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Document Type

Dissertation - University Access Only

Award Date


Degree Name

Doctor of Philosophy (PhD)


Chemistry and Biochemistry

First Advisor

Adam D. Hoppe


Fluorescence microscopy Protein-protein interactions Cells

Library of Congress Subject Headings

Quantitative Fluorescence Resonance Energy Transfer (FRET) microscopy is capable of imaging the nanoscale associations of proteins in living cells. These methods have opened new windows to visualize and quantify when and where proteins are touching and conducting biochemistry within the cell, thereby providing a powerful tool to translate the current understanding of in vitro biochemistry into its native context. Until recently, FRET microscopy approaches have been limited to the analysis of one protein-protein interaction at a time and in two-dimensions. In this dissertation, FRET microscopy methods are developed which are capable of imaging the interactions of any number of fluorophores in two-dimensions. This dissertation also presents the first image reconstruction approach to improve the three-dimensional optical sectioning of multiple protein-protein interaction. Additionally, an in-depth characterization of fluorescent proteins for multifluorophore FRET was conducted leading to the selection of an optimized fluorescent protein trio that enhances the sensitivity and reliability of the new approaches. Together this work represents a significant advance in methods for studying biochemistry within the context of the living cell.


Includes bibliographical references



Number of Pages



South Dakota State University


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