Atomic Force Microscopy (AFM) has become an indispensable metrology tool for nanoscale surface characterization. Today, research and industry demand faster and more accurate metrology and these demands must be met expediently. Traditional AFM cantilevers and associated actuators (i.e. piezoelectric) are limited in regards to actuation speed and resonance frequency presenting the user with an undesired trade-off of speed versus resolution. Based on a pre-existing technology known as the FIRAT (Force Sensing Integrated Readout and Active Tip) AFM probe, this work aims to remedy actuation and response issues by implementing a cantilever-on-cantilever probe as well as a novel seesaw probe. Both cases implement electrostatic actuation, eliminating the need for piezoelectrics while demonstrating large - micron scale - actuation and sensitive displacement detection. These new probe designs can potentially demonstrate a wide bandwidth frequency response (e.g. 100 kHz) ideal for high-speed video-rate imaging. Unlike traditional AFM cantilevers, this is realized by mechanically coupling two physically separate structures to provide a soft resonator sensor atop a stiff actuator structure. Common surface-micromachining techniques are utilized to solve the logistical challenge of fabricating these stacked structures. By manipulating the viscous damping and mechanical mode coupling it becomes feasible to attain the aforementioned desired dynamic characteristics.

The goal of this thesis was the advanced design, fabrication, and application of combined atomic force microscopy - scanning electrochemical microscopy (AFMSECM) probes for high-resolution topographical and electrochemical imaging. The first part of the thesis describes innovative approaches for the optimization of AFM-SECM probe fabrication with recessed frame electrodes. For this purpose, commercial silicon nitride AFM cantilevers were modified using optimized critical fabrication processes including improved metallization for the deposition of the electrode layer, and novel insulation strategies for ensuring localized electrochemical signals. As a novel approach for the insulation of AFM-SECM probes, sandwiched layers of PECVD SixNy and SiO2, and plasma-deposited PFE films were applied and tested. Using sandwiched PECVD SixNy and SiO2 layers, AFM-SECM probes providing straight (unbent) cantilevers along with excellent insulation characteristics facilitating the functionality of the integrated electrode were reproducibly obtained. Alternatively, PFE thin films were tested according to their utility for serving as a mechanically flexible insulating layer for AFM-SECM probes. The electrochemical characterization of PFEinsulated AFM-SECM probes revealed excellent insulating properties at an insulation thickness of only approx. 400 nm. Finally, AFM-SECM cantilevers prepared via both insulation strategies were successfully tested during AFM-SECM imaging experiments. In the second part of this thesis, disk-shaped nanoelectrodes were for the first time integrated into AFM probes for enabling high-resolution AFM-SECM measurements. Disk electrodes with an electrode radius < 100 nm were realized, which provides a significantly improved lateral resolution for SECM experiments performed in synchronicity with AFM imaging. Furthermore, the developed fabrication scheme enables producing AFM-SECM probes with integrated disk nanoelectrodes at significantly reduced time and cost based on a highly reproducible semi-batch fabrication process providing bifunctional probes at a wafer scale. The development of a detailed processing strategy was accompanied by extensive simulation results for developing a fundamental understanding on the electrochemical properties of AFM-SECM probes with nanoscale electrodes, and for optimizing the associated processing parameters. Thus fabricated probes were electrochemically characterized, and their performance was demonstrated via bifunctional imaging at model samples. The third part of this thesis describes the development and characterization of the first AFM tip-integrated potentiometric sensors based on solid-state electrodes with submicrometer dimensions enabling laterally resolved pH imaging. Antimony and iridium oxides were applied as the pH sensitive electrode material, and have been integrated into the AFM probes via conventional microfabrication strategies. The pH response of such AFM tip-integrated integrated pH microsensors was tested for both material systems, and first studies were performed demonstrating localized pH measurements at a model system.

Atomic force microscopy (AFM) is a powerful technique that enables to study biological macromolecules and dynamic biological processes at different scales. It is an excellent tool for imaging of biological objects under various conditions at a nanometer resolution. Force mode of AFM, so called single molecule force spectroscopy (SMFS), allows for investigation of the strength of molecular interactions of different origins established between and within biological molecules. In the present work, SMFS was used to detect and locate structurally and functionally important interactions of sodium/glycine betaine transporter BetP of Corynebacterium glutamicum, which serves as a model system for this class of proteins. Mechanical pulling of BetP molecules embedded into the lipid membranes resulted in a step-wise unfolding of the protein and revealed insights into its structural stability. Effect of the lipid environment, N- and C-terminal extensions on inramolecular interactions of BetP as well as protein activation and ligand binding were investigated in great detail. In another part of this work, I demonstrate an application of the AFM based technique that can record unfolding of a protein under force-clamp conditions. This method directly measures the kinetics of the protein unfolding, allowing for the use of simple methods to analyze the data. For the first time the force-clamp technique was used to describe in detail unfolding kinetics of the membrane protein, i. e. Na+/H+-antiporter NhaA from Escherichia coli. Performed here experiments on NhaA in its functionally active and inactive states demonstrated the advantages of examining unfolding kinetics at the single-molecule level. It was possible to observe unfolding events for pH-activated conformation of NhaA that due to the low frequency of occurrence were not represented in the ensemble average of the single-molecule measurements. As mechanical unfolding, similarly to bond rupture, is a force-dependent process, force-clamp technique can allow for a more direct way of probing protein unfolding and is anticipated to be also useful to examine the folding/unfolding kinetics of other membrane proteins.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002. / Includes bibliographical references. Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 107-109). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Typescript. Vita. Includes bibliographical references (leaves 107-109). Also available on the Internet.

Two technologies, immersion and imprint lithography, represent important stepping stones for the development of the next generation of lithography tools. However, although the two approaches offer important advantages, both pose many significant technological challenges that must be overcome before they can be successfully implemented. For imprint lithography, special care must be taken when choosing an etch barrier because studies have indicated that some physical material properties may be size dependent. Additionally, regarding immersion lithography, proper image focus requires that the optical path length between the lens and substrate be maintained during the entire writing process. The work described in this document was undertaken to address the two challenges described above. A new mathematical model was developed and used in conjunction with AFM nano-indentation techniques to measure the elastic modulus of adhesive, thin polymer films as a function of the film thickness. It was found that the elastic modulus of the polymer tested did not change appreciably from the value determined using bulk measurement techniques in the thickness range probed. Additionally, a method for monitoring and controlling the optical path length within the gap of a nearly index-matching cavity based on coherent broadband interference was developed. In this method, the spectrum reflected for a cavity illuminated with a modelocked Ti:Sapphire laser was collected and analyzed using Fourier techniques. It was found that this method could determine the optical path length of the cavity, quickly and accurately enough to control a servo-based feedback system to correct deviations in the optical path length in real time when coupled with special computation techniques that minimized unnecessary operations. / text

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This thesis describes the development of a high-throughput approach towards the encapsulation of cancer cells in biopolymer microgels with tunable mechanical properties. In particular, this thesis is focused on: i) the high-throughput generation of biopolymer microgels with tunable mechanical properties ii) the measurement of the mechanical properties of the microgels, and iii) the high-throughput encapsulation of a cancer cell line within biopolymer gels. The microgels will be generated by (i) introducing in a microfluidic device two distinct streams of biopolymer solutions; (ii) mixing the streams; (iii) emulsifying the biopolymer and (iv) using thermosetting to transform the droplets in situ into microgels. By applying a compression force to the hydrogel microbead and measuring its deformation, the Young’s modulus and relaxation time of the microgel can be examined. The properties of cells were examined within the gels using various spectroscopic techniques such as absorption (UV-Vis) and fluorescence microscopy (fluorescent microscopy, confocal microscopy).

This thesis describes the development of a high-throughput approach towards the encapsulation of cancer cells in biopolymer microgels with tunable mechanical properties. In particular, this thesis is focused on: i) the high-throughput generation of biopolymer microgels with tunable mechanical properties ii) the measurement of the mechanical properties of the microgels, and iii) the high-throughput encapsulation of a cancer cell line within biopolymer gels. The microgels will be generated by (i) introducing in a microfluidic device two distinct streams of biopolymer solutions; (ii) mixing the streams; (iii) emulsifying the biopolymer and (iv) using thermosetting to transform the droplets in situ into microgels. By applying a compression force to the hydrogel microbead and measuring its deformation, the Young’s modulus and relaxation time of the microgel can be examined. The properties of cells were examined within the gels using various spectroscopic techniques such as absorption (UV-Vis) and fluorescence microscopy (fluorescent microscopy, confocal microscopy).