Investigating single-molecule interactions and photodynamics in solution by counteracting Brownian motion
Abstract/Contents
- Abstract
- Single-molecule fluorescence methods have become a mature and powerful tool to unravel the fundamental mechanisms of biological phenomena. These approaches enable researchers to investigate individual components on the nanoscale for heterogeneous mixtures and to monitor the dynamics of single biomolecules. Unfortunately, traditional burst-based methods are often limited by short observation time, and the immobilization of single biomolecules that enables long observation time is often undesirably perturbative. To address these limitations, a solution-based technique known as the Anti-Brownian ELectrokinetic (ABEL) trap has been developed. In the ABEL trap, high-speed feedback electronics sense position and compensate Brownian motion of a single molecule by applying electrokinetic forces, which keep the molecule in the trapping region for an extended period of time. This dissertation describes my work on the investigation of single-molecule interactions and photodynamics using the ABEL trap technology. First, I show how to measure and monitor single-molecule rotational diffusivity by combining the ABEL trap and maximum likelihood analysis of time-resolved fluorescence anisotropy based on the information inherent in each detected photon. The rotational diffusivity is extremely sensitive to the size of the molecule, and thus can be used to detect and monitor size-changing events, such as association and dissociation. We demonstrate this approach by resolving a mixture of single- and double-stranded fluorescently-labeled DNA molecules at equilibrium, freely rotating in a native solution environment. Next, we investigate photosynthetic photoprotection mechanisms at the level of individual antenna complexes (LHCII), the fundamental units which green plants use to harvest sunlight. We access the intrinsic conformational dynamics of individual LHCII complexes. In addition to an unquenched state, two partially quenched states of LHCII are observed. One of the quenched conformations significantly increases in relative population under environmental conditions mimicking high light. Our results suggest that there are at least two distinct quenching sites with different molecular compositions, meaning multiple dissipative pathways in the LHCII complex. Last, we describe the theoretical basis of a new method to measure the diffusion coefficient of diffusing objects on the surface of individual nanoscale lipid vesicles. We discuss how to probe diffusion in these highly confined environments using FRET pairs and fluorescence intensity correlation, and illustrate that this method is essentially a fluorescence correlation spectroscopy (FCS) experiment with a nanometer-sized probing volume.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2019; ©2019 |
| Publication date | 2019; 2019 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Yang, Hsiang-Yu | |
|---|---|---|
| Degree supervisor | Moerner, W. E. (William Esco), 1953- | |
| Thesis advisor | Moerner, W. E. (William Esco), 1953- | |
| Thesis advisor | Dai, Hongjie, 1966- | |
| Thesis advisor | Fayer, Michael D | |
| Degree committee member | Dai, Hongjie, 1966- | |
| Degree committee member | Fayer, Michael D | |
| Associated with | Stanford University, Department of Chemistry. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Hsiang-Yu Yang. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis Ph.D. Stanford University 2019. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2019 by Hsiang-Yu Yang
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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