In this dissertation I explain the theoretical and experimental details of nuclear magnetic resonance force microscopy (NMRFM). I report the data that I have collected on ammonium hexafluorophosphate at room temperature using NMRFM. This experiment measured cantilever deflection as a function of applied magnetic field. I also report on the progress of a new dynamical room-temperature NMRFM microscope. I describe the new probe and its advantages over the previous generation probe and I show the current calibration measurements. / text

No description available.

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.

No description available.

Exocytosis is a fundamental cellular process where membrane-bound secretory granules from within the cell fuse with the plasma membrane to form fusion pore openings through which they expel their contents. This mechanism occurs constitutively in all eukaryotic cells and is responsible for the regulation of numerous bodily functions. Despite intensive study on exocytosis the fusion pore is poorly understood. In this research micro-fabrication techniques were integrated with biology to facilitate the study of fusion pores from cells in the anterior pituitary using the atomic force microscope (AFM). In one method cells were chemically fixed to reveal a diverse range of pore morphologies, which were characterised according to generic descriptions and compared to those in literature. The various pore topographies potentially illustrates different fusion mechanisms or artifacts caused from the impact of chemicals and solvents in distorting dynamic cellular events. Studies were performed to investigate changes in fusion pores in response to stimuli along with techniques designed to image membrane topography with nanometre resolution. To circumvent some deficiencies in traditional chemical fixation methodologies, a Bioimprint replication process was designed to create molecular imprints of cells using imprinting and soft moulding techniques with photo and thermal activated elastomers. Motivation for the transfer of cellular ultrastructure was to enable the non-destructive analysis of cells using the AFM while avoiding the need for chemical fixation. Cell replicas produced accurate images of membrane topology and contained certain fusion pore types similar to those in chemically fixed cells. However, replicas were often dehydrated and overall experiments testing stimuli responses were inconclusive. In a preliminary investigation, a soft replication moulding technique using a PDMS-elastomer was tested on human endometrial cancer cells with the aim of highlighting malignant mutations. Finally, a Biochip comprised of a series of interdigitated microelectrodes was used to position single-cells within an array of cavities using positive and negative dielectrophoresis (DEP). Selective sites either between or on the electrode were exposed as cavities designed to trap and incubate pituitary and cancer cells for analysis by atomic force microscopy (AFMy). Results achieved trapping of pituitary and cancer cells within cavities and demonstrated that positive DEP could be used as a force to effectively position living cells. AFM images of replicas created from cells trapped within cavities illustrated the advantage of integrating the Biochip with Bioimprint for cellular analysis.

The Atomic Force Microscope (AFM) is a tool for imaging surfaces at the microand nano meter scale. The microscope senses the force acting between a surfaceand a tip positioned at the end of a micro-cantilever, forming an image of the surface topography. Image contrast however, arises not only from surface topography, but also from variation in material composition. Improved material contrast, and improved interpretation of that contrast are two issues central to the further development of AFM. This thesis studies dynamic AFM where the cantilever is driven at multiple frequencies simultaneously. Due to the nonlinear dependence of the tip-surface force on the tip’s position, the cantilever will oscillate not only at the driven frequencies, but also at harmonics and at mixing frequencies of the drives, so-called intermodulation products. A mode of AFM called Intermodulation AFM (ImAFM) is primarily studied, which aims to make use of intermodulation products centered around the resonance frequency of the cantilever. With proper excitation many intermodulation products are generated near resonance where they can be measured with large signal-to-noise ratio. ImAFM is performed on samples containing two distinct domains of different material composition and a contrast metric is introduced to quantitatively evaluate images obtained at each response frequency. Although force sensitivity is highest on resonance, we found that weak intermodulation response off resonance can show larger material contrast. This result shows that the intermodulation images can be used to improve discrimination of materials. We develop a method to obtain material parameters from multifrequency AFM spectra by fitting a tip-surface force model. Together with ImAFM, this method allows high resolution imaging of material parameters. The method is very generalas it is not limited to a specific force model or particular mode of multifrequency AFM. Several models are discussed and applied to different samples. The parameter images have a direct physical interpretation and, if the model is appropriate, they can be used to relate the measurement to material properties such as the Young’s modulus. Force reconstruction is tested with simulations and on measured data. We use the reconstructed force to define the location of the surface so that we can address the issue of separating topographic contrast and material contrast. / Svepkraftmikroskop (eller atomkraftmikroskop från engelskans atomic forcemicroscope, AFM) är ett instrument för att avbilda ytor på mikro- och nanometer skalan. Mikroskopet känner av kraften som verkar mellan en yta och en spetsplacerad längst ut på ett mikrometerstort fjäderblad och kan därigenom skapa en topografisk bild av ytans form. Bildkontrast uppstår dock inte bara från ytans form utan även från variation i material. Förbättrad materialkontrast och förbättrad tolkning av denna kontrast är två centrala mål i vidareutvecklingen av AFM. Denna avhandling berör dynamisk AFM där fjädern drivs med flera frekvensersamtidigt. På grund av det ickelinjära förhållandet i yt-spets-kraften som funktion av spetsens position så kommer fjädern inte bara att svänga på de drivna frekvenserna utan också på övertoner och blandfrekvenser, så kallade intermodulationsprodukter. Vi undersöker primärt Intermodulation AFM (ImAFM) som ämnar att utnyttja intermodulationsprodukter nära fjäderns resonansfrekvens. Med en lämplig drivsignal genereras många intermodulationsprodukter nära resonansen, där de kan mätas med bra signal till brus förhållande. ImAFM utförs på ytor bestående av två distinkta domäner av olika material ochen kontrastmetrik introduceras för att kvantitativt utvärdera bilderna som skapas vid varje frekvens. Trots att känsligheten för kraftmätningen är högst på resonans-frekvensen, så fann vi att svaga intermodulationsprodukter bortanför resonansen kan visa hög materialkontrast. Detta resultat visar att intermodulationsbilderna kan användas för att bättre särskilja olika material. Vi har utvecklat en metod för att rekonstruera yt-spets-kraften från multifrekventa AFM spektra genom modellanpassning i frekvensrymden. Tillsammans med ImAFM leder detta till högupplösta bilder av materialparametrar. Metoden är generell och är applicerbar för olika kraftmodeller och AFM-varianter. Parametrarna har en direkt fysikalisk tolkning och, om lämpliga modeller används, kan egenskaper så som materialets elasticitetsmodul mätas. Metoden har testats på simulerat såvälsom experimentellt data, och den har också används för att särskilja topografisk kontrast från materialkontrast. / <p>QC 20150209</p>

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.

We introduce a sensor which uses phase shifted dual diffraction gratings in order to increase the detectable range of motion when using phase sensitive diffraction for optical detection of displacement in probe microscopy. The modified FIRAT structure is comprised of a micro-machined bridge structure with integrated diffraction gratings for use in optical detection. With this new design and modified geometry/fabrication process, we will demonstrate force sensing structures with improved displacement detection range and dynamic response. The structure is based upon a previous implementation of force sensor modified for use in AFM imaging. It is built upon a transparent substrate and the bridge / grating serve as an integrated electrostatic actuator. The previously introduced an AFM sensor structure, with integrated actuator, can feasibly obtain Pico Newton force resolution along with increased bandwidth. The advantages of the integrated phase-sensitive diffraction grating and electrostatic actuator over other cantilever based implementations are well developed, yet the FIRAT structure suffered from limited displacement detection range of about ë/4 (167.5 nm for ë = 670 nm), its dynamics were dominated by squeeze film damping and the stiffness was not suitable for many imaging applications. This limitation in range was not due to the device physical structure or actuator, but was inherent to the optical detection scheme used. Modifications to the previous structure design, and sensor detection scheme, are implemented in order to increase the detectable range of the sensor implementation, reduce damping and stiffness, and custom tailor our devices to imaging applications

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.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.