Collagen II and the proteoglycan aggrecan are key extracellular matrix (ECM) proteins in cartilaginous tissues such as the intervertebral disc (IVD). Given the functional role that these structural and functional proteins have in the IVD, ECM in tissue engineered intervertebral disc (TE IVD) constructs needs to recapitulate native tissue. As such, there is a need to understand the structure and mechanical function of these molecules in native tissue to inform TE strategies. The aims here were to characterise aggrecan and collagen II using atomic force microscopy (AFM), size-exclusion chromatography multi angle light scattering (SEC-MALS), histology, quantitative PCR, nanomechanical and computational modelling in: (i) skeletally immature and mature bovine articular cartilage (AC) and nucleus pulposus (NP), (ii) TE IVD constructs cultured in hypoxia or treated with transforming growth factor beta [TGFÎ23] or growth differentiation factor [GDF6]), and (iii) porcine AC and NP tissue. No variation in collagen II structure was observed although the proportion of organised fibrillar collagen varied between tissues. Both intact (containing all three globular domains) and non-intact (fragmented) aggrecan monomers were isolated from both AC and IVD and TE IVD constructs. Mature intact native NP aggrecan was ~60 nm shorter (core protein length) compared to AC. In skeletally mature bovine NP and AC tissue, most aggrecan monomers were fragmented (99% and 95%, respectively) with fragments smaller and more structurally heterogeneous in NP. Similar fragmentation was observed in skeletally immature bovine AC (99.5%), indicating fragmentation occurs developmentally at an early age. Fragmentation was not a result of enhanced gelatinase activity. Aggrecan monomers isolated from notochordal cell rich porcine NP were also highly fragmented, similar to bovine NP. Application of a computational packing model suggested fragmentation may affect porosity and nutrient transfer. The reduced modulus was greater in AC than NP (497 kPa and 76.7 kPa, respectively) with the difference likely due to the organisation and abundance of ECM molecules, rather than individual structure. Growth factors (GDF6 and TGFÎ23), and not oxygen tension treated TE IVD constructs were structurally (with >95% fragmented monomers), histologically and mechanically (GDF6: 60.2 kPa; TGFÎ23; 69.9 kPa) similar to native NP tissue (76.7 kPa) and there was evidence of gelatinase activity. To conclude, these results show that the ultrastructure of intact aggrecan was tissue and cell dependent, and could be modified by manipulation of cell culture conditions, specifically GDF6 which may play a role in aggrecan glycosylation.

This study presents a systematic investigation of two types of soft biomaterials: phospholipid-based microbubbles (MBs) and agarose hydrogels, using atomic force microscopy (AFM) force-distance curves. Microbubbles are used widely in several applications, especially in medical applications, where they are used as ultrasound contrast agents (UCAs) and as vehicles for transporting the drugs and genes to their targets, which is commonly known as drug/gene delivery. Although plenty of attention has been paid to these materials by medical researchers there is a shortage of engineering research on the properties of these materials. The present study tries to address this gap by studying these materials from the engineering perspective; therefore, the aim of this study is to investigate the mechanical properties of MBs and hydrogels. In this research, phospholipid-based microbubbles (MBs), commercially called SonoVue® microbubbles and used as UCAs, were investigated to measure their mechanical properties using an AFM mode of operation called force-distance curves (or force spectroscopy mode); this mode allows for direct mechanical tests to acquire the force-deformation (F-Δ) behaviour of the MBs. The compression tool was a flat (tipless) cantilever moved at constant speed, whereas the variable was MB size. The MBs behaviour was assessed by calculating several mechanical properties, which were the stiffness, Young's modulus (three different models were applied), hysteresis, plasticity, adhesion forces, nonlinearity and instability. The stiffness and the Young's modulus values were measured to be in the same range as found in similar studies. A phenomenon was observed that the local stiffness of the MB increases after each unstable step provided that the MB stays within the linear elastic region. The Young's modulus was calculated applying three models, two for estimating the elastic modulus of the shell and the third for modulus of elasticity of the whole MB. The stretching component of the membrane theory was found to provide the best prediction of the Young's modulus value. To investigate the effect of the tip geometry on the mechanical properties of the MBs, the MBs were studied with different cantilever/tips, including a conical-tipped cantilever. The study concluded that there is no impact of the contact geometry on the mechanical properties of the MBs if the applied forces and the spring constant of the cantilever are the same. The same phenomenon, increasing the local stiffness of the MB after each unstable step, was found however with a higher rate. Hydrogels were also studied in this research using AFM and adopting a nanoindentation technique. The indenter was a conical tip moving toward the sample surface with constant speed and applying similar forces on all samples, where the variable was the gel concentration. In addition to the previous mechanical properties, other properties were investigated, such as hardness, universal hardness and pressure. An effect of the gel concentration on the mechanical properties of the gels was observed. There is a difference in the results compared to those reported in the literature review, where some of the results are in the same range as those found here, while others were either higher or lower, due to the influence of factors such as the indenter geometry, the applied force and the load rate; moreover, it was found that the viscoelastic behaviour of the gels played a significant role.

The adsorbed layer morphology of a series of surfactants under different conditions has been examined primarily using atomic force microscopy (AFM). The morphologies of single and double chained quaternary ammonium surfactants adsorbed to mica have been characterised using AFM at concentrations below the cmc. Mixing these different types of surfactants systematically allowed a detailed examination of the change in adsorbed film curvature from the least curved bilayers through to most curved globules. From this study a novel mesh structure was discovered at curvatures intermediate to bilayers and rods. A mesh was again observed in studies examining the morphology change of adsorbed nonionic surfactant films on silica with variation in temperature. Other surfactant mixtures were also examined including grafting non-adsorbing nonionic surfactants and diblock copolymers into quaternary ammonium surfactant films of different morphologies.

To investigate the ultimate resolution in ion beam lithography (IBL) the resist material poly(methyl methacrylate) PMMA has been modified by single ion impacts. The latent damage tracks have been etched prior to imaging and characterisation. The interest in IBL comes from a unique advantage over more traditional electron beam or optical lithography. An ion with energy of the order of 1 MeV per nucleon evenly deposits its energy over a long range in a straight latent damage path. This gives IBL the ability to create high aspect ratio structures with a resolution in the order of 10 nm. Precise ion counting into a spin coated PMMA film on top of an active substrate enabled control over the exact fluence delivered to the PMMA from homogenously irradiated areas down to separated single ion tracks. Using the homogenous areas it was possible to macroscopically measure the sensitivity of the PMMA as a function of the developing parameters. Separated single ion tracks wer e created in the PMMA using 8 MeV F, 71 MeV Cu and 88 MeV I ions. These ion tracks were etched to create voids in the PMMA film. For characterisation the tracks were imaged primarily with atomic force microscopy (AFM) and also with scanning electron microscopy (SEM). The series of studies presented here show that the sensitivity of the resist-developer combination can be tailored to allow the etching of specific single ion tracks. With the ability to etch only the damage track, and not the bulk material, one may experimentally characterise the damage track of any chosen ion. This offers the scientific community a useful tool in the study and fabrication of etched ion tracks. Finally work has been conducted to allow the precise locating of an ion beam using a nanoscale mask and piezoelectrically driven scanning stage. This method of beam locating has been trailed in conjunction with single ion detection in an effort to test the practical limits of ion beam lithography in the single ion realm.

Methods based on the atomic force microscope (AFM) were implemented or developed to measure and map at the nanoscale the mechanical properties of polymer surfaces and of nanomaterials: force spectroscopy, force modulation, phase detection in intermittent-contact mode. Especially, a technique, referred as resonant contact-AFM, was developed. It is based on the electrostatic excitation of the cantilever vibration and on the measurement of its resonance frequency when the tip contacts the probed sample. A theoretical model was developed to determine the tip-sample contact stiffness from the measurement of the frequency shift. These methods were used to study several questions raised in the fields of polymer surfaces and interfaces and of nanomaterials. Surfaces of toughened polypropylene (PP) with ethylene-propylene copolymer (EP) were studied by force spectroscopy and force modulation microscopy (FMM) to characterise the effect of the blending and the moulding processes and the PP/EP viscosity ratio on the surface distribution of the EP rubber nodules. The contribution of the EP rubber to paint adhesion was also demonstrated. Surfaces of atactic polypropylene photo-grafted with acrylic acid monomers were analysed by intermittent-contact AFM (IC-AFM) with phase detection. The combination of these methods with other analytical techniques allowed characterising the chemical composition of the heterogeneous surface morphology obtained after photo-grafting. The tensile elastic modulus of polymer nanotubes and metallic nanowires was measured with force spectroscopy and resonant contact-AFM. These measurements confirmed the ability of resonant contact-AFM to quantitatively measure the mechanical properties of nanomaterials. Moreover, they showed that the measured modulus increased when the nanowires or nanotubes diameter decreases. This behaviour was explained by taking into account the effect of the surface deformation that added a surface stiffness proportional to the surface tension, or surface stress, of the material. Resonant contact-AFM was also used to characterise the variation of the mechanical properties at the interfaces in polymer blends. It was demonstrated that this technique allows the determination of the interfacial width in incompatible polymer blends. It also allowed characterising the mechanical property gradient that can appear in reactive polymer blends.

Several nanomaterials systems, both inorganic and organic in nature, have been extensively investigated by a number of characterization techniques including atomic force microscopy (AFM), electron microscopy, Fourier transform infrared spectroscopy (FTIR), and energy dispersive x-ray spectroscopy (EDX). The first system consists of boron nitride nanotubes (BNNTs) synthesized via two different methods. The first method, silica-assisted catalytic chemical vapour deposition (SA-CVD), produced boron nitride nanotubes with different morphologies depending on the synthesis temperature. The second method, growth vapour trapping chemical vapour deposition (GVT-CVD), produced multiwall boron nitride nanotubes (MWBNNTs). The bending modulus of individual MWBNNTs was determined using an AFM three-point bending technique, and was found to be diameter-dependent due to the presence of shear effects. The second type of nanomaterial investigated is the biomineralized calcareous shell of the serpulid Hydroides dianthus. This material was found to be an inorganic-organic composite material composed of two different morphologies of CaCO3, collagen, and carboxylated and sulphated polysaccharides. The organic components were demonstrated to mediate the mineralization of CaCO3 in vitro. The final system studied is the proteinaceous cement of the barnacle Amphibalanus amphitrite. The secondary structure of the protein components was investigated via FTIR, revealing the presence of β-sheet conformation, and nanoscale rod-shaped structures within the cement were identified as β-sheet containing amyloid fibrils via chemical staining. These rod-shaped structures exhibited a stiffer nature compared with other structures in the adhesive, as measured by AFM nanoindentation.

Background and aims: It is known that smooth muscle possesses substantial mechanical plasticity in that it is able to adapt to large changes in length without compromising its ability to generate force. It is believed that structural malleability of the contractile apparatus underlies this plasticity. There is strong evidence suggesting that myosin thick filaments of the muscle are relatively labile and their length in vivo is determined by the equilibrium between monomeric and filamentous myosin. The equilibrium in turn is governed by the state of phosphorylation of the 20-kD regulatory myosin light chain (MLC20, or RLC). It is known that phosphorylation of the myosin light chain favors formation of the filaments; it is not known how the light chain phosphorylation affects the lability of the filaments. The major aim of this thesis was to measure the mechanical integrity of the filaments formed from purified myosin molecules from bovine airway smooth muscle, and to determine whether the integrity was influenced by phosphorylation of the myosin light chain. Methods: Myosin was purified from bovine trachealis to form filaments, in ATP containing zero-calcium solution during a slow dialysis that gradually reduced the ionic strength. Sufficient myosin light chain kinase and phosphatase, as well as calmodulin, were retained after the myosin purification and this enabled phosphorylation of RLC within 20-40 s after addition of calcium to the filament suspension. The phosphorylated and non-phosphorylated filaments were then partially disassembled by ultrasonification. The extent of filament disintegration was visualized and quantified by atomic force microscopy. Results: RLC phosphorylation reduced the diameter of the filaments and rendered the filaments more resistant to ultrasonic agitation. Electron microscopy revealed a similar reduction in filament diameter in intact smooth muscle when the cells were activated. Conclusion: Our results suggest that RLC phosphorylation is a key regulatory step in modifying the structural properties of myosin filaments in smooth muscle, where formation and dissolution of the filaments are required in the cells’ adaptation to different cell length.

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The image resolution of atomic force microscopy (AFM) is still less superior to that of the electron microscopy (EM). However AFM operated in liquids complemented by Tapping-mode (TM) detection proves to be more suitable for imaging biomolecules in physiological-like environments. Nevertheless, manipulation of AFM in solution turned out to be non-trivial, several technical difficulties were encountered. In the thesis, I report using divalent cation-containing buffer as a feasible method to immobilize DNA molecules effectively for imaging in liquid media. AFM operating conditions, such as cantilever oscillating drive frequency, setpoint amplitude, feedback control parameters and scan rates were studied to obtain the optimized function. Various AFM images of Ssp I-linearized pUC19 DNA/EcoR I restriction enzyme complexes were captured, revealing the molecular details of their complex machineries. For example, the intermediate stage of the enzyme cleavage action was displayed by images showing that DNA was bent by an acute angle at the active site in the presence of one single EcoR I molecule. Some evidence for a jumping, sliding or intersegmental transfer mechanism is achieved. To trace the enzyme-DNA interaction dynamic in real time, preliminary results were obtained, but further improvements are required.

This study successfully demonstrates the electrical control of the surface wettability of liquid crystal and polymer composite film. The application of external voltages significantly affects the surface wettability of the film. This study uses atomic force microscopy to quantitatively characterize the fundamental mechanism responsible for the structurally driven changes in surface properties at various applied voltages. The surface wettability transitions of the film are electrically driven, as shown by reorganized liquid crystal molecules. Measurements of the voltage-dependent surface wettability of the composite film suggest novel approaches to supporting control applications of future electro-optical nanotechnology devices, including liquid lenses, windshields and displays.