Self-assembled protein nanotubular materials are attractive as putative building blocks for a variety of applications. Knowledge of the three-dimensional structures and the physical properties of these protein nanotubes then becomes a prerequisite for their use in rational materials design. The main purpose of the work presented in this thesis is to investigate both the structural and mechanical properties of protein nanotubes utilizing atomic force microscopy (AFM). Several different protein nanotubes will be used as exemplars to develop AFM methods. AFM is capable of both visualizing and monitoring dynamic processes. Within this thesis, not only could the change in morphology of protein nanotubes be visualized by AFM, but also changes in their mechanical properties were monitored as dynamic processes. For example, changes in the morphology (in chapter 3) and flexibility (in chapter 4) of lysozyme fibrils during fibrillization were investigated. Chapters 4 to 6 describe a range of different methods to obtain the mechanical properties of protein nanotubes: the persistence length method (chapter 4), the adhesive interaction method (chapter 5) and the bending beam method (chapter 6). All of these had their own advantages. However, each method was found only to be suitable for protein nanotubes with elasticities within a defined range. The protein nanotubes investigated by AFM in the thesis included Salmonella flagellar filaments, lysozyme fibrils and diphenylalanine (FF) nanotubes. All of the investigated protein nanotube structures had Young’s moduli lying between that of gelatin and bone. This highlights their potential, in terms of mechanical properties, for a range of applications in drug-delivery systems and tissue-engineering scaffolds. In future, if a database of mechanical properties of protein nanotubes could be built up using the AFM methods developed and utilized within this thesis, the development of the applications of protein nanotubes will be accelerated, as the right protein nanotubes will be selected for appropriate applications.

Scanning probe microscopy allows the investigation and manipulation of matter at the atomic and molecular level, and is crucial in the development of new and novel techniques within nanoscience. However, to understand the information obtained from the various forms of scanning probe microscopy, a thorough theoretical understanding is necessary. Often this theoretical background is provided through density functional theory, which, while incredibly powerful, has limitations with regards to the size and complexity of the systems in which it can investigate. Thus, for more complicated systems, alternative techniques are desirable to be used both independently and alongside density functional theory. In this work, theoretical techniques are constructed that allow the information obtained from both scanning tunnelling microscopy and atomic force microscopy to be investigated for a variety of systems. These techniques are all based around Huckel molecular orbital theory or extended Huckel molecular orbital theory, and use a simple linear combination of atomic orbital basis, that allows rapid analysis of various systems. The main focus of the work is the scanning probe microscopy of the C60 fullerene molecule. Theoretical scanning tunnelling microscopy images are constructed for the cases where C60 is adsorbed on both the substrate and the scanning probe in the form of a functionalised tip, as well as when a tip-adsorbed molecule interacts with a sample-adsorbed molecule. The atomic force microscopy images of surface adsorbed C60 are considered, with the main focus centred on the repulsive interaction observed due to the Pauli exclusion principle. The structure of the scanning probe, and the effect this has on this imaging is examined, as well as considering the atomic force microscopy images obtained when two C60s interact. Molecules other than C60 are also considered, with the techniques developed used to interpret and understand the atomic force microscopy images obtained when a pentacene and a PTCDA molecule interact with a carbon monoxide functionalised tip. The theoretical work is accompanied throughout by a variety of experimental work, both from previously published work, and from unpublished work obtained by the University of Nottingham nanoscience group. Much focus is given to the interaction between C60 and the Si(111)-(7x7) reconstruction, both in the sense of a functionalised tip interacting with the surface, and with the interactions present where a C60 is adsorbed onto a surface. In doing so, previously postulated bonding sites for C60 on this surface have been verified.

The class of instruments considered in this thesis, scanning probe microscopes (SPM), raster scan a sensory probe over a surface to form both high resolution images and quantitative interaction measurements. Understanding and extracting information from SPM data has been considered extensively in the three decades since the first SPM. Major developments tend to be greeted with their own theory and data analysis techniques. The more gradual evolution of equipment has not, however, attracted the same level of theoretical consideration. In this thesis we consider the SPM from an instrumentation perspective, concentrating on two specific types of microscope: the scanning tunnelling microscope (STM) and the atomic force microscope (AFM). Both of these microscopes rely on a sensory probe or sensor to induce and measure the desired interaction. We have carefully considered a range of experiments from a `sensor-eye-view', both theoretically and experimentally. We first consider the effect of the geometry of AFM sensors on quantitative force measurements, identifying that the length of tips that the length of tips can induce an unwanted coupling of lateral and normal forces. We go further by developing methods to experimentally correct these force measurements along with designing a sensor which exploits symmetry to separate lateral and normal forces. We also consider the ways to automatically optimise the apex of the sensory probe of an STM to give the desired imaging resolution using a combination of prescribed routines and genetic algorithms. Image analysis techniques developed for this work have been developed into an open-source toolbox to automatically process and analyse SPM images. Finally, we use control theory to analyse the feedback controlling the SPM probe. We find that the methods used in the literature do not fully consider the method with which the control loop is implemented in SPM. We employ a modified approach which results in more realistic simulated SPM operation.

In this thesis, a study of a range of functional surfaces formed in ultra-high vacuum (UHV) conditions using primarily scanning probe microscopy is presented. The construction of a combined scanning tunnelling and atomic force microscope, and the experiments performed using this instrument, are also detailed. Boron nitride and graphene monolayers were formed on rhodium thin films in UHV and investigated with in-situ and ex-situ (ambient conditions) scanning tunnelling microscopy. Simultaneous scanning tunnelling and atomic force microscopy images were also produced for the graphene monolayers. X-ray photoelectron spectroscopy and diffraction results for graphene monolayers on Rh(111) surfaces, as well as low energy electron diffraction data, are also included. The novel formation of monolayer and few-layer graphene on nickel thin films is also described. Graphene layers were detached from these nickel thin films and isolated on other substrates. The results of characterisation experiments using scanning probe microscopy, X-ray photoelectron spectroscopy, X-ray diffraction and electron microscopy techniques are detailed. Graphene layers with approximately 75% monolayer graphene coverage and an increased electronic quality, when compared to many other methods of graphene production, were revealed. Different organic molecules were adsorbed on both the boron nitride and graphene monolayers formed on rhodium thin films in UHV conditions. Perylene tetracarboxylic diimide (PTCDI) and di(propylthio)-PTCDI molecules were investigated on these surfaces and compared with the adsorption of PTCDI on a graphite surface. Furthermore, dibutyl-coronene tetra-carboxylic diimide was deposited on the graphene (on rhodium) surface, in UHV. Although the boron nitride and graphene surfaces were similar, it was discovered that very contrasting molecular formations were formed on the dierent surfaces. The positioning of these nanostructures was determined by the Moire superstructure formed due to the mismatch between the monolayers and the Rh(111) surface. Additionally, different hydrogen-bonded molecular junctions were formed depending on the length of the side chains of the adsorbed organic molecules.

This work is focused on exploring a unique integration of techniques, Raman micro-spectroscopy and atomic force microscopy (AFM), which when combined offer more than the sum of their respective parts. The non-invasive chemical specificity afforded by Raman spectroscopy, combined with the nanoscale-resolution topographic imaging of AFM offer much individually. The physics underlying the practical application of each technique is very general; Raman spectroscopy detects molecular vibrational shifts using light, and AFM uses a physical probe to interact with a surface to provide topographic (and mechanical) information. As a result, there are few restrictions to the possible samples that can be studied with these techniques, from semiconductors and geological crystals, through to simple organic chemical structures all the way to complex biological molecules and systems such as cells and tissue. In this work, a synthetic biomaterial composed of diphenylalanine (FF) peptide units which self-assemble into strong tubular structures is used as a sample of interest when exploring the different possibilities available from a combined Raman-AFM instrument. First, the combined system was set up in order to perform tip-enhanced Raman spectroscopy (TERS), a technique promising Raman spectroscopic imaging at the resolution of AFM imaging. A relatively young technique, TERS has huge potential in extending the reach of Raman spectroscopic imaging to the nanoscale, at a regime where a great deal of structure exists, but is usually blurred by conventional diffraction-limited Raman microspectroscopy. A major focus in this work is addressing a current problem with TERS: the fabrication of suitable probes. TERS typically utilizes AFM tips modified to have a silver nanoparticle, capable of locally enhancing the Raman signal, attached at the probe apex. A new method is presented here that promises several improvements over existing approaches, as the entire fabrication can be performed in-situ on the instrument. Tips produced in this way are then characterized by electron microscopy and tested on FF nanotubes. Following this, several techniques for the synthesis of silver nanoparticles are explored for use in TERS. Here, the focus is particularly on decahedral nanoparticles, which can be grown into rod shaped particles with well- defined shapes and sizes. These are important considerations for obtaining the desired enhancing properties for TERS probes. Finally, the AFM-Raman instrument is used to investigate the mechanical properties of FF tubes using several methods. AFM force spectroscopy of tubes suspended across a gap can be used in conjunction with a bending beam theory to measure the Young's modulus of individual tubes. A new type of co-localized experiment using polarized Raman spectroscopy on a suspended tube under various forces from the AFM is tested, and subsequently information relating to the hydrogen bonding network is used, in conjunction with existing X-ray data, to determine the molecular contributions to the modulus using a simple model for amyloid fibrils. Each experiment operates at the single fibril level, with the same fibrils being used, such that different methods can be compared for a single FF tube.

Scanning probe microscopy (SPM) allows us to directly measure the interactions between a probe and a sample at the atomic scale. Techniques such as non-contact atomic force microscopy (NC-AFM), allows us to to characterize the forces present on a surface, resolve the atomic structure of molecules or examine their chemical properties, while scanning tunneling microscopy (STM) allows their electronic properties to be characterized. As the interactions take place at the atomic scale, the atomistic state of the probe apex plays a crucial role. In AFM, it is the atomic scale forces between the outermost atoms of the probe and surface that are dominant, while for STM the density of states (DOS) that contribute to tunneling are crucial. Therefore, understanding and controlling the tip termination is crucial to derive meaningful interpretations from experimental data. In this thesis, the role of the tip termination is examined for various surfaces and situations. We find that determining the "right" tip state depends critically on the experiment and several general strategies for shaping the tip apex into a preferred state are therefore outlined. H:Si(100) surfaces were used as a substrate for lithographic patterning using STM. We have successfully implemented an automated extraction routine for performing large scale patterning with high fidelity and single atom specificity. Our ultimate goal is to combine the extraction routine with SPM image recognition software to allow analysis and manipulation of atomic scale features without human intervention. To perform manipulations reliably, the tip influence on "what we see" (tip imaging states), or specifically on what the recognition software can identify, needs to be considered. We find, counter-intuitively, that atomic scale manipulation with the highest fidelity occurs when silicon dimers are observed as rows as opposed to when atomic resolution imaging occurs. The tip state influence on measuring surface diffusion of PTCDA on Ag(110) surfaces, was also investigated. We find that the adsorption kinetics of diffusing molecules can only be detected for specific tip imaging states. To allow examination with no-human intervention, the tip state needs to be carefully considered, and a combination of analytical and spectroscopic tools needs to be implemented in conjunction with the experiment. Additionally, characterization of the tip apex was investigated at the tunnel junction between a STM tip and a metal surface using field emission measurements. Our results suggest that field emission measurements performed at the tunnel junction are sensitive to changes in the nanoscopic/mesoscopic tip apex structure, thus opening up the possibility of automating the process of characterization the tip apex.

When a polymeric biomaterial is employed within a living system an interface is created between the solid surface of the polymer and an aqueous environment. The processes that occur at this interface will determine if the biomaterial is accepted by the patient and often will determine if the specific function of the biomaterial can be achieved. Increasingly, novel biomaterials are expected to perform more sophisticated functions and, therefore, their surfaces must be designed to realize precise interfacial events, such as specific interactions with proteins and cells or controlled biodegradation. To design polymeric biomaterials with specific surface properties it is necessary to develop surface analytical techniques that can accurately characterize these properties. The work described in this thesis has aimed to investigate the potential contribution of the atomic force microscope (AFM) to this characterization. The advantages of utilizing AFM in the study of polymeric biomaterials lie in the ability of the instrument to visualize insulating surfaces at a high resolution within a variety of environments, including gaseous and liquid environments. Therefore, it is possible to image the nanoscopic organization of polymeric biomaterials within environmental conditions that are similar to the conditions encountered within living systems. Initial studies have concentrated on imaging the surface morphology of poly(ethylane oxide) (PEG) samples in air. These studies highlighted the high resolution capability of the AFM on untreated polymer samples. On sphemlitic samples, the AFM has visualized the lamellar organization of crystalline fibres. These lamellae had widths of between 10 and 30 nm and height variations of less than 15 nm. The ability of the AFM to resolve such structures, without the introduction of an etching or staining procedure required by transmission electron microscopy, relies on the sensitivity of the instrument to changes in the height of the topography. This sensitivity has been further utilized to image polymer strands with recorded widths of 8 nm. This width represents an overestimation of the true dimensions of the strand due to the finite size of the AFM probe apex and using the circular probe model it has been calculated that the strands have true widths of less than 0.8 nm, indicating that they are composed of one or two PEG molecules. Further studies on PEG have demonstrated the ability to control polymer surface morphology through changes in the temperature of thin film preparation and changes in the method of polymer solution deposition. The work on PEG surface morphology acts as the foundation for the remaining studies, which employ the AFM to study biodegradable polymers within aqueous environments. This in situ application of the AFM has recorded the changes in surface morphology that occur to poly(sebacic anhydride) (PSA) during surface erosion in alkaline conditions. These studies have visualized the preferential degradation of amorphous regions of sphemlites over the crystalline fibres for solution cast and melt-crystallized samples. It has been found that rapid cooling during the solidification of PSA increases the amount of amorphous material at the surface of samples. However, once this outer layer has been eroded the underlying material is dominated by crystalline fibres. In situ AFM studies have also demonstrated the pH dependence of the rate of PSA surface erosion. The AFM techniques developed to visualize the evolution of surface changes during PSA erosion have then been employed to investigate the degradation of immiscible blends of PSA and the polyester poly(DL-lactic acid) (PLA). PLA degrades at a slower rate than PSA and therefore, as these blends eroded the surface morphology became dominated by PLA, revealing the phase separation of the material. For solution cast samples on mica substrates it was found that at high PSA content the PSA formed a continuous network around islands of PLA. However, as the relative content of PLA increased the morphology reversed and the PLA formed the network around islands of PSA. The interest in studying biodegradable polymers is derived from their application in surface eroding drug delivery systems. Having demonstrated the potential of the AFM to visualize dynamic interfacial changes occurring to these polymeric biomaterials, the in situ studies were extended to investigate the release of a model protein drug from a degrading polymer film. The system under investigation was a poly(ortho ester) film containing particles of bovine serum albumin. The AFM visualized the initiation of dissolution of some protein particles within minutes of the exposure of the sample to a pH 6 environment. Other particles, however, displayed retarded dissolution behaviour and did not appear to dissolve until the sample had been exposed to the pH 6 environment for over 1 hour. To assist the interpretation of these studies computational methods of calculating changes in volume during polymer degradation and protein dissolution have been developed on the Genesis II system. In the final experiments of this thesis, the application of a novel combined atomic force microscopy/surface plasmon resonance instrument is described. This instrument allows the simultaneous acquisition of topographical data by the AFM and kinetic data by the surface plasmon resonance instrument (SPR). The instrument is first applied to a simple poly(ortho ester) system to demonstrate that the changes surface morphology and polymer film thickness can be simultaneously monitored. Then, the PSA/PLA blends were re-analysed. This analysis highlighted the synergistic information obtained by the combined AFM/SPR and revealed new data on the relationship between polymer phase separation and biodegradation kinetics. NB. This ethesis has been created by scanning the typescript original and may contain inaccuracies. In case of difficulty, please refer to the original text.

In this thesis we go through a journey of atomic manipulation of Pb dimers on Si(100), then we examine the limitations of AFM in inter- molecular resolution and we take a closer look at a very promising technique named simultaneous STM/AFM which opens new horizons in the field but is part of an ongoing debate as it is still under development. NC-AFM imaging of adsorbed Pb dimers on Si(100) provided detailed information of the Pb configuration, which agrees with previous STM studies. The lateral force required to move a Pb dimer is related to the adsorption of the dimer on the surface, the associated potential energy land scape and in some cases the interaction of the Pb dimer with its neighbouring dimers. In the next part of this thesis, we examine the adsorption of a small organic molecule (NTCDI) on Si(111)-(7×7) and we report two energetically preferable geometries among others. In the last part of this thesis, we present an analysis of the crosstalk effect using an Omicron commercial qPlus sensor and a home built sensor with two system configurations and we compare them with a Createc commercial system. The results show that in order to avoid crosstalk the range of the STM operational pre-amplifier and the optimal elec- tronics wiring play a major role.

Scanning tunnelling microscopy (STM) and atomic force microscopy (AFM) can produce images of molecules with extremely high resolution. However, Claims that dynamic force microscopy has the capability to resolve intermolecular bonds in real space continue to be vigorously debated. Several studies have now shown that tip flexibility, especially at very close tip-sample separations, is responsible for the striking intra- and intermolecular resolution observed with various scanning probe microscopy techniques. The apparent intermolecular features can be observed with dynamic force microscopy even when no bonding interaction is present, suggesting that such features are in fact an artefact and cannot be interpreted as a real-space image of an intermolecular bond. We have studied the interaction between fullerene (C60) molecules using a sum of pairwise Lennard-Jones (12-6) potentials, and investigated how flexibility in the tip can produce a bond like feature between the molecules in a C60 island where there is no chemical bond present except the weak van der Waals force. We also investigate how the potential between the molecules is dependent on their relative orientations. For a given configuration of the tip and the sample molecules, our results allow us to predict the form of the intermolecular potential that would be observed using non contact atomic force microscopy (NC-AFM). Our study on the Si(111)-(7x7) reconstructed surface using the same model provides a better understating on the origin of ‘sub-atomic’ contrast observed in experiment suggesting that the contrast can arise from a flexible tip exploring an asymmetric potential created due to the positioning of the surrounding surface atoms. We have also simulated NC-AFM images of 2D bi-isonicotinic acid lattice using the same model. The geometry of the lattice have been optimized using DFT before simulating AFM images. Simulation results are in a good agreement with the experiment. The theoretical work is accompanied by a variety of experimental results obtained by the group of Prof Philip Moriarty at the University of Nottingham.

Scanning Tunnelling Microscopy (STM) is the fastest possible method of imaging the molecular orbitals of the C[subscript]60 anions with resolution at the single atom level. For the particular anions of fullerene C[subscript]60, the splitting of the molecular orbitals due to the internal Jahn-Teller effects (JT) add further difficulties in understanding the published experimental images. In the current work, the effect of JT interaction on STM recorded images is studied. For higher charged states, the Coulomb interaction affects the distribution of electrons around the ion, and then as a consequence, the STM current. The external interaction between the molecule and the surface substrate is equally important. Symmetry analysis using group theory and Hückel molecular orbital (HMO) theory are applied in order to describe the influence of the surface interactions on JT minima associated with D[subscript]3d, D[subscript]5d, D[subscript]2h, and C[subscript]2h symmetries. It represents some fullerene anions, which are adsorbed to the surface with different orientations, such as pentagon, hexagon, and double-bond prone toward the surface. Several ions with higher charges are investigated, such as C2−60, C3−60, and C4−60. In case of high symmetry orientations, the JT minima of the ions on a surface are split into subgroups with equal energies, depending on the type of orientation. The interpretation of the experimental observations is always possible for any orientation from the JT minima distribution and the contribution to the images from different components of the degenerate molecular orbitals.