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

A versatile scanning probe microscope controller has been constructed. Its suitability for the control of a range of different scanning probe microscope heads has been demonstrated. These include an ultra high vacuum scanning tunnelling microscope, with which atomic resolution images of Si surfaces was obtained, a custom-built atomic force microscope, and a custom-built photon emission scanning tunnelling microscope. The controller has been designed specifically to facilitate data acquisition during molecular manipulation experiments. Using the controller, the fullerene molecule C60 has been successfully manipulated on Si(100)-2x1 surfaces and detailed data has been acquired during the manipulation process. Evidence for two distinct modes of manipulation have been observed. A repulsive mode with success rates up to 90% was found to occur with tunnel gap impedances below 2GΩ, while between 2GΩ and 8GΩ attractive manipulation events were observed, with a maximum success rate of ~8%. It was also found that the step size between feedback updates had a significant effect on tip stability, and that dwell time of the STM tip at each data point had a critical effect on manipulation probability. A multi-function scanning probe microscope head has been developed capable of operation as a scanning tunnelling microscope and an atomic force microscope in vacuum and a magnetic field of 7T. The custom-built controller also presented here was used to control the head. A three-axis inertial sliding motor was developed for the head, capable of reproducible step sizes of <1000Å. In addition, an optical fibre interferometer was constructed with a sensitivity of 0.2Å/ √Hz. Preliminary development of a magnetic resonance force microscope mode has also been performed, with initial results showing such a system to be feasible.

With a proven ability to uncover fundamental biological processes, the atomic force microscope (AFM) represents one of the most valuable and versatile tools available to the biophysical sciences. We study the unsteady small-scale flows generated within the AFM by its sensing probe (a long thin cantilever), which have received relatively little attention to date, yet which are increasingly relevant in an age of microdevices. The early parts of this thesis investigate some canonical two-dimensional flows driven by oscillations of an infinite-length rigid cantilever. These prove amenable to analysis and enable us to investigate many of the important physical phenomena and compile a comprehensive collection of asymptotic expressions for the drag. The corresponding results lay out the influence of a nearby wall, geometry and oscillation frequency. The limitations of a two-dimensional approach are then explored through the development of a novel unsteady slender-body theory (USBT) for finite-length cylinders, an asymptotic treatment of which offers corrections to traditional resistive-force-theory (RFT) methods by accounting for geometric factors and flow inertia. These ideas are then extended to the study of thin rectangular plates. Two key parameters are identified which promote two-dimensionality in the flow, namely the frequency of oscillation and the proximity of a nearby boundary. We then examine flexible cylinders and plates by coupling the hydrodynamics to linearized elastic beam and plate equations, which simulate the hydrodynamically-damped high-speed deformable motion of the AFM's cantilever, when driven either externally or by Brownian motion. In the latter case, we adopt an approach which offers notable improvements over the most advanced method currently available to the AFM community.

Confocal scanning microscopy (CSM) is a much used and advantageous form of microscopy. Although CSM is superior to conventional microscopy in many respects, a major disadvantage is the complexity of the scanning process and the sometimes long time to perform the scan. In this thesis a novel non-scanning fluorescence confocal microscopy is investigated. The method uses a random time-varying speckle pattern to illuminate the specimen, recording a large number of independent full-field frames without the need for a scanning system. The recorded frames are then processed in a suitable way to give a confocal image. The goal of this research project is to confirm the effectiveness and practicality of speckle-illumination microscopy and to develop this proposal into a functioning microscope system. The issues to be addressed include modelling of the system performance, setting up experiments, computer control and image processing. This work makes the following contributions to knowledge: * The development of criteria for system performance evaluation * The development of methods for speckle processing, whereby the number of frames required for an image of acceptable quality can be reduced * The implementation of non-scanning fluorescence confocal microscopy based upon separate recording of the speckle patterns and the fluorescence frames, demonstrating the practicality and effectiveness of this method * The realisation of real-time image processing by optically addressed spatial light modulator, showing how this new form of optical arrangement may be used in practice The thesis is organised into three main segments. Chapters 1-2 review related work and introduce the concepts of fluorescence confocal microscopy. Chapters 3-5 discuss system modelling and present results of performance evaluation. Chapters 6-8 present experimental results based upon the separate recording scheme and the spatial light modulation scheme, draw conclusions and offer some speculative suggestions for future research.

Cotton is a vital material for the textile industry, providing the fundamental raw component for the manufacture of numerous and varied garments. It has been thoroughly characterised both in terms of its constitution; behaviour under a variety of environmental and manufacturing conditions; and several mechanisms by which it takes up dyestuffs. Recently the availability of a range of high-performance surface analysis tools has allowed researchers to begin to assess the contribution of the surface interface to the overall properties of cellulose in cotton. In particular, these approaches offer considerable potential to address the current lack of fundamental experimental data in support of the proposed dye-uptake mechanisms in cotton fibres. The absence of a detailed molecular model for the process makes it difficult to predict dye performance and to identify the key characteristics of the cotton which influence dyeing. For example, the existence and location of dye binding sites is still unclear. It has been postulated that the occurrence of crystalline or amorphous regions in cotton may play a role in such binding and in dye uptake. A deeper understanding of the dyeing mechanisms therefore requires knowledge of the interplay between the physical chemistry of the dye, its adsorption/diffusion onto the surface of and within cotton fibres, and the related physical and chemical characteristics of the cotton itself. Here we begin to address these broad questions through the application of atomic force microscopy (AFM) and other complimentary surface analytical techniques, to analyse a range of dyed and undyed cellulose based textiles. We provide high-resolution surface morphological image data, which show nanometre scale detail of the surface of dyed and undyed cotton fibres. It is believed that the dyeing process induces an increase in crystallinity, due to breakages in hydrogen bonds between cellulose chain molecules during swelling of fibres within the dyebath, thus allowing dye molecules to enter and become entrapped within the fibre matrix. We provide evidence in the form of image data that suggest a difference in the crystal structure between undyed and dyed cellulose fibres to support this theory. We also reveal possible crystalline and amorphous regions within the substrate through AFM phase imaging using modified tips and successfully fingerprint regions of dyed and undyed cellulose fibres. Complimentary surface chemical analysis of dyed and undyed fibres, provide qualitative and quantitative data to show the presence of dye molecules on the surface of dyed cellulose textiles. Novel investigations of dyed fibres through X-ray photoelectron spectroscopy (XPS), determine the amount of dye present at the surface of cotton fibres. Using the N 1s atomic orbital region as a diagnostic peak, the level of dye loading could be directly attributed to the concentration levels of dye within the dyebath. The XPS data also provided strong evidence for possible dye-uptake mechanisms. We observed that certain stages of the dyeing process directly influenced the amount of dye entering cotton fibres. XPS showed that an increase in salt (NaCl) content within the dyebath produced dyed fibres with increased presence of dye compound at the surface. The knowledge obtained from these studies will help to improve the dyes and dyeing mechanisms for cotton and other textiles, thus improving the quality of dyed garments offered to the consumer.

The ability of short peptide fragments to self-assemble in isolation as amyloid and amyloid-like structures has prompted their use as model systems for the study of amyloid formation and recently also for their utilisation as novel nanofibrillar material. The atomic force microscope (AFM) is used here to investigate the self-assembly of two peptide systems and the development of strategies to directly manipulate and control the structures they form. The studies presented in Chapter 2 address the self-assembly of a peptide fragment of the human amylin polypeptide; amylin (20-29). In the opening study we use ex situ AFM imaging to characterise the early stages of amylin (20-29) fibril formation. High-resolution images reveal that following an initial lag phase, fibrils displaying a globular appearance are formed, which over time are replaced by flat ribbon-like fibrils with no periodicity displaying a range of polymorphic structures and assemblies. Following on from these findings, we investigate the influence of solution conditions on amylin (20-29) fibril formation utilising in situ AFM imaging. Altering the pH and electrolyte composition affords a range of morphologies including, truncated and long branched or unbranched flexible fibrils and globular aggregates. Following on from this characterisation chapter, in Chapter 3 a strategy to assemble specifically functionalised fibrillar material from chemically modified amylin (20-29) peptides was investigated. Azide and alkyne moieties were successfully coupled to the amylin (20-29) peptides. Ex situ AFM imaging and Congo red binding confirmed that the additional steric bulk had no detrimental effects on the fibril forming capacity of the peptides. Finally, in Chapter 4 the focus turns to the self-assembly of a dipeptide of phenylalanine which corresponds to the core recognition motif of the beta-amyloid polypeptide. Here, the AFM is used to study the physical properties of the well-ordered, discrete, hollow nanotubes which are formed. Their chemical stability in organic solvents and considerable thermal stability under both dry and wet heating conditions is revealed. Finally, the use of strong magnetic fields to directly control and orientate the diphenylalanine nanotubes was examined by AFM. The results presented throughout this thesis demonstrate the versatility of self-assembling peptides for the generation of fibrillar nanostructures that can be directly modified and controlled to generate novel architectures and functionalised well ordered nanomaterials.

The plant cuticle is located on most surfaces of the plant from seeds to leaves from stem to petal, this is to allow a direct interface between the plant and its environment. These cuticles act as a barrier to prevent waters loss from the plant to the environment and penetration of compounds through the cuticle, like agrochemicals and formulations. The leaf cuticle provides an ideal surface to try to penetrate agrochemicals through. The leaf surface has a larger surface area then most surfaces of the plant allowing ease of application of formulation via spraying. This makes the study of the leaf and its cuticle important, with the interaction of the formulation and the cuticle an area of interest. The main purpose of this thesis is to investigate the applications of a relatively new imaging technique called scanning ion conductance microscopy (SICM). This new technique is utilised to image live cells with the intention to characterise the living processors on the surface. SICM has not been used to image leaf surface before, but its non-contact nature and large z axis range makes it ideal for surface analysis. The first part of this thesis is to describe the comparison of SICM with other conventional techniques used to image leaf surfaces. For example, atomic force microscopy (AFM) and scanning electron microscopy (SEM) and asses the strengths and weaknesses of the technique for leaf imaging. This was achieved by imaging various leaf surfaces and surface features like epicuticular wax (EW) crystals and stomata. Also the possible research routes for the SICM were identified and experiments conducted to ascertain the abilities to perform them. This resulted in wetting being imaged and imaging the drying of a formulation. The other purpose of this thesis is to investigate the possibility of live leaf imaging and characterisation, and the implications of adjuvants on live leaves. This was achieved by thermal characterisation of different leaf surfaces in two states, them being live and intact (but dried). This allowed the understanding of the impact water has on the cuticle and the importance of studying live leaves. This shows that water has a plasticizing effect on the cuticle waxes, and also effects the structure of the cuticle. AFM with scanning thermal microscope (SThM) with local thermal analysis (LTA) were also utilised to investigate the impact of two adjuvants on the surface of live leaf cuticle. These were Brij 98 and Tris (2-ethylhexyl) phosphate (TEHP), Brij 98 in a non-ionic ethoxylated surfactant, while TEHP is a phosphoric acid ester known for its properties has a plasticizer. Both AFM and LTA showed that both resulted in the plasticizing of the cuticle with the area affected showing depression in melting transition compared with that of the native leaf surface. The thesis also shows that it is possible to characterise the impact of adjuvants on live leaf cuticles. This thesis has shown the importance of new techniques being used to image and characterise the leaf surface, showing that image wetting as a possible research route for SICM. The new techniques have resulted in new experiments being performed that provide insight into the interactions of the cuticle with formulations and components of formulations. Also the importance of water in understanding the structure of the cuticle.

A culmination of theory, techniques and devices stemming from a wide variety of sources and disciplines, optical microscopy presents vast possibilities for visualisation of small structures. One of the most fundamental yet significant optical microscopy techniques is Confocal Fluorescence Microscopy (CFM). CFM is studied here by analysing its performance with respect to the two most important metrics - Signal-to-noise ratio and 3D optical resolution. Several authors have commented on the inherent inefficiency of imaging systems such as CFM to utilise the available light when providing resolution beyond the well-known diffraction limit, primarily due to the precise mechanisms that help realise the resolution gain in the first place. In CFM, the detection pinhole is the key mechanism that helps realise up to 1.4 times resolution improvement over conventional wide-field microscopy techniques by trading off SNR. First, an investigation of the inherent SNR-resolution trade-off in a CFM system is studied; the impact of the detection pinhole geometry on the performance of a CFM is examined by means of an effective trade-off curve. Using alternative pinhole geometries in conjunction with new detection schemes, it is next shown how performance gains are realised in both the lateral and axial directions. Examined next is a recently developed detection scheme called subtractive imaging; wherein a special annular pinhole is used to divide the confocal point spread function signal into two detectors. By using fast point detectors in place of CCD arrays, it is shown how using numerical optimisation yields an optimum “differential pinhole” to achieve considerable 3D resolution gains over conventional (circular pinhole based) CFM systems. By examining the trade-off curves it is also shown that the proposed design is able to offer simultaneous and maximum performance gains up to a considerably high SNR in comparison to conventional (circular pinhole) based CFM systems. Lastly, the work will propose the use of a deconvolution technique and an alternative detection scheme to demonstrate substantially higher improvements in the quality of images acquired by a CFM system. Image reconstruction is a tried and tested image post processing strategy to realise super resolution. An image reconstruction technique, based on an expectation maximisation maximum likelihood (EM-ML) algorithm is used in conjunction with array detectors to demonstrate enhanced resolution and noise performance of a CFM system. The point scan method used here renders the algorithm slow with long run times. To mitigate this, structured illumination is used to show how similar resolution gains in the array detector based CFM systems could be realised but in a much shorter time.

The purpose of this study was to develop novel optical microscopy techniques in order to investigate colloidal drug particle endocytosis by mammalian cells. A total internal reflection microscope (TIRM) was initially developed for high resolution cellular imaging. TIRM is a non-fluorescent imaging technique based on the principle of ‘scattering’ of the evanescent field created when a light beam undergoes total internal reflection at an interface between two media with different refractive indices, such as glass and air. The key design considerations with respect to development of a TIRM instrument are discussed. The technique is also compared and contrasted to the more commonly known non-fluorescent RICM (Reflection Interference Contrast Microscopy) technique using computer simulations. Time-lapse video TIRM is applied to imaging the interaction between A549 and 3T3 cells, and a polylysine coated substrate. Real-time label-free visualisation of 0.5 and 1 m polystyrene particle endocytosis by living cells is then demonstrated. Modifications to the TIRM system to include a dual-colour fluorescent TIRF (Total Internal Reflection Fluorescence) microscope are described in detail. Results are shown which demonstrate the ability of a combined TIRM/TIRF instrument to selectively image the basal cell membrane both label-free and fluorescently. 3T3 fibroblast cells were genetically modified using standard molecular biology protocols to express the fluorescent fusion protein EGFP-Clathrin LCa (enhanced green fluorescent protein clathrin light chain a). Finally, colloidal particle endocytosis by the genetically modified cell was imaged using the TIRM/TIRF microscope. Direct visualisation of the internalisation of 500 nm particles via clathrin coated pits in 3T3 cells was shown for the first time.