Doctor of Philosophy / Department of Chemistry / Daniel A. Higgins / This dissertation describes the application of single molecule spectroscopy and tracking to investigations of the nanoscale properties of thin-film chemical gradients and the transport dynamics of molecules dispersed within and upon these gradients. Chemical gradients are surface bound materials that incorporate gradually changing chemical and/or physical properties. A continuous and gradual change in the properties of gradients are expected and often required for their intended applications, which range from directed growth of cell colonies to combinatorial materials science. In reality, such conditions are almost never met due to spontaneous demixing and dewetting processes that can lead to properties variations on microscopic length scales. A better understanding on the properties of chemical gradients on microscopic length scales will aid in the production of better engineered materials. Single molecule spectroscopy (SMS) allows for gradient properties to be probed on nanometer-to-micrometer length scales. In this dissertation, quantitative measurements of gradient polarity (i.e., dielectric properties) are made along a sol-gel derived thin film that incorporates a macroscopic polarity gradient. These measurements report on the microscopic heterogeneity of the gradient film, and point to the occurrence of phase separation of the polar and nonpolar components along the gradient. Single molecule tracking (SMT) provides an important means to examine the dynamics of molecular mass transport in thin films and on surfaces. In this dissertation, SMT is employed to study mass transport in thin water films condensed over monolayer wettability gradients under ambient environments. The results show that the rate and the mechanism of molecular transport depend on the surface wettability, and on the ambient relative humidity. Finally, wettability gradients have been broadly used to drive the transport of liquid droplets. In this dissertation, these applications are extended to achieve spontaneous stretching of DNA by the propulsion of liquid droplets along the gradient. Single molecule fluorescence imaging of DNA stretched along these gradients demonstrates that hydrophobic surfaces play an important role in DNA stretching. The study also shows the surface tension force acting at the gradient-droplet contact line (interface) to be responsible for DNA elongation and alignment. Overall, single molecule methods have been shown to be highly useful for better understanding the properties of chemical gradients as described in this dissertation.

This paper demonstrates that it is possible to generate a reasonably accurate coordinate system on randomly distributed processors, using only local information and local communication. By coordinate systems we imply that each element assigns itself a logical coordinate that maps to its global physical location, starting with no apriori knowledge of position or orientation. The algorithm presented is inspired by biological systems that use chemical gradients to determine the position of cells. Extensive analysis and simulation results are presented. Two key results are: there is a critical minimum average neighborhood size of 15 for good accuracy and there is a fundamental limit on the resolution of any coordinate system determined strictly from local communication. We also demonstrate that using this algorithm, random distributions of processors produce significantly better accuracy than regular processor grids - such as those used by cellular automata. This has implications for discrete models of biology as well as for building smart sensor arrays.

A multitude of biophysical signals, including chemical, mechanical, and contact guidance cues, are embedded within the extracellular matrix (ECM) to dictate cell behavior and determine cell fate. To understand the complexity of the cell-matrix interaction and how changes to the ECM contribute to the development of tissues or diseases, three-dimensional (3D), culture systems that can decouple the effects of these cues on cell behavior are required. This dissertation describes the development and characterization of approaches based on multiphoton excitation (MPE) to control the chemical, mechanical, and topographical presentation of micro-3D-printed (μ-3DP) protein hydrogels independently. Protein hydrogels were chemically functionalized via the MPE-induced conjugation of benzophenone-biotin without altering the physical properties of the matrix. Complex, immobilized patterns and chemical gradients were generated within protein hydrogels with a high degree of spatial resolution in all axes. Hydrogel surfaces were also labeled with adhesive moieties to promote localized Schwann cell adhesion and polarization. Laser shrinking, a method based on MPE to manipulate the topographical and mechanical presentation of protein hydrogels after fabrication, is also presented. Topographical features on an originally flat substrate are created with depths approaching 6 μm. The Young’s modulus of protein hydrogels can also be increased by 6-fold (~15 – ~90 kPa) using laser shrinking, and parameters can be adjusted to create continuous gradient profiles for studying durotaxis. At determined scan conditions, the two properties can be adjusted independently of each other. Most importantly, the physical properties of the hydrogels can be manipulated in situ to study the effects of dynamic changes to the substrates on cells. As a potential tool to monitor cellular responses to presented cues, fluorescent probes that detect nitric oxide are characterized. Collectively, these technologies represent a key advance in hydrogel tunability, as the platforms presented offer independent, dynamic, and spatiotemporal control of the chemical, mechanical, and topographical features of protein hydrogels. The introduced technologies expand the possibilities of protein hydrogels to clarify underlying factors of cell-matrix interactions that drive morphogenesis and pathogenesis, and are broadly applicable to a multitude of physiological systems. / text

Interactions of cells with their chemical microenvironments are critical to many polarized processes, including differentiation, migration, and pathfinding. To investigate such cellular events, tools are required that can rapidly reshape the microscopic chemical landscapes presented to cultured cells. Existing chemical dosing technologies rely on use of pre-fabricated chemical gradients, thus offering static cell-reagent interactions. Such interactions are particularly limiting for studying migration and chemotaxis, during which cells undergo rapid changes in position, morphology, and intracellular signaling. This dissertation describes the use of laminar streams, containing cellular effector molecules, for precise delivery of effectors to selected subcellular regions. In this approach, cells are grown on an ultra-thin polymer membrane that serves as a barrier to an underlying reagent reservoir. By using a tightly-focused pulsed laser beam, micron-diameter pores can be ablated in the membrane upstream of desired subcellular dosing sites. Emerging through these pores are well-defined reagent streams, which dose the targeted regions. Multiple pores can be ablated to allow parallel delivery of effector molecules to an arbitrary number of targets. Importantly, both the directionality and the composition of the reagent streams can be changed on-the-fly under a second to present dynamically changing chemical signals to cells undergoing migration. These methods are applied to study the chemotactic responses of neutrophil precursor cells. The subcellular localization of the chemical signals emerging through pores is found to influence the morphological evolution of these motile cells as they polarize and migrate in response to rapidly altered effector gradients. / text