The goal of this project was to develop techniques and instrumentation to measure the elastic properties of the lens and lens capsule in situ and their changes with age using Atomic Force Microscopy (AFM). The studies include the construction, characterization, and calibration of laboratory-based Atomic Force Microscope (AFM) to measure mechanical properties of ophthalmic tissues. Atomic Force Microscopy is a nanoscale imaging technique that has been applied to mechanical property measurement through nanoindentation. Young's modulus of elasticity is determined by monitoring the cantilever deflections when it contacts the sample. The studies also include the development of tissue preparation techniques to enable measurement of the lens elasticity using AFM. This study found that lens capsule elasticity decreases with age, outer lens cortex elasticity remains constant with age, and the inner lens cortex is stiffer than the outer lens cortex. The effect of the changing biometry and mechanical properties with age was investigated by developing a mathematical model of accommodation. These changes will be the limiting factor to accommodative amplitude. Changes in lens capsule mechanical properties will affect the maximal accommodative amplitude in older eyes.

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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Atomic force microscopy (AFM) is a particularly useful tool, for obtaining structural information about drug-nucleic acid interactions. The mode of drug binding intercalation versus groove binding can be determined from images acquired on individual DNA molecules as the length of a DNA molecule increases in direct proportion to the number of intercalators bound to it. The efforts of this research were directed toward elucidating the mode of binding of a series of drugs based on polymers of naphthalenetetracarboxyl diimide (NDI) interacting with a linearized DNA plasmid. During the course of the investigation of these drugs, DNA intercalation was confirmed as the mode of binding and the binding affinity estimated. Unexpectedly, concentration-dependent formation of secondary DNA structures including condensates was observed. DNA toroids, spheres, and rods were imaged and measured. Conformations that are believed to be intermediate condensate forms were also identified at lower poly-NDI concentrations. Models for the DNA condensation process have been proposed. Ultimately, this research furthers the understanding of DNA condensation which can be applied to gene delivery systems and anti-viral agents. It may also help direct the development of better drugs based on the insight of poly-intercalators interactions with DNA.

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.

High-resolution lattice periodicity images of a variety of well-defined surfaces, including graphite, mica, and Au(111), validated the good stability of our atomic force microscope (AFM) system. Combining self-assembled monolayer (SAM) and AFM technology, we demonstrated the capabilities of pattern fabrication as well as modification of surface functionality. AFM-based nanolithography operating conditions, such as scan rate, deflection setpoint, and number of scan were studied to obtain the optimized quality of the fabricated patterns. Thiolated-DNA probe molecules could be patterned at a nanometer scale on a gold substrate. However, we found that the surface coverage began to drop notably with the probe length (number of DNA bases). Therefore, the displaced DNA molecules during nanoshaving were reversibly adsorbed, and patterning became unreliable. We were unsuccessful in detecting the subsequent hybridization reactions at these nanopatterns from AFM measurements. To realize the DNA hybridization, further studies on the incubation temperature, probe length and even DNA sequences are required to demonstrate that this AFM-based gene diagnostic method is truly operational.

The fabrication characteristic of focused ion beam (FIB) for fused quartz was investigated. With the progress of nanotechnology, new technologies and devices are invented constantly. In nanofabrication, FIB has several advantages such as high material removal rate, high resolving power and direct fabrication in some selected areas without etching mask. Therefore, it had been studied in detail to fabricate nano-structures by FIB. In this study, we found out the effect of nano-machining by adjusting the parameters of FIB system such as: beam current, overlap, and dwell time. The fabricated features together with their surface morphology and profile were investigated by scanning electron microscope (SEM) and atomic force microscope (AFM). Results show that when beam current was smaller, overlap was 50% and dwell time was 10£gs could get best performance by FIB.

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.