Stimulated emission depletion super-resolution fluorescence microscopy : addressing biophysical questions from bacteria to eukaryotic cells
Abstract/Contents
- Abstract
- Fluorescence microscopy is a widely used tool in biological studies, allowing specific labeling and imaging of proteins and structures within living cells. As reviewed in Chapter 1, conventional fluorescence microscopy is unable to distinguish structures below ~250 nm in size due to the diffraction limit of light. Over the last two decades, the field of super-resolution microscopy has overcome this diffraction limit, allowing fluorescence imaging of structures just a few tens of nanometers in size. This thesis discusses one super-resolution technique, stimulated emission depletion (STED) microscopy, and its application to various cellular and molecular biophysics questions. In Chapter 2, the bespoke STED microscope used for much of this thesis is described in detail. Some guiding principles behind STED microscopes and their design are discussed. We next discuss the major improvements to the STED microscope: fast scanning and two-color. Fast scanning protects fluorophores from photobleaching during imaging, improving image quality and expanding the range of available fluorophores for STED imaging. Two-color imaging allows the examination of two different protein species, a universal requirement in many biological studies. In Chapter 3, we apply STED microscopy to study how the bacteria Caulobacter crescentus assembles a 2D paracrystalline surface layer (S-layer) composed of the protein RsaA. We show that S-layer assembly is localized to highly curved regions of the cell, and that this localized assembly depends upon the presence of a pre-existing crystalline S-layer. Together, our results suggest the topology of the cell surface combined with continuous protein crystallization localizes S-layer assembly. In Chapter 5, 2-color STED microscopy is used to examine protein interactions important for the 3D organization of the genome in eukaryotic cells. We quantify clustering of the DNA binding protein CCCTC-binding factor (CTCF), as well as the molecular coupling between CTCF and cohesin, two proteins hypothesized to play a major role in organizing topologically associating domains (TADs). We show that cohesin plays a regulatory role in CTCF clustering by perturbing its activity and monitoring cluster size. Finally, we discuss several applications of STED imaging to support larger studies. Chapter 4 discusses applying a genetically encoded fluoromodule for STED imaging in live bacteria cells. In the first half of Chapter 6, STED reveals the localization of the centriole protein Cby1 to the transition zone, supporting a role for Cby1 in recruiting Ahi1, another transition zone protein. Finally, the second half of Chapter 6 uses two-color STED microscopy to demonstrate that deletion of the intracellular cargo transport protein IFT25 causes simultaneous defects in the cilium cytoskeleton as well as ciliary membrane morphology
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 | Comerci, Colin James |
|---|---|
| Degree supervisor | Moerner, W. E. (William Esco), 1953- |
| Thesis advisor | Moerner, W. E. (William Esco), 1953- |
| Thesis advisor | Huang, Kerwyn Casey, 1979- |
| Thesis advisor | Shapiro, Lucy |
| Degree committee member | Huang, Kerwyn Casey, 1979- |
| Degree committee member | Shapiro, Lucy |
| Associated with | Stanford University, Biophysics Program. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Colin J. Comerci |
|---|---|
| Note | Submitted to the Biophysics Program |
| Thesis | Thesis Ph.D. Stanford University 2019 |
| Location | electronic resource |
Access conditions
- Copyright
- © 2019 by Colin James Comerci
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
Also listed in
Loading usage metrics...