Phospholipid coated, inert gas filled microbubbles (MBs) are currently in widespread use in medical applications for the enhancement of diagnostic ultrasound images, and they are promising candidates for use in the area of targeted drug/gene delivery and uptake. As phospholipid coated MBs were developed for use with diagnostic ultrasound, their behaviour under acoustic loading is well investigated, however much less is known about their response to direct mechanical loading, which will potentially prove important as the range of uses of MBs expands. This is particularly true of the existing commercially available MB products. In this thesis, atomic force microscopy (AFM) was used to investigate the mechanical behaviour of three types of commercially produced phospholipid coated MBs, Definity®, BR14 and Sonovue®, at small deformations. Force spectroscopy was used to produce force-deformation (F-Δ) curves showing how the MBs deform under mechanical loading. Definity® MBs were deformed with tipless cantilevers at high deformations (though still less than 30% of the initial height of the MB); BR14 and Sonovue® MBs were probed with both tipless and tipped cantilevers to investigate both whole-bubble deformation and also shell indentation. BR14 was limited to low deformations; Sonovue® included both low and high deformations. The F-Δ curves were used to evaluate MB stiffness and also in combination with up to four mechanical models to predict the Young’s modulus of the MBs. The suitability of Reissner, Hertz, Elastic Membrane and De Jong theories for the prediction of the Young’s modulus of the MBs was explored. In the case of Definity® MBs no correlation between MB size and stiffness was observed; however an unexpected size dependence was observed in the Young’s modulus values, possibly due to variations in the thickness of the phospholipid shell. The membrane stretching component of elastic membrane theory was found to be the most applicable model on these MBs in this higher deformation regime. However, in this regime, gas compressibility could play a role and this is not included in the model. We studied the mechanical properties of BR14 MBs at very low deformations using ‘soft’ cantilevers. In this regime, gas compressibility should play a minimal role and there are several mechanical models which may be used. These MBs demonstrated decreasing stiffness with increasing diameter, and little variation in Young’s modulus with diameter. Hertz and De Jong theories showed more realistic Young’s modulus values (compared to other models) with little observable trend. Sonovue® MBs were used for a more comprehensive study of the small and very small deformation regimes using ‘soft’, ‘hard’ and tipped cantilevers. They showed no definitive trend in MB stiffness with MB diameter. Hertz and De Jong theory were again found to be most suitable. Analysis of curves acquired with tipped cantilevers indicated that the stiffness of a localised area of the shell membrane is similar to the overall stiffness of the MB and that the apparent Young’s modulus of the membrane according to the Hertz theory is also similar to that of the MB as a whole. Generally, considering all systems, Reissner theory was found to produce large overestimates of Young’s modulus, exceeding expected values by several orders of magnitude. Hertz and De Jong theories produced underestimates, though by a much smaller margin. Elastic membrane theory worked well and produced realistic Young’s modulus values only at relatively high deformation (the stretching term) in spite of the fact that gas compressibility is not taken into account. The suitability of the models is therefore very dependent on the deformation regime of the experiment. It seems that there is scope for better models at low deformation taking into account the soft shell of the MB and possibly its specific structure. Precise structural information of the MB shells does not exist; it is not trivial to attain and should certainly be a topic of future work with additional instrumentation.

Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Nanoscale visualisation of live cells and cellular components under physiological conditions has long been a goal in microscopy. The objective of this study was to validate the use of Atomic Force Microscopy (AFM) as a new tool in unravelling the mysteries of antimicrobial peptide mechanism of action. Using the simplest AFM imaging technique, we were able to analyse the influence of haemolytic melittin and anti-bacterial magainin 2 on different target membranes at nanometer resolution, without using fixing agents. First, magainin 2 was synthesised and purified by gel permeation chromatography and high performance liquid chromatography (HPLC). The purity of magainin 2 and melittin, isolated from bee venom (Sigma-Aldrich), was verified with electro spray ionisation mass spectrometry (ESI-MS). Second, dose-response experiments were used to determine the optimum peptide/target cell ratio that would allow interaction with the membrane without causing lysis. Third, peptide/target-cell samples were placed on silica plates and visualised using contact mode AFM. Images obtained of the cells before and after peptide treatment, showed distinct changes in cell membrane surface topology. We observed grooves, lesions, membrane collapse and vesiculation depending on the concentration, type of peptide and target-cell used, allowing us to make conclusions regarding the mechanism of action of melittin and magainin 2. In comparison with model membrane studies, our AFM results show that a peptide can function by more than one mechanism of action depending on the structural composition of the membrane, which appears to have specific segregated lateral organisation. Magainin 2 (non-toxic) selectively targets cell membranes using different mechanisms of action. In this way it can lyse bacterial membranes (anti-bacterial agent) using one mechanism, while using another mechanism to interact with mammalian cells at physiological concentrations, without destroying them. In contrast, melittin (toxic) is non-selective, and uses the same mechanism of interaction with bacterial and mammalian cells. In conclusion, we propose a new holistic model for the mechanism of action of antimicrobial peptides. / AFRIKAANSE OPSOMMING: Nanoskaal visualiseering van lewende selle en sellulêre komponente onder fisiologiese toestande is al 'n geruime tyd 'n mikpunt in mikroskopie. Die doel van hierdie studie was om antimikrobiese peptiede se meganisme van werking op teikenselle op nanoskaalvlak met AFM te visualiseer. Sonder om fikseermiddels by te voeg, het ons die eenvoudigste AFM tegniek gebruik om die effek van hemolitiese melittien en anti-bakteriële magainin 2 op verskillende teikenselle, in nanometer resolusie, waar te neem. Eerstens is Magainin 2 gesintesiseer en gesuiwer met behulp van gelpermeasie chromatografie en hoë doeltreffenheid vloeistof chromatografie (HPLC). Die suiwerheid van magainin 2 en kommersiële bye gif melittien, is bevestig met behulp van elektrosproei-ionisasie massaspektrometrie (ESI-MS). Tweedens, is dosis-respons eksperimente gebruik om die optimale peptied/teikensel verhouding te bepaal voordat membraanliese plaasvind. Derdens, is peptied/teikensel monsters op silika plate gevisualiseer met gebruik van kontak AFM. Die beelde van die selle, voor en na peptied behandeling, het duidelike veranderinge in seltopologie getoon. Ons het groewe, letsels, membraaninstorting en vesikulasie, afhangende van die konsentrasie peptied en teikensel gebruik, waargeneem. Dit het ons toegelaat om tot gevolgtrekkings te kom aangaande die meganisme van werking van melittien en magainin 2. In ooreenstemming met model membraan studies, het ons AFM resultate gewys dat 'n peptied veelvoudige meganismes van werking kan hê, afhangend van die strukturele samestelling van die membraan, wat klaarblyklik laterale segregasie toon. Magainin 2 (nie-giftig) is selektief ten opsigte van teikenselle omdat dit gebruik maak van verskillende meganismes van werking op bakteriële en soogdier selle. In teenstelling is melittien (giftig) nie-selektief, en gebruik dieselfde meganisme van werking op bakteriële en soogdierselle. Ten slotte, stel ons 'n nuwe model vir die meganisme van werking voor.

Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2006. / Dyeing has an influence on the characteristic properties of wool fibres. This can result in changes in the final properties of the fibre including fibre elasticity, fibre strength and breaking elongation of the wool fibres, especially in the case of dark colours. Damage that occurs to the fibre surface due to the action of acid, alkali, dyestuff, water, heat, and mechanical stress during the dyeing process can thus have an affect on the fibre breaking elongation and the fibre strength. The aim of this project was to assess the effect of dyeing with different colours (ranging from light to dark) on the surface of wool fibres using atomic force microscopy (AFM). The results indicated that four different types of surface damage can be discerned: scale raising, scale chipping, fluting and pitting. The findings also indicate that the surface damage to the fibres was greater and more noticeable (especially the scale raising) on fibres dyed with dark colours than to fibres dyed with light colours. The same applied to the fibre strength and elongation, where generally the fibre breaking extension and the breaking load was smaller in the case of darker colours. The effect of dyeing with different colour shades on the fibre surface was observed with AFM. Several characteristic scale parameters of a statistical significant number of dyed and undyed fibres were measured in order to determine the surface damage caused by dyeing. The correlation between the surface damage, observed by AFM, and the loss in tensile strength and fibre elongation, determined by tensile tests, were investigated. This was done in order to determine the effect of the cuticle damage on the tensile stress/strain behaviour. The results show that AFM is a viable tool to study the effects of different dyes on the fibre surface and for detecting surface modifications with great accuracy. It was found that dyeing with dark colours caused greater damage to the surface than light colours, and it was possible to distinguish different lightness areas, which could be regarded as light and dark colours.

Thesis (MEng (Electrical and Electronic Engineering))--University of Stellenbosch, 2006. / The Josephson effect of high-temperature superconducting nanobridge structures is studied worldwide. Until now, nanobridges are generally fabricated with focused ion beam milling on planar thin films. These nanobridges are employed as weak links in superconducting quantum interference devices (SQUIDs) and used in superconducting flux flow transistors and devices. This project had two main objectives: to improve the sidewall angle of photoresist lines, with the aid of atomic force microscopy (AFM) nanolithography; and to fabricate hightemperature Josephson junctions by constricting superconductive YBCO lines on MgO substrates with AFM nanolithography. The entire fabrication process is explained including photolithography, deposition of the YBCO thin films with pulsed laser deposition (PLD), nanolithography and wet etching. Although the testing of the junctions did not show any Josephson behaviour, it could be demonstrated that nanobridge structures can successfully be created by AFM nanolithography. The entire fabrication process has been demonstrated in detail for the benefit of future research.

This dissertation serves to study a protein's conformation-function relationship since immobilized proteins often behave differently from their solution-state counterparts. Therefore, this study is important to the application of protein-based biodevices. Another aim of this dissertation is to explore a new approach to realize low voltage electrowetting without the help of oil bath. Utilizing this approach, a protein micro-separation was realized. Additionally, the interfacial properties of ionic liquid (IL) solid-like layer, which played a key role in electrowetting, was studied for further developments of IL-based applications. Atomic Force Microscopy (AFM) was utilized in the study and played multiple roles in this dissertation. First, AFM was used as a fabrication tool. In the contact mode, conductive AFM tip was used to conduct the electrochemical oxidation to create a chemical pattern or to conduct an electrowetting experiment. Subsequently, AFM was used as a characterization tool in the tapping mode to characterize the surface structure, the thickness, and the surface potential. Furthermore, AFM in the contact mode was used as a measurement tool to measure the tribological force properties of sample. The results of the study concerning the conformational change in immobilized calmodulin showed that the immobilized CaM retained its activity. Additionally, the immobilization of CaM on a solid support did not interfere with the ability of the protein to bind calcium, as well as CaM kinase binding domain. For the electrowetting experiment, our data suggested that the ultra-high capacitance density of the IL dielectric layer leads to the low voltage electrowetting. We also successfully demonstrated the streptavidin and GFP proteins separation by Electrowetting-on-Dielectric (EWOD) force. The results of the surface properties study indicated that the charge and dipole of the substrate can influence the structures and properties of the IL interfacial layer. Our study would be beneficial in research and assay work involving engineered proteins, as well as the study and development of electrowetting applications.

Spreading plays an important role in coating, lubrication, printing and etc. During the spreading process, a liquid thin film forms prior to the expansion of a liquid drop. This thin film is called a precursor film. It not only changes the spreading mechanism, but impacts the wettability of a liquid. Early studies on the precursor films showed the films were stacked in a terraced structure, and the radius of each layer of the films was proportional to the square root of time. Optical techniques such as ellipsometry, X-ray diffraction and X-ray reflectivity solved the conformations of liquid molecules at the interfaces. However, the conformations of the interfacial molecules have rarely been correlated with their positions at the interface. In addition, the properties of the precursor films have not been fully studied yet. In this dissertation, two kinds of organic compounds, hexatriacontane (C36) and 1-butyl-3-methylimidazolium ([Bmim][Cl]), are proposed to be spread over octadecyltrichlorosilane partially degraded (OTSpd) patterned surfaces. Once organic molecules flow over such OTSpd surfaces, the liquids are limited within the patterned area. Characterized by atomic force microscopy (AFM), the structures and chemical identities and the formation mechanism of the precursor films are resolved thereafter. The precursor films formed by both compounds, C36 and [Bmim][Cl], were observed in a bilayer structure in that the molecules close to the solid substrate had different orientation from the molecules close to the air. They were called parallel layers and standing-up layers, respectively. The parallel layers of C36 formed prior to the standing-up layers through the vapor phase transport. In addition, the parallel layers were found more stable thermodynamically and the standing-up layers were more stable mechanically. The frictional study of C36 showed the standing-up layers could hold 0.49GPa pressure. The orientation of [Bmim][Cl] molecules were impacted by the polarities of the solid substrates. The achievements in this dissertation not only resolve the properties of the precursor films of two organic compounds, but provide a general method for the further studies of the precursor films.

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Understanding the mechanisms by which proteins act on membrane surfaces is fundamental if we are to exploit their capabilities or halt the progression of the diseases they are associated with. Arguably, the best way to study these interactions is by using techniques that can obtain molecular-scale information, in real time and under physiologically relevant conditions. Studying supported lipid bilayer systems with high spatial resolution tools, such as atomic force microscopy (AFM), and high temporal resolution techniques, such as polarized total internal reflection fluorescence microscopy (pTIRFM), allows us to meet these requirements [1]. The goal of this project is to use methods that are currently available and further their applications and capabilities to provide insight into the mechanisms by which amyloidogenic and antimicrobial peptides act on membranes.