Radial-field magnetic electron lenses

This investigation looks critically at conventional magnetic lenses in the light of present-day technology with the aim of advancing electron microscopy in its broadest sense. By optimising the cooling arrangements and heat transfer characteristics of lens windings it was possible to increase substantially the current density in the winding, and achieve a large reduction in the size of conventional magnetic electron lenses. Following investigations into the properties of solenoidal lenses, a new type of lens with only one pole-piece was developed. The focal properties of such lenses, which differ considerably from those.of conventional lenses, have been derived from a combination of mathematical models and experimentally measured axial flux density distributions. These properties can be profitably discussed with reference to "half-lenses". Miniature conventional twin pole-piece lenses and the proposed radial field single pole-piece lenses have been designed and constructed and both types of lenses have been evaluated by constructing miniature electron optical columns. A miniature experimental transmission electron microscope (TEM), a miniature scanning electron microscope (SEM) and a scanning transmission microscope (STEM) have been built. A single pole-piece miniature one million volt projector lens of only lOcm diameter and weighing 2.lkg was designed, built and tested at 1 million volts in a commercial electron microscope. iii. Preliminary experiments indicate that in single pole lenses it is possible to extract secondary electrons from the specimen in spite of the presence of the magnetic field of the probe-forming lens. This may well be relevant for the SEM in which it is desirable to examine a large specimen at a moderately good resolution.

Investigating the structure of biomass-derived non-graphitizing mesoporous carbons by electron energy loss spectroscopy in the transmission electron microscope and X-ray photoelectron spectroscopy

We have investigated the microstructure and bonding of two biomass-based porous carbon chromatographic stationary phase materials (alginic acid-derived Starbon® and calcium alginate-derived mesoporous carbon spheres (AMCS) and a commercial porous graphitic carbon (PGC), using high resolution transmission electron microscopy, electron energy loss spectroscopy (EELS), N2 porosimetry and X-ray photoelectron spectroscopy (XPS). The planar carbon sp -content of all three material types is similar to that of traditional nongraphitizing carbon although, both biomass-based carbon types contain a greater percentage of fullerene character (i.e. curved graphene sheets) than a non-graphitizing carbon pyrolyzed at the same temperature. This is thought to arise during the pyrolytic breakdown of hexauronic acid residues into C5 intermediates. Energy dispersive X-ray and XPS analysis reveals a homogeneous distribution of calcium in the AMCS and a calcium catalysis mechanism is discussed. That both Starbon® and AMCS, with high-fullerene character, show chromatographic properties similar to those of a commercial PGC material with extended graphitic stacks, suggests that, for separations at the molecular level, curved fullerene- like and planar graphitic sheets are equivalent in PGC chromatography. In addition, variation in the number of graphitic layers suggests that stack depth has minimal effect on the retention mechanism in PGC chromatography. © 2013 Elsevier Ltd. All rights reserved.

High-resolution cyclostratigraphy of the Cenomanian-Turonian interval: Paleoceanographic implications, a comparison between deep sea drilling project site 386 and 144

The purpose of this study was to determine the extent to which oceanic anoxic events (OAE's) are recorded in deep-water deposits of the former western Tethyan Sea, by investigating the Cenomanian-Turonian time interval characterized by the worldwide OAE 2 event. The study improved our knowledge of the possible controlling mechanisms that triggered this event at these sites, and furthered our understanding of this global phenomenon. This was examined by high-resolution, multi-proxy analyses of sediments at DSDP Sites 386 and 144, including sedimentology, scanning electron microscopy, stable isotopes, bulk and clay mineralogy, major and trace element geochemistry, biomarkers, and paleontological data. ^ The results provide a better stratigraphic resolution for the Cenomanian-Turonian, which allowed for more precise determination of chronologic boundaries, sedimentation rates at DSDP Site 386, and a more accurate calculation of the frequency of the cycles recorded in the sequence, which fall predominantly within the precession (∼23 kyr) and short eccentricity (∼100 kyr) ranges. The combined proxies allow assessment of the correlation of δ13Corg, and major and trace elements with the predominance of cyanobacteria. These organisms were the main producers of the organic matter during the dysoxic and euxinic conditions of OAE 2 at DSDP Site 386. A huge amount of microcrystalline quartz of eolian origin is also associated with OAE 2. The geochemical proxies further provide evidence that OAE 2 was linked to increased volcanism outside the deep water of the proto-Atlantic Ocean. The clays in the Turonian sediments are terrigenous and derived predominantly from eolian transport. Comparing DSDP Site 386 and 144 with stratotype sections, the δ13C org and TOC data indicate that OAE 2 seems diachronous throughout the proto-Atlantic Ocean. ^ This study concludes that the development of anoxic conditions in the deep water of the Atlantic during the latest Cenomanian-Turonian resulted from a combination of factors related to local oceanic setting and mitigated by global tectonism and climate. The data provide a more comprehensive view of the interacting factors that led to sustained high productivity of the cyanobacteria and photosynthetic protists that produced organic-carbon-rich deposits in the world's oceans. ^

Paleoenvironmental implications of the Indidura Formation (Cenomanian/Turonian), northeastern Mexico: A high resolution stratigraphic study

High-resolution lithostratigraphic data from rock sequences known as the Indidura Formation near Parras de La Fuente, Coahuila, NE Mexico, led to achieve a significant improvement of our knowledge of that Formation. The results of this study indicate for the first time that the sequence at Parras de La Fuente developed from the deposition of calcareous cyanobacterial microspheroids that accumulated under perennial blooms during the Late Cenomanian through the Middle Turonian. Multi-proxy analyses included sedimentological, petrographical, scanning electron microscopy, stable isotope, trace element geochemistry, and paleontological data. The combined results allowed the correlation of δ13C and anomalies in Mo, V, and Cr with the abundance and predominance of calcareous cyanobacterial microspheroids, which were the main suppliers of the carbonate components and the organic matter throughout deposition of the Indidura Formation in the Parras de la Fuente area, under dysoxic/anoxic conditions. Conspicuous interbeds of dark and light-gray laminated marly calcilutites, and dark-gray marlstones that characterize the stratigraphic sequence formed in response to external forcing climatic factors of millennial-scale Milankovitch cycles (ca. 20 ka precession). At the microscopic level, the prominent dark and light-gray laminae were formed during cycles similar to the 10 to 15 years solar irradiance maximum, and represent alternating periods of high and low calcareous cyanobacterial microspheroids productivity.

Paleoenvironmental Implications of the Indidura Formation (Cenomanian/Turonian), Northeastern Mexico: a High Resolution Stratigraphic Study

High-resolution lithostratigraphic data from rock sequences known as the Indidura Formation near Parras de La Fuente, Coahuila, NE Mexico, led to achieve a significant improvement of our knowledge of that Formation. The results of this study indicate for the first time that the sequence at Parras de La Fuente developed from the deposition of calcareous cyanobacterial microspheroids that accumulated under perennial blooms during the Late Cenomanian through the Middle Turonian. Multi-proxy analyses included sedimentological, petrographical, scanning electron microscopy, stable isotope, trace element geochemistry, and paleontological data. The combined results allowed the correlation of δ13C and anomalies in Mo, V, and Cr with the abundance and predominance of calcareous cyanobacterial microspheroids, which were the main suppliers of the carbonate components and the organic matter throughout deposition of the Indidura Formation in the Parras de la Fuente area, under dysoxic/anoxic conditions. Conspicuous interbeds of dark and light-gray laminated marly calcilutites, and dark-gray marlstones that characterize the stratigraphic sequence formed in response to external forcing climatic factors of millennial-scale Milankovitch cycles (ca. 20 ka precession). At the microscopic level, the prominent dark and light-gray laminae were formed during cycles similar to the 10 to 15 years solar irradiance maximum, and represent alternating periods of high and low calcareous cyanobacterial microspheroids productivity.

The morphology of ordered block copolymer patterns as probed by high resolution imaging

The microphase separation of block copolymer (BCP) thin films can afford a simple and cost-effective means to studying nanopattern surfaces, and especially the fabrication of nanocircuitry. However, because of complex interface effects and other complications, their 3D morphology, which is often critical for application, can be more complex than first thought. Here, we describe how emerging microscopic methods may be used to study complex BCP patterns and reveal their rich detail. These methods include helium ion microscopy (HIM) and high resolution x-section transmission electron microscopy (XTEM), and complement conventional secondary electron and atomic force microscopies (SEM and TEM). These techniques reveal that these structures are quite different to what might be expected. We illustrate the advances in the understanding of BCP thin film morphology in several systems, which result from this characterization. The systems described include symmetric, lamellar forming polystyrene-b-polymethylmethacrylate (PS-b-PMMA), cylinder forming polystyrene-b-polydimethylsiloxane (PS-b-PDMS), as well as lamellar and cylinder forming patterns of polystyrene-b-polyethylene oxide (PS-b-PEO) and polystyrene-b-poly-4-vinylpyridine (PS-b-P4VP). Each of these systems exhibits more complex arrangements than might be first thought. Finding and developing techniques whereby complex morphologies, particularly at very small dimensions, can be determined is critical to the practical use of these materials in many applications. The importance of quantifying these complex morphologies has implications for their use in integrated circuit manufacture, where they are being explored as alternative pattern forming methods to conventional UV lithography.

Atomic resolution studies of oxide superlattices and ultrathin films

The ability to grow ultrathin films layer-by-layer with well-defined epitaxial relationships has allowed research groups worldwide to grow a range of artificial films and superlattices, first for semiconductors, and now with oxides. In the oxides thin film research community, there have been concerted efforts recently to develop a number of epitaxial oxide systems grown on single crystal oxide substrates that display a wide variety of novel interfacial functionality, such as enhanced ferromagnetic ordering, increased charge carrier density, increased optical absorption, etc, at interfaces. The magnitude of these novel properties is dependent upon the structure of thin films, especially interface sharpness, intermixing, defects, and strain, layering sequence in the case of superlattices and the density of interfaces relative to the film thicknesses. To understand the relationship between the interfacial thin film oxide atomic structure and its properties, atomic scale characterization is required. Transmission electron microscopy (TEM) offers the ability to study interfaces of films at high resolution. Scanning transmission electron microscopy (STEM) allows for real space imaging of materials with directly interpretable atomic number contrast. Electron energy loss spectroscopy (EELS), together with STEM, can probe the local chemical composition as well as local electronic states of transition metals and oxygen. Both techniques have been significantly improved by aberration correctors, which reduce the probe size to 1 Å, or less. Aberration correctors have thus made it possible to resolve individual atomic columns, and possibly probe the electronic structure at atomic scales. Separately, using electron probe forming lenses, structural information such as the crystal structure, strain, lattice mismatches, and superlattice ordering can be measured by nanoarea electron diffraction (NED). The combination of STEM, EELS, and NED techniques allows us to gain a fundamental understanding of the properties of oxide superlattices and ultrathin films and their relationship with the corresponding atomic and electronic structure. In this dissertation, I use the aforementioned electron microscopy techniques to investigate several oxide superlattice and ultrathin film systems. The major findings are summarized below. These results were obtained with stringent specimen preparation methods that I developed for high resolution studies, which are described in Chapter 2. The essential materials background and description of electron microscopy techniques are given in Chapter 1 and 2. In a LaMnO3-SrMnO3 superlattice, we demonstrate the interface of LaMnO3-SrMnO3 is sharper than the SrMnO3-LaMnO3 interface. Extra spectral weights in EELS are confined to the sharp interface, whereas at the rougher interface, the extra states are either not present or are not confined to the interface. Both the structural and electronic asymmetries correspond to asymmetric magnetic ordering at low temperature. In a short period LaMnO3-SrTiO3 superlattice for optical applications, we discovered a modified band structure in SrTiO3 ultrathin films relative to thick films and a SrTiO3 substrate, due to charge leakage from LaMnO3 in SrTiO3. This was measured by chemical shifts of the Ti L and O K edges using atomic scale EELS. The interfacial sharpness of LaAlO3 films grown on SrTiO3 was investigated by the STEM/EELS technique together with electron diffraction. This interface, when prepared under specific conditions, is conductive with high carrier mobility. Several suggestions for the conductive interface have been proposed, including a polar catastrophe model, where a large built-in electric field in LaAlO3 films results in electron charge transfer into the SrTiO3 substrate. Other suggested possibilities include oxygen vacancies at the interface and/or oxygen vacancies in the substrate. The abruptness of the interface as well as extent of intermixing has not been thoroughly investigated at high resolution, even though this can strongly influence the electrical transport properties. We found clear evidence for cation intermixing through the LaAlO3-SrTiO3 interface with high spatial resolution EELS and STEM, which contributes to the conduction at the interface. We also found structural defects, such as misfit dislocations, which leads to increased intermixing over coherent interfaces.

Transmission electron tomography: quality assessment and enhancement for three-dimensional imaging of nanostructures

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.

Raman microscopy of the molybdate minerals koechlinite, iriginite and lindgrenite

A series of molybdate bearing minerals including wulfenite, powellite, lindgrenite and iriginite have been analysed by Raman microscopy. These minerals are closely related and often have related paragenesis. Raman microscopy enables the selection of individual crystals of these minerals for spectroscopic analysis even though several of the minerals can be found in the same matrix because of the paragenetic relationships between the minerals. The molybdenum bearing minerals lindgrenite, iriginite and koechlinite were studied by scanning electron microscopy and compositionally analysed by EDX methods using an electron probe before Raman spectroscopic analyses. The Raman spectra are assigned according to factor group analysis and related to the structure of the minerals. These minerals have characteristically different Raman spectra.

Characterization of commercial double-walled carbon nanotube material : composition, structure, and heat capacity

A purified commercial double-walled carbon nanotube (DWCNT) sample was investigated by transmission electron microscopy (TEM), thermogravimetry (TG), and Raman spectroscopy. Moreover, the heat capacity of the DWCNT sample was determined by temperature-modulated differential scanning calorimetry in the range of temperature between -50 and 290 °C. The main thermo-oxidation characterized by TG occurred at 474 °C with the loss of 90 wt% of the sample. Thermo-oxidation of the sample was also investigated by high-resolution TG, which indicated that a fraction rich in carbon nanotube represents more than 80 wt% of the material. Other carbonaceous fractions rich in amorphous coating and graphitic particles were identified by the deconvolution procedure applied to the derivative of TG curve. Complementary structural data were provided by TEM and Raman studies. The information obtained allows the optimization of composites based on this nanomaterial with reliable characteristics.

Synthesis and characterisation of cobalt hydroxide, cobalt oxyhydroxide and cobalt oxide nanodiscs

Cobalt hydroxide, cobalt oxyhydroxide and cobalt oxide nanomaterials were synthesized through simple soft chemistry. The cobalt hydroxide displays hexagonal morphology with clear edges 20 nm long. This morphology and nanosize is retained through to cobalt oxide Co3O4 through a topotactical relationship. Cobalt oxyhydroxide and cobalt oxide nanomaterials were synthesized through oxidation and low temperature calcination from the as-prepared cobalt hydroxide. Characterisation of these cobalt-based nanomaterials were fully developed, including X-ray diffraction, transmission electron microscopy combined with selected area electron diffraction, scanning electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy, and thermal gravimetric analysis. Bonding of the divalent cobalt hydroxide from the oxyhydroxide and oxides by studying their high resolution XPS spectra for Co 2p3/2 and O 1s. Raman spectroscopy of the as-prepared Co(OH)2, CoO(OH) and Co3O4 nanomaterials characterised each material. The thermal stability of the materials Co(OH)2 and CoO(OH) were established. This research has developed methodology for the synthesis of cobalt oxide and cobalt oxyhydroxide nanodiscs at low temperatures.

Microstructural characterization of electron beam evaporated tungsten oxide films for gas sensing applications

Tungsten trioxide is one of the potential semiconducting materials used for sensing NH3, CO, CH4 and acetaldehyde gases. The current research aims at development, microstructural characterization and gas sensing properties of thin films of Tungsten trioxide (WO3). In this paper, we intend to present the microstructural characterization of these films as a function of post annealing heat treatment. Microstructural and elemental analysis of electron beam evaporated WO3 thin films and iron doped WO3 films (WO3:Fe) have been carried out using analytical techniques such as Transmission electron microscopy, Rutherford Backscattered Spectroscopy and XPS analysis. TEM analysis revealed that annealing at 300oC for 1 hour improves cyrstallinity of WO3 film. Both WO3 and WO3:Fe films had uniform thickness and the values corresponded to those measured during deposition. RBS results show a fairly high concentration of oxygen at the film surface as well as in the bulk for both films, which might be due to adsorption of oxygen from atmosphere or lattice oxygen vacancy inherent in WO3 structure. XPS results indicate that tungsten exists in 4d electronic state on the surface but at a depth of 10 nm, both 4d and 4f electronic states were observed. Atomic force microscopy reveals nanosize particles and porous structure of the film. This study shows e-beam evaporation technique produces nanoaparticles and porous WO3 films suitable for gas sensing applications and doping with iron decreases the porosity and particle size which can help improve the gas selectivity.

Thin film deposition and characterization of pure and iron-doped electron-beam evaporated tungsten oxide for gas sensors

Pure Tungsten Oxide (WO3) and Iron-doped (10 at%) Tungsten Oxide (WO3:Fe) nanostructured thin films were prepared using a dual crucible Electron Beam Evaporation techniques. The films were deposited at room temperature in high vacuum condition on glass substrate and post-heat treated at 300 oC for 1 hour. From the study of X-ray diffraction and Raman the characteristics of the as-deposited WO3 and WO3:Fe films indicated non-crystalline nature. The surface roughness of all the films showed in the order of 2.5 nm as observed using Atomic Force Microscopy (AFM). X-Ray Photoelectron Spectroscopy (XPS) analysis revealed tungsten oxide films with stoichiometry close to WO3. The addition of Fe to WO3 produced a smaller particle size and lower porosity as observed using Transmission Electron Microscopy (TEM). A slight difference in optical band gap energies of 3.22 eV and 3.12 eV were found between the as-deposited WO3 and WO3:Fe films, respectively. However, the difference in the band gap energies of the annealed films were significantly higher having values of 3.12 eV and 2.61 eV for the WO3 and WO3:Fe films, respectively. The heat treated samples were investigated for gas sensing applications using noise spectroscopy and doping of Fe to WO3 reduced the sensitivity to certain gasses. Detailed study of the WO3 and WO3:Fe films gas sensing properties is the subject of another paper.

Pathogenicity of Plesiomonas shigelloides : interactions with eukaryotic host cells in vitro

Diarrhoea is one of the leading causes of morbidity and mortality in populations in developing countries and is a significant health issue throughout the world. Despite the frequency and the severity of the diarrhoeal disease, mechanisms of pathogenesis for many of the causative agents have been poorly characterised. Although implicated in a number of intestinal and extra-intestinal infections in humans, Plesiomonas shigelloides generally has been dismissed as an enteropathogen due to the lack of clearly demonstrated virulence-associated properties such as production of cytotoxins and enterotoxins or invasive abilities. However, evidence from a number of sources has indicated that this species may be the cause of a number of clinical infections. The work described in this thesis seeks to resolve this discrepancy by investigating the pathogenic potential of P. shigelloides using in vitro cell models. The focus of this research centres on how this organism interacts with human host cells in an experimental model. Very little is known about the pathogenic potential of P. shigel/oides and its mechanisms in human infections and disease. However, disease manifestations mimic those of other related microorganisms. Chapter 2 reviews microbial pathogenesis in general, with an emphasis on understanding the mechanisms resulting from infection with bacterial pathogens and the alterations in host cell biology. In addition, this review analyses the pathogenic status of a poorly-defined enteropathogen, P. shigelloides. Key stages of pathogenicity must occur in order for a bacterial pathogen to cause disease. Such stages include bacterial adherence to host tissue, bacterial entry into host tissues (usually required), multiplication within host tissues, evasion of host defence mechanisms and the causation of damage. In this study, these key strategies in infection and disease were sought to help assess the pathogenic potential of P. shigelloides (Chapter 3). Twelve isolates of P. shigelloides, obtained from clinical cases of gastroenteritis, were used to infect monolayers of human intestinal epithelial cells in vitro. Ultrastructural analysis demonstrated that P. shigelloides was able to adhere to the microvilli at the apical surface of the epithelial cells and also to the plasma membranes of both apical and basal surfaces. Furthermore, it was demonstrated that these isolates were able to enter intestinal epithelial cells. Internalised bacteria often were confined within vacuoles surrounded by single or multiple membranes. Observation of bacteria within membranebound vacuoles suggests that uptake of P. shigelloides into intestinal epithelial cells occurs via a process morphologically comparable to phagocytosis. Bacterial cells also were observed free in the host cell cytoplasm, indicating that P. shige/loides is able to escape from the surrounding vacuolar membrane and exist within the cytosol of the host. Plesiomonas shigelloides has not only been implicated in gastrointestinal infections, but also in a range of non-intestinal infections such as cholecystitis, proctitis, septicaemia and meningitis. The mechanisms by which P. shigelloides causes these infections are not understood. Previous research was unable to ascertain the pathogenic potential of P. shigel/oides using cells of non-intestinal origin (HEp-2 cells derived from a human larynx carcinoma and Hela cells derived from a cervical carcinoma). However, with the recent findings (from this study) that P. shigelloides can adhere to and enter intestinal cells, it was hypothesised, that P. shigel/oides would be able to enter Hela and HEp-2 cells. Six clinical isolates of P. shigelloides, which previously have been shown to be invasive to intestinally derived Caco-2 cells (Chapter 3) were used to study interactions with Hela and HEp-2 cells (Chapter 4). These isolates were shown to adhere to and enter both nonintestinal host cell lines. Plesiomonas shigelloides were observed within vacuoles surrounded by single and multiple membranes, as well as free in the host cell cytosol, similar to infection by P. shigelloides of Caco-2 cells. Comparisons of the number of bacteria adhered to and present intracellularly within Hela, HEp-2 and Caco-2 cells revealed a preference of P. shigelloides for Caco-2 cells. This study conclusively showed for the first time that P. shigelloides is able to enter HEp-2 and Hela cells, demonstrating the potential ability to cause an infection and/or disease of extra-intestinal sites in humans. Further high resolution ultrastructural analysis of the mechanisms involved in P. shigelloides adherence to intestinal epithelial cells (Chapter 5) revealed numerous prominent surface features which appeared to be involved in the binding of P. shige/loides to host cells. These surface structures varied in morphology from small bumps across the bacterial cell surface to much longer filaments. Evidence that flagella might play a role in bacterial adherence also was found. The hypothesis that filamentous appendages are morphologically expressed when in contact with host cells also was tested. Observations of bacteria free in the host cell cytosol suggests that P. shigelloides is able to lyse free from the initial vacuolar compartment. The vacuoles containing P. shigel/oides within host cells have not been characterised and the point at which P. shigelloides escapes from the surrounding vacuolar compartment has not been determined. A cytochemical detection assay for acid phosphatase, an enzymatic marker for lysosomes, was used to analyse the co-localisation of bacteria-containing vacuoles and acid phosphatase activity (Chapter 6). Acid phosphatase activity was not detected in these bacteria-containing vacuoles. However, the surface of many intracellular and extracellular bacteria demonstrated high levels of acid phosphatase activity, leading to the proposal of a new virulence factor for P. shigelloides. For many pathogens, the efficiency with which they adhere to and enter host cells is dependant upon the bacterial phase of growth. Such dependency reflects the timing of expression of particular virulence factors important for bacterial pathogenesis. In previous studies (Chapter 3 to Chapter 6), an overnight culture of P. shigelloides was used to investigate a number of interactions, however, it was unknown whether this allowed expression of bacterial factors to permit efficient P. shigelloides attachment and entry into human cells. In this study (Chapter 7), a number of clinical and environmental P. shigelloides isolates were investigated to determine whether adherence and entry into host cells in vitro was more efficient during exponential-phase or stationary-phase bacterial growth. An increase in the number of adherent and intracellular bacteria was demonstrated when bacteria were inoculated into host cell cultures in exponential phase cultures. This was demonstrated clearly for 3 out of 4 isolates examined. In addition, an increase in the morphological expression of filamentous appendages, a suggested virulence factor for P. shigel/oides, was observed for bacteria in exponential growth phase. These observations suggest that virulence determinants for P. shigel/oides may be more efficiently expressed when bacteria are in exponential growth phase. This study demonstrated also, for the first time, that environmental water isolates of P. shigelloides were able to adhere to and enter human intestinal cells in vitro. These isolates were seen to enter Caco-2 host cells through a process comparable to the clinical isolates examined. These findings support the hypothesis of a water transmission route for P. shigelloides infections. The results presented in this thesis contribute significantly to our understanding of the pathogenic mechanisms involved in P. shigelloides infections and disease. Several of the factors involved in P. shigelloides pathogenesis have homologues in other pathogens of the human intestine, namely Vibrio, Aeromonas, Salmonella, Shigella species and diarrhoeaassociated strains of Escherichia coli. This study emphasises the relevance of research into Plesiomonas as a means of furthering our understanding of bacterial virulence in general. As well it provides tantalising clues on normal and pathogenic host cell mechanisms.