The overall objective of this work is to develop novel single molecule microscopy methods that will enable mechanistic studies of the ways in which surface chemistry affects adsorbed protein conformation and intermolecular associations. Although resistance to protein adsorption is often cited as necessary for a particular application (biosensing, biocompatibility, etc.), even the most protein- resistant surfaces permit some protein adsorption. Therefore, this work will test the hypothesis that vicinal surface chemistry indirectly affects protein behavior after adsorption to influence the propensity for intermolecular associations (binding, aggregation, etc.). A mechanistic understanding of post- adsorptive protein behavior and the ability of the surface to mediate this behavior will ultimately lead to better surface coatings for a variety of biomedical technologies. As a relevant and convenient model system, fluorescently-labeled fibronectin (Fn) will be studied on model biocompatible surfaces as well as surfaces that specifically probe electrostatic, hydrophilic and hydrophobic interactions. This work will use single-molecule fluorescence microscopy techniques at the solid-aqueous interface that are capable of simultaneously measuring the molecular conformation of an individual Fn molecule and tracking its effect on dynamic processes such as adsorption, diffusion, desorption, aggregation, and receptor binding. Resonance energy transfer between Fn labels will be used to probe molecular conformation. The first specific aim will examine the ability of different surfaces to influence Fn conformation and the subsequent effect this has on Fn surface affinity and diffusion. Building on this understanding, the second aim will address protein-protein interactions for their propensity to form a stable protein film. The surfac is expected to indirectly influence film formation through the mobility of proteins on the surface and their propensity for cluster formation, properties that likely depend on Fn conformation. PUBLIC HEALTH RELEVANCE: Interactions between surfaces and proteins have widespread relevance to human health, affecting medical diagnostics, therapeutic protein stability, vaccine efficacy/safety, and biomaterial design. A common technological goal involves the preparation of protein- resistant surfaces. However, the mechanisms of protein resistance remain elusive, and even the most biocompatible surfaces fail to prevent adhesion entirely. To resolve this inconsistency, this work will develop new microscopy techniques to test the hypothesis that biocompatible surfaces permit adhesion of proteins that are unlikely to trigger an adverse response and may also influence their post-adhesion behavior to prevent them from adopting unwanted behavior. A more complete understanding of the role of the surface in biocompatibility will lead to the design of improved surfaces for biosensors, implanted devices and equipment for biological experimentation.