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In recent years, second harmonic generation (SHG) microscopy has revolutionised the field of biological imaging by offering a new means of visualising the fine structures of collagen tissues with excellent image penetration while minimising photodamage. This project involves the design and construction of a SHG microscope that is built around a compact femtosecond fibre laser for collagen imaging. Operating at 1032 nm, the microscope has demonstrated a penetration depth of beyond 320 microns in collagen, which is considerably superior to depths of 250 to 300 microns achievable with a conventional SHG microscope coupled to a Ti:sapphire excitation laser. The imaging characteristics of the microscope have been tested with a modified sample of bovine pericardium. The results indicate the microscope is polarisation-sensitive to the tissue structure and is capable to detecting signal changes at 10 μm resolution. This thesis will describe in detail, to our best knowledge, the first SHG microscope equipped with a compact and robust all-fibre femtosecond 1032 nm laser source.

Second Harmonic Generation (SHG) microscopy has undergone rapid transformation as an imaging tool for research due to its noninvasive properties that provides high resolution images for quantitative analysis. Extensive research has been performed to study the type of materials compatible with SHG but the effects of varying laser wavelengths on SHG efficiency is still poorly understood. The aim of this project is to design and build a scanning laser SHG microscope that is optimised for a 1030 nm giant chirp oscillator (GCO) designed by the University of Auckland. The project has shown that this wavelength is capable of producing SHG in collagen tissues of thickness up to 100 µm. We used nonlinear crystals and collagen samples to acquire signals and high resolution 2D images. The images were found to be of excellent quality with resolutions of up to a few microns to allow accurate measurements of the size and orientation of the fibrillar structures.

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HREM imaging was performed using the Jeol 4000ex microscope on specimens prepared from an as-grown ingot of semi-insulating Gallium Arsenide. Various low angle grain boundaries were imaged in the [110] orientation, misorientations varying between 4°-13°. Detailed study of a grain boundary of 11.5° misorientation about the [110] rotation axis has been carried out. Burgers vector analysis showed the presence of perfect 60° and [001] dislocations. Modelling of the [001] dislocation has been carried out using the Tersoff potential, Bond Order Potential and a tight binding Hamiltonian for GaAs, using Chadi (1984) parameters. The dislocation core was associated with an 8-membered and two 5-membered rings. Assum- ing there is a minimum of wrong bonds, we predict that the core has two wrong bonds, one being Ga-Ga, and the other As-As, both in equivalent positions where the two 5-membered rings were appended to the 8-membered ring. The Ga-Ga bond is considerably shorter and hence stronger than the As-As bond. Band structure calculations performed using a Vogl (1983) sp³s* Hamiltonian revealed deep states in the gap, which are associated with atoms in the core only. Using Stadelmann's (1987) EMS program, successful image matching of calculated images of the [001] dislocation has been achieved with the experimental image, using the atomic structure generated by tight binding relaxation. Ga and As being only two atomic numbers apart have similar scattering factors and cannot be easily distinguished in the experimental image. The equivalence of the position of the two wrong bonds greatly eases image matching as it is no longer necessary to know which is the Ga-Ga , and which is the As-As bond. This is the first suggested model of the [001] dislocation in GaAs, to the best of my knowledge. It is found to be similar to the atomic structure of the 90° partial dislocation in silicon (Bigger et al., 1992). No account of segregation of impurities to the grain boundary, or the [001] dislocation core is taken here, though it is very likely that an impurity atom would sit itself in this large space. The relaxed atomic structure for the 60° dislocation showed a doubling of periodicity along the dislocation line, similar to that found in the 30° partial in Si. The core consists of a 7-membered and a 5-membered ring with a minimum of two wrong bonds. In addition to this, quantitative comparisons of the [001] HREM image and simulated structures have been made and an iterative structure refinement carried out in order to achieve the best image matching. The resultant 'experimental-best-fit' structure was not found to be physically or chemically plausible.

The fundamental atomic and electronic behaviour of clean silicon surfaces has been studied within a simple tight-binding picture of bonding in solids. Of the various contributions to the surface binding energy, the lowering in the promotion energy (i.e. rehybridization) which accompanies localized Jahn-Teller distortions has been identified as a major electronic driving force underlying the stability of silicon surfaces. The structure of Si(113) has been experimentally determined by the technique of scanning tunnelling microscopy (STM). Despite its high index, the Si(113) surface is found to be highly stable. STM images of both empty and filled states provide strong evidence for a particular structural model with a 3x2 unit cell. The STM results are explained in terms of a general rehybridization principle, suggested by the earlier theoretical study, which accounts for the low surface energy as well as the observed spatial distribution of empty and filled states. In addition, the STM images reveal a high density of domain boundaries which introduce energy states that pin the Fermi level and explain earlier reports of a 3x1 reconstruction for this surface. Voltage-dependent STM image simulations for the Si(113)3x2 surface have been carried out using a simple tight-binding description of surface electronic structure. Quantitative agreement with experiment is obtained confirming the qualitative rehybridization arguments used previously. The local barrier for tunnelling electrons is shown to have an important effect on the interpretation of STM images. The high stability of clean Si(l 13) is shown by STM to be disrupted by adsorption of submonolayer amounts of atomic hydrogen which saturates dangling bonds. Mass transport of silicon occurs and structural models are proposed for the resultant mixed 2x2 and 2x3 surface.

This thesis concerns the development of simple, compact scanning optical microscopes which can obtain confocal and interference images. The effects of feeding the reflected signal back into the laser cavity of a confocal microscope are investigated and exploited. Monomode optical fibres are used to perform the spatial filtering required for confocal microscopy and, later, as the source of reference beams for interferometry. The theory describing the basic operation of the microscopes is developed. The optical systems are modelled using scalar diffraction theory and the effects of optical feedback into the laser cavity are described, with the practical implications emphasised. A fully reciprocal arrangement of the microscope is developed, in which a single mode optical fibre both launches the signal towards the object and then collects the reflected signal. The fibre is shown to exhibit the spatial filtering properties required for the source and detector in a confocal microscope. It is shown that a semiconductor laser can be used as a detector of the amplitude of the object signal. This is first demonstrated by directing the microscope signal back into the laser cavity and measuring the variation of the optical intensity in the cavity itself. Comparable results are obtained when the variation of the junction voltage across the cavity is measured. It is also shown that the optical fibre is redundant in this system, since the lasing mode of the cavity itself is sufficiently small to adequately spatially filter the reflected signal. When a Helium-Neon laser is used as the source of illumination the effect of the feedback on the laser is seen to be very different, resulting in interferometry. It is shown that high frequency modulation techniques can be used to obtain both confocal images and surface profiles from the same system. This is first demonstrated using an optical feedback scheme in which the modulation of the optical path length of the object beam is controlled electrooptically. In an alternative scheme the images are obtained by calculation, rather than by using a control loop system. In this case the modulation is achieved mechanically. The theoretical limits for the resolutions of the systems described are discussed. It is shown that the lateral resolution of the surface profile systems is inherently non-linear with feature height. Finally, a semiconductor laser based microscope is developed which can obtain confocal images and surface profiles independently. The dependence of the wavelength on the injection current is exploited as a convenient means of introducing a phase shift into the feedback signal by which profilometry can be achieved. All the systems are described theoretically and demonstrated experimentally.

Transmission Electron Microscope (TEM) studies have been carried out of emitter regions in polysilicon contacted emitter bipolar transistors. The preparation of suitable TEM thin foils is described. In addition a technique is developed for the observation and quant jtative interpretation of the break-up of the interfacial oxide layers observed in these samples. The effect of annealing the samples prior to emitter dopant implantation (pre-annealing) is investigated for phosphorus and arsenic doped samples, implanted into a polysilicon layer 0.4μm thick, with a dose of 1x10¹⁶cm². Two wafer pre-cleans have been used prior to polysilicon deposition to produce a thin oxide (0-8Å) and a thicker oxide (14Å). In the presence of the thinner oxide, the phosphorus doped samples enhance epitaxial regrowth of the polysilicon layer compared with the arsenic doped or undoped samples. In the presence of the thicker oxide, no difference is observed between the samples. A mechanism is proposed to explain this. The mechanisms controlling the gain of a phosphorus doped device are investigated and a model proposed to explain the observed electrical characteristics. It is concluded that there are two mechanisms responsible for the observed supression of hole current. Firstly tunnelling through the interfacial oxide and secondly some blocking effect of the interface. Carrier transport in the polysilicon is not believed to contribute to this supression. A dopant sensitive etch has been applied to TEM thin foils containing fully processed emitters in state-of-the-art devices. The shape of the emitter dopant distribution is revealed in such devices for the first time, and a 2-D profile is obtained from the emitter. It is shown that reduction in the emitter depth to 8OOÅ or less does not alter the emitter dopant geometry. The technique is demonstrated to be capable of resolving spatial separations of dopant iso-concentration contours of 100Å or less.

This thesis describes an investigation into the folding behaviour of hen lysozyme by characterisation of the structure and the dynamics of different conformational states of the protein. The native state, a partially structured state generated by the addition of a cosolvent, trifluoroethanol (TFE), and a highly denatured state of the protein in presence of urea at low pH, have been studied at equilibrium by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The principal methods utilised in-fchis thesis are the measurement and interpretation of amide hydrogen exchange and ¹⁵ N relaxation data. Amide hydrogen exchange rates from a highly denatured state of hen lysozyme in 8 M urea, at pH 2.0, are well approximated by recent estimates for intrinsic exchange rates from unstructured polypeptides, implying that residual .structures cannot be persistent in this state. In the native protein, by contrast, protection factors are measured to be of the order of 10⁴-10⁸ and can be rationalised by the involvement of amides in secondary structure and burial from the protein surface. Surface accessibility has also been found to be an important determinant of the dynamics of main and sidechain groups monitored by ¹⁵N relaxation. For this purpose ¹⁵N resonances of native lysozyme were assigned and order parameters derived. The dynamic behaviour was considered in the light of features of the crystal and NMR structures, suggesting that a lack of van der Waals contacts is a major determinant for mobility. A denatured state of hen lysozyme, formed by addition of TFE cosolvent, has been developed as a model for a partially ordered conformation of the protein. Amide hydrogen exchange measurements show that sites with the highest protection factors, up to 250, are located in stable native-like helices. Near complete assignment of ¹⁵N-edited NMR spectra allowed a detailed description of secondary structure in the TFE denatured state. Non-native α-helical structure is present as extensions to the native helices and in a region of the polypeptide which forms part of the β-sheet in the native state. These structures have been shown to be in accord with the helical propensities of the primary sequence. Preliminary structure calculations suggest that, despite the absence of extensive and persistent tertiary interactions, topological restraints exist in parts of the TFE denatured state, resulting in considerable propensities for native-like arrangements of the secondary structure. Mainchain dynamics determined by ¹⁵N relaxation, although increased in magnitude, appear to be related to that of the native state and are dominated by the position of disulphide bonds and by chain hydrophobicity. Thus the structural and the dynamic behaviour of the polypeptide chain in the TFE denatured state could be rationalised, at least in part, by consideration of features of the primary sequence.