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Reconstructing the surface microtopography through data fusion from a range of microscopies offers a way to provide considerably more information than any one technique alone. This thesis explores several methods for combining the info rmation from different kinds of microscopes including height data, electron images and optical images. Each microscopy has distinct advantages and disadvantages that can be considered in terms of data leakage in the transfer function from the actual sampl e topography to any individual dataset. These leakage paths are identified for a range of microscopies including atomic force microscopy, scanning electron microscopy and white light interferometry. Existing techniques for reconstruction from multiple image datasets, such as shape-from-shading and stereomicroscopy are evaluated, again in terms of information leakage. A new approach is presented which attempts to minimize leakage by identifying a pathway of data fusion which first isolates the most reliable information in each dataset, and then maintains this information as the datasets are combined. The identification of the most trusted data requires knowledge of each imaging mechanism, while the final reconstruction is based on iterative matching between the datasets and a simulated image. Hence the final reconstruction incorporates both the datasets from the microscopes and their imaging physics to suppress data leakage. This approach is first validated with a model micromachined sample using a combination of height interferometer data together with back-scattered and secondary scanning electron microscope images. It is then applied to particulate samples to demonstrate the capabilities of the approach.

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An experimental study of buckministerfullerene on the Au(111) surface and of chlorobenzene and oxygen molecules on the Si(111)-7x7 surface has been conducted with variable and room temperature (RT) scanning tunneling microscopes, respectively. First, the formation of hybrid clusters, (C\(_{60}\))\(_n\)-(Au)\(_m\), from 110 K to RT has been studied at different C\(_{60}\) molecule coverages. The properties of the hybrid clusters, such as rotation, transformation and diffusion, were observed at RT. Mechanical manipulation of C60 molecules in the hybrid cluster was performed in order to explore the production of a single type of hybrid cluster. Cascade manipulation was achieved by downsizing (C\(_{60}\))\(_{14}\)-(Au)\(_{63}\) clusters to (C\(_{60}\))\(_{7}\)-(Au)\(_{19}\) clusters at RT. The manipulation of the hybrid clusters was performed at 110 K in addition to RT. A comparative study of the non-local manipulation of chlorobenzene molecules and oxygen on the Si(111)-7x7 surface was performed with the RT STM via electron induced from the STM tip. It is found that a suppression region (~40 Å) for both molecules is quite universal in the STM experiments. A local desorption threshold of +1.4 V was found for the chlorobenzene molecule. Local manipulation of bright and dark sites of oxygen were induced six different transformations on the molecular sites.

This thesis presents advances made to the scanning ion conductance microscope (SICM), a tool predominantly used to date for topographical imaging of biological samples. This technique is demonstrated to be a powerful tool for non-invasive surface charge mapping as well, through probing of the diffuse double layer formed at charged interfaces. Surface charge mapping with SICM is demonstrated for a range of samples, including biological systems, and it is shown that through the use of a novel feedback technique, also introduced herein, and newly implemented scanning regimes, that the surface charge information can be elucidated unambiguously, together with topography. Through adopting a characterisation protocol presented in this work, which helps provide a fuller understanding of the used nanopipette probe, the SICM response to charged interfaces and also in bulk solution can become quantitative, allowing for surface charge values for cell membranes and other substrates to be determined. This combination of: SICM experiments, complete probe characterisation and FEM simulations serves as a robust platform for investigating biological and other charged interfaces. The surface charge mapping protocols used allow for unseen surface charge heterogeneities, presented on cell membranes, to be identified and are amenable to future studies, performed in combination with other microscopy techniques, that could help correlate charged domains with physiological function. Finally, the nanopipette probe is also used as a reaction centre for driving the crystallisation of calcium carbonate, as an exemplar system. Through partitioning the constituent ions of calcium carbonate, with calcium present in a bath solution, and carbonate ions in a nanopipette, a bias can subsequently be applied to drive the ions together, leading to the formation of a crystalline entity, which blocks the nanopipette. Changes in the nanopipette conductance can then provide information about the growth process or subsequently the dissolution as the applied bias is reversed. FEM simulations can allow for an understanding of the underlying mixing problem and the technique is shown to be powerful for the screening of growth additives.

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.

Detectors for Electron Microscopy have traditionally used a scintillator to generate photons from fast electrons, which are then detected by a sensor. However, in recent years direct detection has become an area of interest due to the potential improvements to detector performance. In this thesis various aspects of direct detection are presented. I will begin with simulations of direct detectors based on Joy’s model of straight trajectories between Rutherford scattering events, where signal is generated by inelastic scattering events. The effects of microscope operating voltage, detector thickness, a surface electrically dead layer and diode depth on detector performance are presented. A prototype detector was developed using the DUOS sensor, two thicknesses of the sensor were produced a 50μm thick detector and a 20μm thick detector. EBSD results are presented which show how the use of a reactive ion etch to reduce the dead layer thickness of a mechanically thinned sensor improve the detection efficiency of a sensor allowing EBSD work to be carried out at operating voltages as low as 5keV. The MTF and DQE of both thicknesses of DUOS sensor are measured at 80kV and 200kV, which show that there is little difference between the two thicknesses at 80kV, but at 200kV the thinner detector shows an improved MTF. The results are then and compared with the equivalent simulated detectors. I show how the high frame rate of a detector and rigid and non-rigid registration can be used to improve image quality, resolving the {331} lattice spacing which is not visible with a simple summation of frames. Detectors using gallium nitride rather than silicon as the base semiconductor are simulated. The MTF at the Nyquist frequency for a GaN detector is double that of a Si detector at an operating voltages of 80kV due to the smaller interaction volume of an electron in GaN. However, at higher voltages the improvement is much smaller as most electrons pass through the detector.

Fluorescence imaging has become a vital tool for understanding cardiac cell function. With the advent of calcium-sensitive and fast response voltage-sensitive dyes, it is now possible to gain complex physiological recordings from various cardiac muscle preparations, while simultaneously optically sectioning the samples. However, due to the complexity of these experimental set-ups, it is difficult to set up control samples that allow the user to predict how their sample is affecting the quality of the data being captured. The aim of this thesis is to assess the strengths and weaknesses of the different types of optical sectioning microscope methods that are currently available in many biological labs. These include confocal, two-photon and selective illumination microscopes. The resolution capabilities of these systems and the associated objective lenses were investigated to determine the optimum conditions for their use. This work includes investigations of the contribution of signal noise and performance of motorised z-stage systems used in these microscope systems to the ability of assess optical resolution. The optical components of cardiac muscle are investigated individually. This includes the scattering properties of the myocardium and the effects of the refractive index mismatch, as well as the intrinsic fluorescent substances found in cardiac muscle. Microstructures are used to generate novel ways of overcoming some of the problems faced when imaging cardiac muscle in order to improve the sensitivity and resolution of these systems. Finally, a method is investigated to enhance the the relative concentration of live cardiac:dead cells in after dissociation from intact hearts. This approach would aid the study of isolated cardiac cells be reducing the interference from damaged cells.