Exploring cellular structures on the nanoscale with stimulated emission depletion microscopy
Abstract/Contents
- Abstract
- Super-resolution fluorescence microscopy has proven to be a powerful tool to study biology. STimulated Emission Depletion (STED) microscopy is a super-resolution technique that is compatible with a variety of biological applications including imaging in multicolor and with live cells. In this thesis, I describe three such applications from imaging cell samples to whole tissue, beginning with a short primer on the scientific concepts that are fundamental to my thesis work. In Chapter 2, I then lay the foundation for the super-resolution projects I expound on in the rest of the thesis. In Chapter 3, I detail work done in collaboration with Dr. Colin Comerci, Dr. Bo Gu, and Dr. Saumya Saurabh to study proteins involved in genomic organization. CCCTC-binding factor (CTCF) and cohesin had been previously shown to be involved in organizing the so-called topologically associated domains (TADs) within chromatin in mammalian cells. Two-color STED microscopy combined with quantitative image analysis of endogenously labeled cells provides insight into the relationship between CTCF and cohesin in the nucleus. We demonstrate that CTCF and cohesion are organized into clusters, and a fraction of these clusters are coupled with a characteristic distance, which could perhaps indicate a molecular interaction. Through additional biochemical perturbations, we provide further insight into how transcription and other processes influence this relationship. In Chapter 4, I explain my contribution to Dr. Damien Garbett's larger study of actin and an ancient actin bundling protein, T-plastin. Dr. Garbett probed how cells are able to migrate across large gaps in the extracellular matrix (ECM). He found that T-plastin helps facilitate cell migration especially in the context of discontinuous ECMs. Here, I contributed super-resolution STED images to explore in greater detail the relationship between actin and T-plastin in protrusive areas as compared to the main cell body. In Chapter 5, I recount a preliminary study done in collaboration with doctors at California Pacific Medical Center studying immune checkpoint proteins that are the targets of a new immunotherapy treatment. Although promising, immune checkpoint immunotherapy is currently only effective in fraction of cancer patients. The variation in sub-cellular structure and density of immune checkpoint proteins in the tumor microenvironment could perhaps correlate with response to immunotherapy treatment. We utilize two-color STED microscopy to probe the nano-scale organization of the immune checkpoint protein Programmed Death-1 (PD-1) in metastatic melanoma tissue. We observe various degrees of nanoscale clustering of PD-1 on cells within the tumor microenvironment. Here, I also describe some challenges to two-color imaging in tissue samples.
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 | 2021; ©2021 |
| Publication date | 2021; 2021 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | McCarthy, Dannielle Grace |
|---|---|
| Degree supervisor | Moerner, W. E. (William Esco), 1953- |
| Thesis advisor | Moerner, W. E. (William Esco), 1953- |
| Thesis advisor | Bertozzi, Carolyn R, 1966- |
| Thesis advisor | Cui, Bianxiao |
| Degree committee member | Bertozzi, Carolyn R, 1966- |
| Degree committee member | Cui, Bianxiao |
| Associated with | Stanford University, Department of Chemistry |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Dannielle Grace McCarthy. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Thesis Ph.D. Stanford University 2021. |
| Location | https://purl.stanford.edu/rk048mc4872 |
Access conditions
- Copyright
- © 2021 by Dannielle Grace McCarthy
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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