Fluorescence microscopy has emerged as a popular and powerful tool within biology research, owing to its exceptional signal contrast, specificity, and the versatility of the various microscope designs. Fluorescence microscopy has been used to study samples across orders of magnitude in physical scale ranging from tissues to cells, down to single-molecules, and as such has led to breakthroughs and new knowledge in a wide variety of research areas. In particular, single-molecule techniques are somewhat unique in their ability to study biomolecules in their native state, which enables the visualization of short-lived interactions and rare events which can be highly relevant in clinical applications. For example, single-molecule real-time DNA sequencing has become a workhorse in genomics and personalized medicine. However, there have been few other analytical tools based on single-molecule fluorescence microscopy that have become popular in biomedical applications. This dissertation describes work performed in an effort to transition single-molecule techniques from a research setting to a clinical setting. There were two main goals throughout: to develop quantitative single-molecule assays for data-rich analysis, and to make those assays more user-friendly to facilitate their adoption as standardized techniques. An initial study demonstrated the practicality of single-molecule analysis as a diagnostic tool by measuring differences in protein content between healthy patients and patients with Parkinson's disease. From there, the assay was improved through various methods of beam shaping, which enabled more quantitative analysis of the detected biomolecules. A passivation scheme and sample preparation protocol were developed that reduce the time to perform a single-molecule assay by more than half while improving the assay sensitivity. Additionally, work performed to control the fluorescent labeling of the target protein is described, with a goal of determining the stoichiometry of protein complexes, which is highly relevant to the pathology of Parkinson's disease and other neurodegenerative diseases. The report concludes with prospective projects that could extend the work completed thus far. An alternative labeling approach is outlined that may achieve one-to-one labeling between the proteins and fluorophores, as well as a project that shifts away from fluorescence microscopy and moves to a label-free scattering-based microscope design.
If this is your thesis or dissertation, and want to learn how to access it or for more information about readership statistics, contact us at STARS@ucf.edu
Han, Kyu Young
Doctor of Philosophy (Ph.D.)
College of Optics and Photonics
Optics and Photonics
Optics and Photonics
Length of Campus-only Access
Doctoral Dissertation (Open Access)
Croop, Benjamin, "Development of Quantitative Intensity-Based Single-Molecule Assays" (2021). Electronic Theses and Dissertations, 2020-. 1130.