFM system for the imaging of biomolecules at subzero temperatures was executed. Preliminary results show that such a system has the potential to reduce the two main intrinsic effects limiting current AFM imaging; sample softness and thermal motion. The application of AFM in this thesis shows its strength as a tool in molecular biology not only for the high resolution imaging of proteins and protein assemblies but also as a technique that can be uniquely applied to solve biomolecular problems. This thesis also shows for the first time that AFM can be applied to generate sub-molecular resolution of protein assemblies. The strength of the AFM data when combined with crystallography data shows that AFM is a very powerful tool for the imaging of protein assemblies; it could even become the technique of choice

Transmission electron microscopy (TEM) is a powerful and versatile tool for investigating nanomaterials. In this thesis, various transmission electron microscopy techniques are used to study the chemical and structural features of different types of inorganic nanoparticles of well-defined morphologies as well as their assemblies. The synthesis of spherical and anisotropic nanoparticles (iron oxide nanocubes and other morphologies, gadolinium orthophosphate nanorods, tungsten oxide nanowires and nanorods, palladium nanospheres, and facetted iron-manganese oxides hybrid nanoparticles) using thermal decomposition of metal complex precursors in high-boiling point organic solvents and hydrothermal process are described in details. Electron diffraction tomography (3D EDT) is a recently developed technique that is used to investigate the 3D structure of crystalline materials. Reciprocal space volume reconstruction of 3D EDT data of thin WO3 nanowires assembled into nanorods revealed single crystal domains of hexagonal symmetry. Moreover, the use of 3D EDT enabled to identify and solve the structures of individual GdPO4 nanorods in a mixed phase powder. The use of 3D EDT was extended using small-angle diffraction mode to investigate the packing arrangements and defects in nanoparticle assemblies. A high concentration of planar defects found in different nanoparticle assemblies highlights the competition between the fcc and hcp arrangements during the assembly process. Iron-manganese oxides hybrid nanoparticles with different three-dimensional configurations, i.e. core|shell and asymmetric facetted dimers, were investigated using a combination of several electron microscopy techniques (HRTEM, SAED, STEM-HAADF, EFTEM, EELS). The growth of the facetted cubic MnO phase onto preformed Fe3O4 seed particles occurs preferentially along the Fe3O4 nanocube edges forming a well-oriented crystalline interface despite the lattice mismatch and defects. Atomic resolution monitoring of the structural changes in Mn3O4|Fe3O4 and Fe3O4|Mn3O4 core|shell nanoparticles induced by the electron beam revealed a strain relief mechanism at the interface involving inhomogeneous diffusion of cations and defects creation. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 4: Manuscript. Paper 5: Manuscript.</p>

Although many multi-component polymer systems are well characterised, the surface properties of polymers mixed with low surface energy additives have received little attention. In addition, the new branches of scanning probe microscopy that enable high resolution mapping and modification of surface charge distributions have been infrequently applied to polymer surfaces. The surface segregation of a fluorochemical additive directly from a polypropylene host matrix has been investigated by AFM and other surface analysis techniques. The level of surface enrichment was found to be governed by the temperature and duration of annealing. Further investigation revealed that the speed and extent of surface enrichment of the additive increases with polymer molecular weight. The effect of additive structure on surface segregation has also reported. A method of depositing charge onto polypropylene substrates from a high potential scanning AFM tip was developed. The relation between AFM tip- voltage and the level of charge deposited on the substrate suggested that a localised corona discharge was generated. AFM scanning parameters were found to effect the deposition of charge. The charging behavior of fluorochemical doped polypropylene surfaces was investigated on macroscopic scales using a scanning electrometer probe, and on microscopic scales using EFM. Fluorochemical domains at the surface have been found to preferentially accumulate both positive and negative charge. Surface charge distributions were found to become more uniform during annealing. Sub-micron particle capture by charged surfaces was investigated using EFM. In addition, spatially confined amine beads were deposited onto a patch of localised charge and subsequently functionalised to produce a metallic gold coating.

The genes of eukaryotes exist as DNA-RNA-protein complexes known as chromatin. The structure of chromatin fluctuates to allow controlled access to genetic information while maintaining its important packaging function. Recent improvements in optics, image acquisition electronics, and live imaging techniques, as well as the introduction of fluorescent fusion proteins, have made it possible to use fluorescence light microscopy to study the dynamic nature of chromatin compaction in cells. Here we report the application of advanced fluorescence microscopy to characterize the effects of transcription on chromatin compaction in living yeast cells. Repressor protein-GFP fusion proteins which recognize specific operator sequences were used to fluorescently tag specific gene loci, and an OMX fluorescence light microscope was then used to track their positions in three dimensions. It was determined that image acquisition with the OMX microscope is rapid enough to track fluorescently tagged genomic loci in live yeast cells in 3D, and that it does so with a root mean squared (RMS) measurement error of 162 nanometers (nm). It was also determined that the OMX microscope can distinguish between strains with fluorescent spots separated by 40 or 70 kb genomic distances. Additionally, it was found that chromatin compaction of a 15 kb gene driven by the Gal1 promoter is correlated with the carbon source on which the cells are fed, and that three different carbon sources produce three different transcription-dependent chromatin structures. Reversible changes in end-to-end distance of ~500 nm within two seconds were detected in the induced strain. These findings indicate that improvements in light microscopy enable chromatin to be studied in living cells on a scale not previously possible.

Over the past few decades, fluorescence microscopy has proven to become the most widely used imaging technique in the field of life sciences. Unfortunately, all classical optical microscopy techniques have one thing in common: their resolution is limited by the diffraction. Thankfully, due to the very strong interest, development of fluorescent microscopy techniques is very intense, with novel solutions surfacing repeatedly. The major breakthrough came with the appearance of super-resolution microscopy techniques, enabling imaging well below the diffraction barrier and opening the new era of nanoscopy. Among the fluorescent super-resolution techniques, Stimulated Emission Depletion (STED) microscopy has been particularly interesting, as it is a purely optical technique which does not require post image processing. STED microscopy has proven to resolve structures down to the molecular resolution. However, super-resolution microscopy is not a cure to all the problems and it also has its limits. What has shown to be particularly challenging, was the super-resolution imaging of thick samples. With increased thickness of biological structures, the aberrations increase and signal-to-noise (SNR) decreases. This becomes even more evident in the super-resolution imaging, as the nanoscopic techniques are especially sensitive to aberrations and low SNR. The aim of this work is to propose and develop a 3D STED microscope that can successfully image thick biological samples with nanoscopic resolution. In order to achieve that, adaptive optics (AO) has been employed for correcting the aberrations, using the indirect wavefront sensing approach. This thesis presents a custom built 3D STED microscope with the AO correction and the resulting images of thick samples with resolution beyond diffraction barrier. The developed STED microscope achieved the resolution of 60nm in lateral and 160nm in axial direction. What is more, it enabled super-resolution imaging of thick, aberrating samples. HeLa, RPE-1 cells and dopaminergic neuron differentiated from human IPS cells were imaged using the microscope. The results shown in this thesis present 3D STED imaging of thick biological samples and, what is particularly worth to highlight, 3D STED imaging at the 80μm depth, where the excitation and depletion beams have to propagate through the thick layer of tissue. 3D STED images at such depth has not been reported up to date.

Este trabalho tem como objetivo principal, o estudo de técnicas de microscopia para a caracterização estrutural de semicondutores, visando o desenvolvimento das técnicas de preparação de amostras, visto que a caracterização estrutural é de suma importância para a obtenção de melhores resultados no processo de produção de filmes de semicondutores do grupo III-V. Dentre as técnicas mais utilizadas na caracterização estrutural, destacam-se as técnicas de microscopia eletrônica de varredura e de transmissão, juntamente com a microscopia de força atômica. Foram utilizadas amostras semicondutoras de InGaAs/GaAs e InAs/GaAs, crescidas pela técnica de MBE (epitaxia por feixe molecular), contendo pontos quânticos, estruturas estas ricas em detalhes. Tais amostras foram preparadas e caracterizadas em cada uma das técnicas em estudo. A microscopia de varredura e de força atômica apresentam fácil preparação. Os resultados obtidos, porém mostram que a técnica de microscopia eletrônica de varredura não oferece resolução suficiente para visualização das heteroestruturas; já a técnica de microscopia de força atômica mostra resultados excelentes da topografia dos pontos quânticos. Para a microscopia de transmissão a preparação de amostras mostra-se muito difícil e demorada, entretanto, o resultado obtido foi muito satisfatório. O processo de preparação passa por etapas de clivagem, \"dimpling\" e \"ion milling\". As imagens obtidas revelam com clareza a estrutura de pontos quântico. Com o estudo realizado, foi possível determinar as principais características de cada técnica, assim como determinar uma metodologia que pode vir a ser aplicada a outros tipos de heteroestruturas semicondutoras / This work has as main objective, the study of microscopy techniques for structural characterization of semiconductors and the development of the techniques of sample preparation, because the structural characterization is of highest importance for the obtaining of better results in the process of production of semiconductors thin films. The techniques more used in the structural characterization, are the techniques of electronic microscopy (Scanning and Transmission), together with the Atomic Force Microscopy. Samples of InGaAs/GaAs and InAs/GaAs were used, grown by the technique of MBE (Molecular Beam Epitaxy), with quantum dots, structures these rich ones in details. Such samples were prepared and characterized in each one of the techniques in study. The Scanning Microscopy and Atomic Force present easy preparation. The obtained results even so they show that the technique of Scanning Microscopy doesn\'t offer enough resolution for visualization of the heteroestructures; already the technique of Atomic Force shows excellent results of the topography of the quantum dots. For the Transmission Microscopy the preparation of samples is shown very difficult and delayed, however, the obtained result was very satisfactory. The preparation process goes by cutting stages, dimpling and ion milling. The obtained images reveal with clarity the quantum structure of points. With the accomplished study, it was possible to determine the main characteristics of each technique, as well as determining a methodology that can come to be applied to the other types of semiconductors heterostructures

Dans ce travail de thèse, nous développons des protocoles d'imagerie optique pour l'observation des nanoparticules sur des coupes de tissus afin de relier leur localisation et leur «comportement» à l'environnement biologique, en particulier son éventuel état pathologique. Nous avons synthétisé des agents de contraste bimodaux, sous forme de nanoparticules -NP- visibles en résonance magnétique et en optique, à base d'oxydes de fer et de zinc (Zn(Fe)O) avec une nouvelle méthode de polyol azéotropique dans des solvants glycoliques (DEG et PG). L'élimination de l'eau à l'aide de l'appareil Dean-Stark est une nouvelle stratégie pour la synthèse de NP dans une solution de polyol, avec un rendement élevé et produisant des particules de petite taille. Les NP les plus visibles, selon leur contraste IRM, ont été revêtus de carboxyméthyl pullulane, de polyéthylène glycol, de carboxyméthyl dextrane et de fucoïdane, ce dernier étant un polysaccharide capable de se lier spécifiquement à la paroi vasculaire. Les NPs montrent de bonnes propriétés magnétiques et optiques à température ambiante. Les NP recouvertes ont été injectées dans un modèle de rat d’athérothrombose pour localiser le thrombus par IRM avant sacrifice et collecte des tissus pour étude des coupes histologiques par microscopie optique. La différence entre les images IRM avant et après l'injection de fucoïdane-NPs et de CMD-NPs est claire. Les résultats montrent que les NP recouvertes de fucoïdane sont liées au thrombus. Certains types de microscopies, tels que la microscopie de fluorescence, la microscopie en champ sombre, la microscopie hyperspectrale à champ sombre et la microscopie interférentielle à champ sombre ont été développés pour la détection des NPs en milieu liquide et dans les tissus. En analysant le spectre de chaque pixel et en le comparant au spectre des matériaux de référence, la microscopie hyperspectrale peut détecter la présence de NPs sur des coupes de tissus, les localiser, les identifier et les caractériser. Zn(Fe)O NPs constituerait donc un agent de contraste bimodal potentiel pour l’IRM et l’imagerie optique. Cependant, bien que de nombreux outils optiques avancés aient été développés, nous avons constaté qu'il est toujours difficile d'identifier de manière fiable les NP dans le tissu. / In this thesis work, we develop optical imaging protocols for the observation of then anoparticles on tissue slices in order to further link their localization and their “behaviour” to the biological pathological environment. Bimodal zinc and iron oxide-based MRI/optical nanoparticle contrast agents (Zn(Fe)O) have been synthesised with a novel azeotropicpolyol method in glycol solvents (DEG and PG). The most potent NPs, as regard to their MR contrast power, have been coated with carboxymethyl pullulan, polyethylene glycol,carboxymethyl dextran (CMD) and fucoidan, the latter being a polysaccharide able to specifically bind to the vascular wall. The coated NPs were injected into rat to locate atherothrombosis by MRI. Then the histological slices of harvested diseased tissue were imaged with our homemade optical microscope. Water removal using Dean-Stark apparatus is a novel strategy for the synthesis of NPs in polyol solution with high yield and small size.The NPs show the good magnetic and optical properties at room temperature. The coated nanoparticles were injected into an atherothrombotic rat model to locate the thrombus by MRI prior to sacrifice of the animals and tissue collection for histological study by optical microscopy. The difference of MRI images between before and after injection with Fucoidan-NPs and CMD-NPs is clear. The results indicated that fucoidan-NPs are linked to the thrombus. Some type of microscopies, such as fluorescent microscopy, dark field microscopy, hyperspectral dark field microscopy and interference dark field microscopy have been developed for the detection of NPs in liquid medium and in the histological tissue. By analyzing the spectrum of every pixel and comparing to the spectrum of reference materials, hyperspectral microscopy can detect the presence of nanomaterial on exposed tissue slices, locate, identify, and characterize them. Zn(Fe)O NPs would therefore constitute a potential bimodal contrast agent for MRI and optical imaging. Although many advance optical tools have been developed, but we found it is still a challenge to identify reliably the NPs in the tissue.