Date Thesis Awarded

5-2024

Access Type

Honors Thesis -- Access Restricted On-Campus Only

Degree Name

Bachelors of Science (BS)

Department

Chemistry

Advisor

Kristin Wustholz

Committee Members

J.C. Poutsma

Andrew Tobolowsky

Christopher Abelt

Abstract

The visualization of biological systems can provide insight into the structure of sub-cellular components. By overcoming the diffraction limit of light, super-resolution microscopy can image structures on a nano-scale level with incredible resolution. Multicolor super-resolution imaging with fluorescent probes is a popular method for analyzing the interactions of different facets of biological systems. Typically, experiments involving multiple probes within a single system require fluorescent molecules with distinguishable emission spectra in order to provide multicolor images. The small number of available and compatible probes of different spectra further limits the production of these images. To address this limitation, we describe Blinking-Based Multiplexing (BBM), a method for identifying probes based not on their spectra, but on their intrinsic fluctuations in emission intensity under continuous excitation (so-called blinking). This thesis describes the development of BBM, and its applications for distinguishing three spectrally-overlapped emitters, Rhodamine 6G (R6G), CdSe/ZnS quantum dots (QD), and pyrromethene 605 (PM605), a fluorescent probe from the promising BODIPY class of molecules. As a first step, the blinking mechanisms of all three emitters are determined by analyzing the on- and off-time durations for emitters on glass under continuous excitation. Using change point detection and logistic regression analyses, we determine that R6G can be distinguished from QD and PM605 with minimum classification accuracies of 92.5% and 76.1% respectively. To understand photophysical properties responsible for low R6G/PM605 classification accuracy, we next investigate the underlying mechanism for PM605 emission at two excitation powers, 1.05 µW and 0.787 µW. First determine the functional form that best represents the on- and off-time distributions using goodness-of-fit tests based on the Kolmogorov-Smirnov statistic and maximum likelihood estimation. We find that the data fits a power-law distribution and a log-normal distribution, which is consistent with the Albery model for dispersive electron transfer. In addition, using fit parameters, we quantify the rates of entry to and exit from the dark state and determine that PM605 blinking is a dispersive process. This study reveals the blinking mechanism of PM605 and demonstrates BBM to be an effective method for distinguishing spectrally-overlapped, small-molecule fluorophores.

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