Advances in single molecule spectroscopy and microscopy for biological imaging and polymer characterization

single molecule fluorescence;fluorescent probe development;self-healing fluorophores;magnetic tweezers;photostabilization;Trolox

Reilly, Daniel Timothy

关键词: single molecule fluorescence;    fluorescent probe development;    self-healing fluorophores;    magnetic tweezers;    photostabilization;    Trolox;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/90560/REILLY-DISSERTATION-2016.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Single molecule fluorescence microscopy and single molecule spectroscopy provide tremendously powerful methods for studying the behavior of a wide variety of biological systems. In this way, single molecule techniques can be used to gain an increased understanding of molecular mechanisms underlying basic phenomena in biology, materials science, and soft matter. In general, these approaches allow for detailed molecular information to be obtained when compared to bulk level methods performed using macroscopic techniques. In the first part of this thesis, we use single molecule fluorescence microscopy (SMFM) to develop and characterize a new class of fluorescent probes. Using SMFM, target biomolecules are commonly labeled with single fluorescent dyes allowing for real-time observation of dynamics and transient events. However, single molecule fluorescence imaging critically relies on bright dyes for robust signal detection above a noisy cellular background. Additionally, photostable dyes are desired to allow for continuous imaging of long time scale biological processes. To address this challenge, we developed a new class of fluorescent probes for SMFM using a two primary strategies designed to increase brightness and photostability. First, we developed fluorescent dendrimer nanoconjugates (FDN) consisting of multiple individual Cy5 dye molecules conjugated to a polymer dendrimer scaffold, which allows for increases in the total brightness of the molecules. In addition, we designed a series of 'self-healing' dendrimers that have a photoprotective molecule, Trolox, covalently attached to the probes, thereby resulting in increased photostability. Specifically, we designed the 'self-healing' FDNs using two complementary synthetic strategies, termed 'random addition' and 'controlled addition' allowing for control over the average stoichiometric ratio between Trolox and Cy5, and exact conjugation of Trolox and Cy5 with a precise one-to-one ratio. In all FDNs synthesized with the 'self-healing' strategy, we observe increases in probe photostability. In the second part of this thesis, we use single molecule force spectroscopy (SMFS) to study the mechanical properties of polymer systems. Here, we specifically examine the effect of the photostabilizer Trolox on the physical properties of the biopolymer, DNA. Using this technique, we exert force on a single polymer and observe the response of the molecule (typically increased extension under a stretching force). Using this method, we are able to determine polymer physical properties such as persistence length and contour length. We use magnetic tweezers for SMFS, a commonly used technique where a paramagnetic bead is attached to a tethered polymer, allowing for an external magnetic field to pull on the polymer. By observing the bead position over time, we can extract information on the polymer physical properties. We compare how these properties change with the addition of commonly used solution additives, primarily Trolox, used in fluorescence studies in polymer physics to provide enhanced photostability. In particular, we show that the addition of Trolox into solution with DNA induces an increase in the total contour length, consistent with results from our lab on DNA stretching experiments with SMFM. In summary, my work applies a consistent theme of addressing dye photostability and the photophysical properties of fluorescent probes for single molecule techniques. We demonstrate the development of a new class of photostable probes for fluorescence microscopy, and we determine the effect of commonly used photostabilizer on the physical properties of a model polymer system, DNA. Overall, this work will help advance the techniques available in single molecule imaging by increasing our understanding of the photophysical mechanisms underlying multi-dye conjugates and the possible physical changes to a system that can occur when using photostabilizing agents.