Electron cryomicroscopy reveals biological structure in a native, frozen– hydrated state. Images are recorded under low dose conditions in order to reduce radiation damage. They are therefore low in contrast and require computational analysis to extract structural information. This thesis describes the development of computational methods for structure determination by single particle analysis and tomography and their application to several biologically important specimens. The reliable determination of reconstruction parameters in single particle analysis is the first subject addressed. To determine a structure from projection images, relative orientations must be assigned to each view of the specimen as well as microscope parameters describing magnification and contrast transfer. A new method of magnification determination is described that provides an automatic estimate of magnification using apoferritin as a reference. Modelbased refinement of particle orientation parameters is susceptible to “model bias” in the case of noisy experimental images and can result in incorrect orientation determination. A new implementation of tilt pair cross-validation was created in order to validate orientation parameters. The software has been implemented as a fully automatic web server toward the goal of providing a standard cross-validation tool. The latter part of the thesis is devoted to problems in the computation and interpretation of low contrast, sparsely sampled tomographic reconstructions. The first model of an intact Weibel-Palade Body (WPB) was built based on tomograms computed from tilt series of frozen-hydrated endothelial cells. A template matching procedure was introduced in order to facilitate the analysis of von Willebrand Factor packaging in WPBs leading to an understanding of the morphology and physical properties of WPBs. New analytical tools were also required to facilitate the interpretation of the spatial organization of glycoproteins on the surface of influenza A virus that may have important consequences for influenza biology. A three dimensional model of the influenza haemagglutinin positions on the virion surface was constructed based on analysis of cylindrical projections.

Probe based confocal laser endomicroscopy (pCLE) is an in vivo device which acquires high resolution superficial optical biopsies. Despite encouraging data on neoplasia identification in colorectal polyps and Barrett's oesophagus, there are many hurdles to pCLE advancement beyond the research arena. This thesis defines these challenges, and via 5 experimental chapters expounds four key requirements: 1) the effect of probe contact force upon the tissue needs assessing and harnessing; 2) the probe's diagnostic accuracy needs to be improved; 3) the probe's site of optical biopsy acquisition needs improved tracking; and 4) the ergonomics of pCLE optical biopsy need development. These challenges are elucidated and elaborated upon using two short clinical studies. A library is created describing the differences between normal, inflammatory and neoplastic endobronchial pCLE images from 16 pCLE bronchoscopies, but an inability to distinguish between endobronchial carcinoma and carcinoma-in-situ is shown. The tolerability of pCLE imaging of the colorectum over five sessions during chemoradiotherapy for advanced rectal cancer is explored, and possible morphological changes are described. The accuracy of bronchoscopic pCLE for diagnosing peripheral lung diseases is investigated. In 116 bronchopulmonary segments in 43 subjects pCLE safely provided video sequences for comparison with computed tomography, transbronchial biopsy, and bronchoalveolar lavage. With pCLE, non-emphysematous peripheral diseases tend to create diminishment of the distinct elastin alveolar microstructure, and distinction between microvessels and septal walls becomes more difficult. Alveolar septal wall density is reduced in severe emphysema, and the pleura is demonstrated beyond bullae in two patients. Though assessors have some consistency, they are shown to be unable to accurately judge the category of emphysema severity in this small study. Intravenous fluorescein is shown to cause 'white-out' and thus provide no advantage for pCLE imaging of the pulmonary lobule. At least in the laboratory, this thesis demonstrates that mucosal distension/colonic inflation and probe contact force significantly affect the consistency of pCLE images in terms of gland diameter and intergland separation. It also introduces the concept of pCLE elastography: if a device attaches to the probe to enable it to produce a sinusoidal force upon the tissue, the amount with which the glands individually expand/shrink and separate is altered if the mucosa is artificially stiffened to mimic disease. It suggests there is potential for improving pCLE ergonomics, and tracking (at least within peripheral lung). A transvaginal flexible endoscopic robot is predicted to reach 88% of a human peritoneal cavity for pCLE periteoneoscopy; a porcine feasibility study provides interpretable abdominal cavity images. Probe tracking in the peripheral lung is especially difficult and important in heterogeneously distributed disease. A study using ex vivo porcine lung assesses the predictability of probe tip destination; it shows that only half of the probe deployments beyond the most distally visible bronchus have a single possible path. Finally, a role for pCLE evaluation of chronic radiation proctopathy is proposed. 26 patients with symptomatic disease are pCLE sigmoidoscoped with correlative pinch biopsy. Macroscopically severe disease is associated with pCLE sequences showing glandular disruption and vasculopathy (dilated or absent vessels; 'dilabscence'). A score from 16 histological features correlates with pCLE vessel dilabscence and glandular disruption suggesting pCLE may have a role in the longitudinal monitoring of this unpleasant disease. Overall, the thesis approaches pCLE challenges from several angles in both laboratory and clinical settings, and the data are often descriptive and preliminary. However, it significantly expands upon the understanding of pCLE in peripheral and endobronchial lung disease, describes two novel clinical roles for pCLE (radiation proctopathy and evaluation of neoadjuvant therapy response), and contains the first description of two technical pCLE developments (elastographic interrogation and robotic actuation). The time is fast approaching when one of these clinical niches capitalises on pCLEs enormous potential for mainstream deployment.

No description available.

The Atomic Force Microscope (AFM) is an invention that has enabled a significant number of studies and discoveries in the field of nanotechnology. It is well-known that the resolution of AFM-based applications is critically dependent on the tip bluntness of the probe utilised. Numerous researchers have proposed different approaches to assess the condition of AFM probe tips. In spite of these efforts, further advances are still needed for the express analysis of the bluntness of such tips. In this context, the overall aim of the research work presented in this Thesis was to investigate a novel in-situ technique for assessing the apex condition of AFM tips. In particular, this technique relies on the analysis of depth-sensing data obtained from the nanoindentation of the probe tip into a soft elastic sample. Nanoindentation is a process that is readily implemented on AFM devices. For this reason, the proposed technique could be a fast an efficient approach for deciding when AFM probes should be replaced. The theoretical argument on which the technique is based is that the current shape of the tip apex in its working position within an AFM device can be approximated as a power-law function and that the exponent of this function can be used as a quantitative measure of the tip bluntness. Based on this approximation and the use of the self-similar (scaling) approach to depth-sensing indentation, it is possible to extract this bluntness parameter, herein also referred to as the degree of tip bluntness, from AFM nanoindentation data. The practical implementation of this technique was realised using a commercial AFM device and commercial probes. The actual geometry of the apex of these probe was also studied in details using additional experimental methods via the use of Scanning Electron Microscopy and also via the so-called “reverse imaging” method to obtain two- and three-dimensional data about the tip apex of these probes. Among the different iv contributions made from the work carried out in this research, the most important conclusion is that a good agreement was found between values of the bluntness parameter evaluated by the proposed technique and the effective bluntness obtained from analysing the actual three dimensional geometry of the AFM tips. Thus, it can be argued that the technique put forward in this work for the express analysis of the bluntness of AFM probe tips using depth-sensing nanoindentation can be considered as a valid method when assessing the condition of AFM probes.

Super-resolution microscopy is a relatively new and rapidly growing field. Development has been largely technology-driven, with high power lasers, higher resolution CCD cameras, and increasing computing power all enabling new biological questions to be explored. Single-molecule imaging is the tool of choice for studying systems where heterogeneity is present; ensemble methods can average away the interesting behaviour and lead to false conclusions. This thesis develops and optimises bespoke fluorescence microscopy for application to three biological questions, each pushing a limit of super-resolution imaging. Super-resolution imaging of lambda DNA labelled with the intercalating dye YOYO-1 and the minor groove binder SYTO-13 at localisation precisions of 40nm and 62nm respectively has been achieved in preparation for combined fluorescence imaging and magneto-optical tweezers experiments. The combination of these two methods is challenging as both operate with low tolerances.\ Single-molecule tracking was used to measure the diffusion coefficients of the chemokines CXCL13 and CCL19 at extremely high temporal resolution. Single-molecule imaging was found to have advantages over the ensemble techniques of FRAP and FCS for measuring the diffusion coefficient of the test molecule; Alexa Fluor 647 labelled bovine serum albumin. The diffusion coefficients of the two chemokines, CXCL13 and CCL19 were found by single particle tracking at sub-millisecond timescales in a collagen matrix to be 6.2±0.3µm2s-1 and 8.4±0.2µm2s-1. Further, CXCL13 was tracked in B cell follicle regions of ex vivo lymph node tissue sections at ~2 millisecond timescales, giving a diffusion coefficient of 6.6±0.4µm2s-1.\ Fluorescence microscopy was used to elucidate the stoichiometry of YOYO-1 on DNA origami tiles after treatment with low temperature plasma. Undamaged tiles were found to have a mean stoichiometry of 67.4±25.2 YOYO-1 molecules and a model of LTP damage to DNA origami tiles was proposed.

The cell is a fundamental yet complicated unit of biology; a dynamic system of interacting processes. To observe these processes, it is necessary to develop a contrast mechanism that isolates the behaviour under study from the natural cell contrast. Commonly this is achieved by labelling with single fluorescent entities; however, due to the inherent photochemical properties of single quantum emitters the photon rate is fundamentally limited and furthermore prone to blinking and bleaching. Recently, metallic nanoparticles have gained significant interest as biological labels as they do not suffer from these limitations as they scatter light proportional to the incident power. However, the applied power can not be infinitely increased as cells are sensitive to photodamage. As such, it is important to develop a technique capable of detecting weakly scattered signals at low incident optical powers. We have recently shown the detection of 5 nm gold nanoparticles at low incident power (< 1μW) under a technique we have termed Interferometric Cross-Polarization Microscopy (ICPM). Toward the goal of metallic nanoparticle detection in biological material, herein we characterise the complex Point Spread Function (cPSF) of ICPM and show its capacity for optical sectioning. We develop a model for the detection sensitivity of ICPM to elucidate the detection capabilities and areas of potential application. Building from these chapters, we show how the technique may be applied to colocalise weak scattering signals of individual nanodiamonds against their fluorescent signatures from embedded NV- centres. And finally, we apply ICPM to the detection of metallic nanoparticles in HeLa cells.

Super-resolution microscopy has become an important tool to image cells at ever increasing resolution. Specifically, localization microscopy, in which single emitters are localized with high accuracy, has allowed us to follow and track single-molecules in live cells. The technique has revealed aspects in the proteins' real-time function that were impossible to study previously. Despite their extensive use in recent years, there are still possibilities to improve and validate the technique and the resulting data. This thesis presents the first tracking photo-activation localization microscopy (tracking PALM) on three transcription factors (TFs): the lac repressor (LacI), araC protein and the cAMP receptor protein (CRP). With this work I expand the range of applicability of localization methods and increase in detail an important part of cellular function. TFs control the expression of genes so that cells can adapt to external conditions. In Escherichia coli (E. coli), previous studies have shown that more than 60% of TFs have less than 100 monomers per genome copy. For example LacI, involved in the lactose utilization control, exists in E. coli in around 40 monomers in contrast to other proteins that exist in the tens of thousands. To study such low-copy numbers we found it necessary increase the throughput of tracking PALM and this thesis summarises our efforts to increase throughput. After constructing a setup capable of tracking PALM with a 3X increased throughput, I studied the diffusion and localization of LacI and LacI relative to its operators. By studying LacI, I found that there were a number of challenges related to low copy numbers, specifically background noise that leads to localisations that can affect the results. I characterized the background tracks and tested the ability of clustering to find a number of tandem operators. This knowledge allowed us to study other TFs: araC, involved in the control of arabinose utilization which also exists in low-copy numbers but functions in multiple genes; and the CRP, which both regulates multiple sites and exists in large copy numbers. In addition I worked towards the understanding of TF binding by simulating the combination of non-specific binding and free diffusion that emulates the search process of TFs. I obtained insight into the analysis of the data generated from tracking PALM. Finally I focused on creating a strategy to use our existing data to extract blinking rates from the fluorescent protein. Our insights into TFs diffusion and binding have shown that tracking PALM can yield important insights at this regime of protein concentrations. Our work can be applied to other TFs and proteins that function in low copy numbers.

A magnetically coated tip is a fundamental part of the MFM instrument. These tips’ are bought commercially and/or individually manufactured in various shapes and sizes and with various material coatings and thicknesses. The sheer extent of possible combinations and the lack of a truly standard and reproducible tip is perhaps, one of the major contributing factors that prevent a complete understanding of the instrument and its characteristics and a full comprehension of how the tip interacts with a sample. While the MFM instrument is capable of generating qualitative images, a full metrological characterisation of its magnetic probe is one of the major concerns. In this research project, the practical implications of a diagnostic sample in the form of a simple geometrical wire structure have been demonstrated. With the aid of mathematical modelling, the understanding of the interaction between the tip and the sample is improved. In addition, this research explored the effects of systematic reduction of a tip’s magnetic volume and its resulting images. It highlighted the significance of magnetic volume in image capture and provided a comprehensive quantitative insight in image type, reproducibility and quality. This project thus represents a further step towards the characterisation of MFM probes, which has the potential for ultimately benefitting the nano-magneto-electronic and data storage industry.

As the size of the bulk crystal reduces to the nanometre scale, anatase titania exhibits enhanced photocatalytic properties. Nanostructuring of TiO2 involves engineering the crystal facets in a way that speci c types of surfaces dominate the 3D shape. The atomic structure of the surfaces and 3D morphology of the crystal determine the electronic properties of the material, and should be characterized with atomic precision. Due to its high spatial resolution (0.1 nm), aberration-corrected transmission electron microscopy was used to obtain morphological and structural information on anatase nanoplatelets and nanorods. TEM morphological analysis showed that the main 3D shape of the platelets is that of a truncated tetragonal bipyramid, where f001g facets dominate. This 3D shape is accessible via 2D projections of the crystal structure. In the nanorod specimens, the types of edge morphology found link to intermediate or nal stages of growth, occurred via oriented attachment of primary nanocrystals and classical monomer addition. The structural characterization of the nanocrystals was carried out by examining the exit plane wave of the specimen, which was reconstructed from a serial acquisition of aberration-corrected TEM images of di erent defocus. The phase of the reconstructed wave reproduces the atomic potential of the specimen, and provides information with the maximum resolution of the microscope. The optical properties of the platelets and rods were also analysed using a combination of STEM imaging and EELS. Due to the high surface to volume ratio of the platelets, the EELS spectrum is dominated by strong surface features that arise from the polarization of the surface electrons induced by the electron beam. The in uence of the surface excitations on the EELS spectrum is strongly determined by the thickness of the platelets: by modifying the crystal thickness below 20 nm, the frequency of the surface excitations changes, enabling the optical properties of titania to be tuned in the visible and UV range. Finally, preliminary EELS investigations on the nanorods suggest that, unlike metallic nanoparticles, the surface excitations are not in uenced by the morphology of the crystal, but strongly depend on its thickness.

Optical microscopy and scanning probe microscopy techniques have been utilised to acquire high resolution topography and fluorescence images of several biological samples. Applying these techniques to patterned samples and single molecules allow the optical properties of a sample to be investigated near to and below the diffraction limit, allowing emission properties to be correlated with those of topography. Optically active biological samples outside of their cellular environment are prone to photo-degredation and in measuring them a challenge is to ensure that optical measurements can be made before the onset of damage to the fluorophore. In this study two forms of fluorescence microscope have been utilised with scanning probe techniques of AFM and SNOM. These techniques have been used alongside microcontact printed arrays of fluorescent proteins and photosynthetic light harvesting complexes to address the accuracy of the printing technique and it's applicablity to biological components for future bionanotechnological applications. Furthermore, the periodicity associated with the arrays has been applied to the techniques to address the relative resolutions of the microscopes as well as the samples being a drive behind implimenting biologically friendly components/techniques to the microscopes (such as liquid cells). Larger structures from photosynthetic bacteria have also been addressed in this study in the form of chlorosomes which are model structures for light harvesting in low light conditions. Studies on the spectral properties of populations of 3 species have been conducted in this work with fluorescence microscopy and it has been shown that populations show small local variations in fluorescence. Furthermore it has been shown that the developed scanning fluorescence technique can be applied to photo senstitive samples successfully with only a small number of cases where spectral properties were affected by the measurement technique. Using high resolution microscopy techniques this research shows the surface patterning techniques in conjunction with biological samples to have mixed success depending on the sample. It also shows spectral measurements on newly discovered chlorosomes with little photo degredation. It further shows the role that the microscopy techniques have in analysing biological systems in different configurations on substrates.