Chemical and Biological Engineering ETDs

Publication Date

Spring 5-16-2026

Abstract

Single-molecule fluorescence spectroscopy is a powerful technique for resolving transient biomolecular dynamics and quantifying free energy landscapes, kinetics, and binding interactions. However, two fundamental limitations restrict its broader application: slow, labor-intensive data acquisition and the limited observation time imposed by fluorophore photobleaching. These limitations hinder its use in drug discovery and biomedical engineering applications that require both high-throughput and access to long-time dynamics. In this work, both challenges are addressed through technical advancements. First, a simple, generalizable approach is introduced to automate data acquisition, eliminating manual intervention during experiments. This increases the acquisition rate by more than an order of magnitude. Second, a method is introduced to extend observation times by controlling the repetition rate of a pulsed excitation source. Using surface-immobilized Alexa Fluor 350 as a model system, decreasing the repetition rate from 40 MHz to 2.5 MHz extends bleaching time by an order of magnitude. Although photon flux decreases, correlation-based analyses such as fluorescence correlation spectroscopy do not suffer a loss in signal-to-noise ratio. Together, these advances can be applied to any single-molecule spectroscopy setup to improve throughput and extend observation times.

Keywords

Single-Molecule Spectroscopy, Automation, Fluorescence Lifetime Correlation Spectroscopy (FLCS), Pulsed Laser

Document Type

Thesis

Language

English

Degree Name

Chemical Engineering

Level of Degree

Masters

Department Name

Chemical and Biological Engineering

First Committee Member (Chair)

John King

Second Committee Member

Nick Carroll

Third Committee Member

William Bricker

Fourth Committee Member

Rama Gullapalli

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