Year of Publication

2017

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department

Chemistry

First Advisor

Dr. Christopher Isaac Richards

Second Advisor

Dr. Dennis Clouthier

Abstract

Fluorescence microscopy is a powerful tool for interpreting the structure and function of biomolecules, and their interactions with one another. Understanding fundamental biological mechanisms is important to the development of improved treatments for a variety of diseases. Fluorescent tags are used to track the motion and longevity of such events, with the ability to monitor several biomolecules at once. Currently, many of these studies are conducted using bulk measurements, or several biological events at the same time, because the added light from several emitters can more easily overcome the high fluorescence background inherent to biological systems. Although important in their own right, these studies tend to cloud the mechanistic nature of most biological interactions. Single molecule fluorescence studies are preferred over bulk methods to gather information regarding biological interactions as they are capable of extrapolating more intricate details about an event, but the caveat is that fewer fluorescent tags means a greater demand for fluorophores that are very bright and photostable for long periods of time. My projects study the modifications that fluorophores undergo when their external environment is modified by nanoscale metallic apertures, called zero-mode waveguides (ZMWs). To probe these modifications, I characterize multiple combinations of fluorophores that emit in varying regions of the visible spectrum with ZMWs of different compositions. After determining the combinations that invoke the highest fluorescence efficiency for specific biological situations, I used the information to assist in the development of an imaging technique that allowed for the observation of single ligand-receptor interactions. By incorporating hybrid ZMW nanostructures with a novel microfluidic delivery chamber, this technique successfully imaged the binding of single epidermal growth factor (EGF) molecules to their corresponding receptors in living neuroblastoma cells. My final project performed a new characterization study focused on maximizing the fluorescent enhancement capabilities of ZMWs in three dimensions, and use the information to provide a practical guide for choosing ZMWs during future project development. These projects use a combination of fluorescence microscopy, nanofabrication, and single molecule isolation techniques to demonstrate the viability for using ZMWs to single biological interactions in living cells.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2017.389

Available for download on Monday, March 12, 2018

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