In this thesis optical microscopy techniques are used to consider aspects of viral and bacterial infections. In part 1, the physical effects of cytomegalovirus on lipid droplet dynamics in live cells are studied; in part 2, the effects of an antimicrobial peptide on the fluidity of model bacterial membranes are studied. The optical microscopy techniques used to study the effects of murine-cytomegalovirus (mCMV) on lipid droplets in live NIH/3T3 fibroblast cells in real-time are coherent anti- Stokes Raman scattering (CARS), two-photon fluorescence (TPF) and differential interference contrast (DIC) microscopies. Using a multimodal CARS and TPF imaging system, the infection process was monitored by imaging the TPF signal caused by a green fluorescent protein (GFP)-expressing strain of mCMV, where the amount of TPF detected allowed distinct stages of infection to be identified. Meanwhile, changes to lipid droplet configuration were observed using CARS microscopy. Quantitative analysis of lipid droplet numbers and size distributions were obtained from live cells, which showed significant perturbations as the infection progressed. The CARS and TPF images were acquired simultaneously and the experimental design allowed incorporation of an environmental control chamber to maintain cell viability. Photodamage to the live cell population was also assessed, which indicated that alternative imaging methods must be adopted to study a single cell over longer periods of time. To this end, DIC microscopy was used to study the lipid droplet dynamics, allowing lipid droplet motion to be tracked during infection. In this way, the effects of viral infection on the mobility and arrangement of the lipid droplets were analysed and quantified. It was found that the diffusion coefficient of the lipid droplets undergoing diffusive motion increased, and the droplets undergoing directed motion tended to move at greater speeds as the infection progressed. In addition, the droplets were found to accumulate and cluster in infected cells. The second part of this thesis presents a study on the effects of an antimicrobial peptide on model bacterial membranes. Giant unilamellar vesicles (GUVs) were produced as a simple model of E. Coli membrane using a 3:1 mixture of DPPC and POPG lipids. Incorporating Laurdan fluorescent dye into the lipid membrane of the GUVs allowed the membrane fluidity to be probed and visualised using TPF microscopy, whereby the fluidity was quantified by determining the general polarization (GP) values. Studying GUVs comprising single lipid and mixed lipid compositions over a temperature range from 25 C to 55 C enabled the lipid phase bands to be identified on the basis of GP value as gel phase and liquid crystalline phase. As such, the changes in lipid phase as a result of interaction with AMP were quantified, and phase domains were identified. It was found that the amount of liquid crystalline phase domains increased significantly as a result of AMP interaction.

Since its development, the scanning transmission electron microscope has rapidly found uses right across the material sciences. Its use of a finely focussed electron probe rastered across samples offers the microscopist a variety of imaging and spectroscopy signals in parallel. These signals are individually intuitive to interpret, and collectively immensely powerful as a research tool. Unsurprisingly then, much attention is concentrated on the optical quality of the electron probes used. The introduction of multi-pole hardware to correct optical distortions has yielded a step-change in imaging performance; now with spherical and other remnant aberrations greatly reduced, larger probe forming apertures are suddenly available. Probes formed by such apertures exhibit a much improved and routinely sub-Angstrom diffraction-limited resolution, as well as a greatly increased probe current for spectroscopic work. The superb fineness of the electron beams and enormous magnifications now achievable make the STEM instrument one of the most sensitive scientific instruments developed by man, and this thesis will deal with two core issues that suddenly become important in this new aberration-corrected era. With this new found sensitivity comes the risk of imaging-distortion from outside influences such as acoustic or mechanical vibrations. These can corrupt the data in an unsatisfactory manner and counter the natural interpretability of the technique. Methods to identify and diagnose this distortion will be discussed, and a new technique developed to restore the corrupted data presented. Secondly, the subtleties of probe-shape in the multi-pole corrected STEM are extensively evaluated via simulation, with the contrast-transfer capabilities across defocus explored in detail. From this investigation a new technique of STEM focal-series reconstruction (FSR) is developed to compensate for the small remnant aberrations that still persist – recovering the sample object function free from any optical distortion. In both cases the methodologies were developed into automated computer codes and example restorations from the two techniques are shown (separately, although in principal the scan-corrected output is compatible with FSR). The performance of these results has been quantified with respect to several factors including; image resolution, signal-noise ratio, sample-drift, low frequency instability, and quantitative image intensity. The techniques developed are offered as practical tools for the microscopist wishing to push the performance of their instrument just that little bit further.

Aberration corrected transmission electron microscopy is a powerful tool for the structural characterisation of materials at the atomic scale, but the passage of high velocity electrons through the material can often induce structural changes via the transfer of large amounts of energy from the beam. The work in this thesis theoretically considers the nature of this transfer of energy and its impact upon the material being studied. The computational modelling of molecular species encapsulated inside carbon nanotubes and their response to electron irradiation is compared to results from TEM experiments, and used to explain the experimental observations. The high rate of destruction of C-H bonds under the beam is quantified, and its implications for TEM studies of organic materials considered. An effective solution for mitigating this rate of destruction is found, applied to a model system, and then confirmed experimentally. Using the considerations of stability under the beam, two experimentally witnessed reactions are investigated in detail, and careful comparison to intermediate structures observed in TEM allows full reaction mechanisms to be proposed. The dynamic motion of atomic defects in irradiated graphene is considered with the aid of a large library of experimental TEM images. A novel defect structure is observed, and is seen to undergo structural rearrangements on a quicker time-scale than accessible to TEM imaging. This species enables the very quick migration of defect structures across the graphene lattice, and is attributed to a trivacancy structure. The rates of beam induced reactions are considered in the framework of chemical kinetics, and a method for extracting kinetic parameters of a reaction from the statistics of a large number of TEM observations of it occurring is developed. This is used to obtain the first cross-sections for the formation and healing of the irradiation induced Stone-Wales bond rotation, and the first experimental activation energy for its healing. The latter agrees well with a theoretically predicted mechanism of catalysis, while the former demonstrates that the widely assumed process of direct knock-on damage cannot be responsible for the beam induced reaction. An alternative mechanism is proposed, resulting from the electronic excitation of the defect.

Scanning tunnelling microscopy (STM) and atomic force microscopy (AFM) can produce images of molecules with extremely high resolution. However, Claims that dynamic force microscopy has the capability to resolve intermolecular bonds in real space continue to be vigorously debated. Several studies have now shown that tip flexibility, especially at very close tip-sample separations, is responsible for the striking intra- and intermolecular resolution observed with various scanning probe microscopy techniques. The apparent intermolecular features can be observed with dynamic force microscopy even when no bonding interaction is present, suggesting that such features are in fact an artefact and cannot be interpreted as a real-space image of an intermolecular bond. We have studied the interaction between fullerene (C60) molecules using a sum of pairwise Lennard-Jones (12-6) potentials, and investigated how flexibility in the tip can produce a bond like feature between the molecules in a C60 island where there is no chemical bond present except the weak van der Waals force. We also investigate how the potential between the molecules is dependent on their relative orientations. For a given configuration of the tip and the sample molecules, our results allow us to predict the form of the intermolecular potential that would be observed using non contact atomic force microscopy (NC-AFM). Our study on the Si(111)-(7x7) reconstructed surface using the same model provides a better understating on the origin of ‘sub-atomic’ contrast observed in experiment suggesting that the contrast can arise from a flexible tip exploring an asymmetric potential created due to the positioning of the surrounding surface atoms. We have also simulated NC-AFM images of 2D bi-isonicotinic acid lattice using the same model. The geometry of the lattice have been optimized using DFT before simulating AFM images. Simulation results are in a good agreement with the experiment. The theoretical work is accompanied by a variety of experimental results obtained by the group of Prof Philip Moriarty at the University of Nottingham.

Scanning probe microscopy (SPM) nanolithography has been found to be a powerful and low-cost approach for sub-100 nm patterning. In this thesis, the possibility of using a state-of-the-art SPM system to controllably deposit nanoparticles on patterned Si substrates with high positional control has been explored. These nanoparticles have a range of interesting properties and have been characterised by electron microscopy and scanning probe microscopy. The influence of different deposition parameters on the nanoparticle properties was studied. Contact mode atomic force microscopy (AFM)-based local oxidation nanolithography (LON) was used to oxidise sample surfaces. Two different substrates were studied which were native oxide silicon (Si) and molybdenum (Mo). A number of factors that influence the height and width of the oxide features were investigated in order to achieve the optimal oxidation efficiency. The height and width of the oxide structures were found to be strongly dependent on the applied voltage and scan speed. The tunneling AFM (TUNA) technique was used to measure the ultralow currents flowing between the tip and the sample during the oxidation process. It was found that a threshold voltage for our oxidation experiments was -4.0 ± 1.6 V applied to the tip when fabricating geometric patterns as well as 2.9 ± 1.6 V and 2.8 ± 2.2 V applied to the substrate for nanodot fabrication. In addition, comparisons of nanodot-array patterns produced with different AFM tips were studied. The influence of applied voltage, type of AFM tip and substrate, humidity and ramping time has been studied for dot formation providing a comparison between native oxide Si and Mo surfaces. The nanodot sizes were found to be clearly dependent on the applied voltage, type of substrate, relative humidity and ramping time. Dip-pen nanolithography (DPN) was used to study a direct deposition strategy for gold (Au) nanodot fabrication on a native oxide Si substrate. In this process, hydrogen tetrachloroaurate (HAuCl4) molecules were deposited onto the substrate via a molecular diffusion process, in the absence of electrochemical reactions. This approach allowed for the generation of Au dots on the SiO2 substrate without the need for surface modification or additional electrode structures. The dependence of the size of the Au dots on different „scanning coating‟ (SC) times of AFM tips was studied. A thermal annealing process was used to decompose the generated HAuCl4 molecular dots to leave Au (0) metal dots. A stereomicroscope has been used for preliminary observation of different steps of Au deposition treatments. A scanning electron microscope (SEM) was used to characterise the SC AFM tips both before and after the DPN process. SEM energy-dispersive X-ray spectroscopy (EDS) has provided information about the elemental content of deposited particles for different annealing temperatures. Fountain-pen nanolithography (FPN) has also been used to study nanowriting of HAuCl4 salt and a variety of solvents on a native oxide Si surface. In this technique, a nanopipette was mounted within an AFM to deliver appropriate solutions to the silica substrate. We found that an aqueous Au salt solution was the most suitable ink for depositing gold using the FPN technique. In the case of solvents alone, ethanol and toluene were achieved with depositing onto a SiO2 substrate using the FPN technique.

Nanotechnology has revolutionised humanity's capability in building microscopic systems by manipulating materials on a molecular and atomic scale. Nan-osystems are becoming increasingly smaller and more complex from the chemical perspective which increases the demand for microscopic characterisation techniques. Among others, transmission electron microscopy (TEM) is an indispensable tool that is increasingly used to study the structures of nanosystems down to the molecular and atomic scale. However, despite the effectivity of this tool, it can only provide 2-dimensional projection (shadow) images of the 3D structure, leaving the 3-dimensional information hidden which can lead to incomplete or erroneous characterization. One very promising inspection method is Electron Tomography (ET), which is rapidly becoming an important tool to explore the 3D nano-world. ET provides (sub-)nanometer resolution in all three dimensions of the sample under investigation. However, the fidelity of the ET tomogram that is achieved by current ET reconstruction procedures remains a major challenge. This thesis addresses the assessment and advancement of electron tomographic methods to enable high-fidelity three-dimensional investigations. A quality assessment investigation was conducted to provide a quality quantitative analysis of the main established ET reconstruction algorithms and to study the influence of the experimental conditions on the quality of the reconstructed ET tomogram. Regular shaped nanoparticles were used as a ground-truth for this study. It is concluded that the fidelity of the post-reconstruction quantitative analysis and segmentation is limited, mainly by the fidelity of the reconstructed ET tomogram. This motivates the development of an improved tomographic reconstruction process. In this thesis, a novel ET method was proposed, named dictionary learning electron tomography (DLET). DLET is based on the recent mathematical theorem of compressed sensing (CS) which employs the sparsity of ET tomograms to enable accurate reconstruction from undersampled (S)TEM tilt series. DLET learns the sparsifying transform (dictionary) in an adaptive way and reconstructs the tomogram simultaneously from highly undersampled tilt series. In this method, the sparsity is applied on overlapping image patches favouring local structures. Furthermore, the dictionary is adapted to the specific tomogram instance, thereby favouring better sparsity and consequently higher quality reconstructions. The reconstruction algorithm is based on an alternating procedure that learns the sparsifying dictionary and employs it to remove artifacts and noise in one step, and then restores the tomogram data in the other step. Simulation and real ET experiments of several morphologies are performed with a variety of setups. Reconstruction results validate its efficiency in both noiseless and noisy cases and show that it yields an improved reconstruction quality with fast convergence. The proposed method enables the recovery of high-fidelity information without the need to worry about what sparsifying transform to select or whether the images used strictly follow the pre-conditions of a certain transform (e.g. strictly piecewise constant for Total Variation minimisation). This can also avoid artifacts that can be introduced by specific sparsifying transforms (e.g. the staircase artifacts the may result when using Total Variation minimisation). Moreover, this thesis shows how reliable elementally sensitive tomography using EELS is possible with the aid of both appropriate use of Dual electron energy loss spectroscopy (DualEELS) and the DLET compressed sensing algorithm to make the best use of the limited data volume and signal to noise inherent in core-loss electron energy loss spectroscopy (EELS) from nanoparticles of an industrially important material. Taken together, the results presented in this thesis demonstrates how high-fidelity ET reconstructions can be achieved using a compressed sensing approach.

No description available.

Probe microscopy techniques (Atomic Force Microscopy and Kelvin Force Microscopy) have been shown to be instrumental in the analysis of samples; such as resists and nanostructured materials. Through these techniques detailed surface information has been derived, including information such as surface roughness and surface charge distribution. Poly(Methylmethacrylate) (PMMA), remains at the forefront of resists utilised in e-beam lithography in the electronics industry. Surface morphology (specifically roughness) analysis remains a key parameter of investigation, particularly in the examination of polymeric films. This research aimed to investigate PMMA based electron beam resists as well as a novel (SML) resist material in terms of suitability for electron-beam lithography. Various concentrations (5, 7, 8, 9 and 11% w/v) of both PMMA and the novel resist material were spin-coated onto silica substrates. Samples were baked at 180oC for 3 minutes and examined under ultra-high vacuum using Omicron AFM/SPM to derive RMS values in order to assess roughness in addition to thickness measurements taken. SML resists were then utilised in the development of a new digital etch onto InGaAs/InAlAs wafer. The novel, SML resist material was found to offer smoother resist surface even at higher concentrations of polymer, a difference which was observed to be statistically significant (p<0.01). The SML resist was also notably thicker than the comparable PMMA resist (p>0.05) indicating that lower concentrations of the novel resist would be required to achieve the required resist thickness. Digital etching rates were found to be in agreement with previously documented findings. SML was concluded to be a superior resist in terms of thickness and smoothness, with AFM being further established as an essential characterization technique.

Le biogaz est une source d’énergie qui intéresse de plus en plus l’Europe et notamment la France pour ses avantages environnementaux et économiques. Produit de la fermentation de matière organique, il contient du biométhane. Ce dernier est une alternative plus durable aux énergies fossiles. Cependant, à l’état brut, les polluants dans le biogaz peuvent provoquer des dégâts sur la santé et l’environnement, notamment en raison de la présence de siloxanes et des composés soufrés. L’objectif de cette recherche consiste donc à développer des méthodes améliorant à la fois économiquement et écologiquement la désulfuration du biogaz, dans le but de les intégrer aux unités de traitement du biogaz déjà existantes et présentes sur le territoire. A partir d’un état de l’art sur les propriétés du biogaz et ses traitements de purification, deux procédés ont été particulièrement mis en avant et étudiés dans cette thèse. Le premier correspond au traitement de la désulfuration en précombustion consistant à éliminer le H2S et les siloxanes à travers un réacteur polyphasé à barbotage gaz-liquide spécifique, utilisant un nouveau solvant avec des propriétés « superacides ». Le second, quant à lui, correspond au traitement en postcombustion de la désulfuration des fumées provenant de la combustion du gaz, via un réacteur polyphasé à lit fixe gaz-solide. Pour ce faire, un prototype de l’unité de désulfuration est intégralement conçu, construit et testé dans le cadre de la thèse. Cette thèse présente notamment le développement des différents modèles numériques, ainsi que les résultats d’expériences en laboratoire, confirmant l’efficacité de ces procédés innovants. / Biogas. It is an energy source increasingly popular in Europe, remarkably in France, due to its environmental-friendly and economic-saving capabilities. It is produced by the organic matter fermentation, leading to biomethane production, as a sustainable alternative to fossil fuels. Nevertheless, as a raw gas, pollutants in biogas lead to environmental, health and process-related issues, especially because of its unique content on sulfur compounds. The objective of this research is to develop new processes, economically and environmentally feasible, for biogas desulfurization, seeking a process integration in existing biogas treatment units in France. Considering the state of the art on biogas properties and its possible purification treatments, two processes have been identified and studied in this thesis. The first process is a precombustion desulfurization treatment aiming to eliminate the H2S and the siloxanes through a gas-liquid bubbling-typed polyphasic reactor. This reactor is equipped with a new solvent with “superacid” properties. The second process is a postcombution desulfurization treatment for stack gas, through a gas-solid fixed-bed polyphasic reactor. A prototype of this unit was entirely designed, built and tested in the thesis. This thesis describes the applied research method, the developed numerical models, and the experimental results confirming the efficiency of the novel processes.