Fluorophores for single-molecule imaging in living cells : characterizing and optimizing DCDHF photophysics
Abstract/Contents
- Abstract
- The number of reports per year on single-molecule imaging experiments has grown roughly exponentially since the first successful efforts to optically detect a single molecule were completed over two decades ago. Single-molecule spectroscopy has developed into a field that includes a wealth of experiments at room temperature and inside living cells. The fast growth of single-molecule biophysics has resulted from its benefits in probing heterogeneous populations, one molecule at a time, as well as from advances in microscopes and detectors. There is a need for new fluorophores that can be used for single-molecule imaging in biological media, because imaging in cells and in organisms require emitters that are bright and photostable, red-shifted to avoid pumping cellular autofluorescence, and chemically and photophysically tunable. To this end, we have designed and characterized fluorescent probes based on a class of nonlinear-optical chromophores termed DCDHFs. This dissertation describes various physical and optical studies on these emitters, from sensing local environment to photoactivation. Chapter 1 is a general introduction to fluorescence and single-molecule spectroscopy and imaging. Single-molecule experiments in living cells are discussed and probes used for such experiments are summarized and compared. Chapter 2 explores the basic photophysics of the DCDHF fluorophores and some general methods of measuring relevant spectroscopic parameters, including photostability. Chapter 3 discusses the various approaches we have taken to modify particular properties by changing the fluorophore's structure. We have redesigned the DCDHF fluorophore into a photoactivatable fluorogen--a chromophore that is nonfluorescent until converted to a fluorescent form using light--described in Chapter 4. Finally, a different, chemical route to fluorescence activation is presented in Chapter 5. The remainder of the Dissertation is the Appendix and a full Bibliography. The Appendix includes a table of photophysical parameter for DCDHF fluorophore, various protocols used in the experiments discussed, MatLab codes, and NMR spectra.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2010 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Lord, Samuel Joseph | |
|---|---|---|
| Associated with | Stanford University, Department of Chemistry | |
| Primary advisor | Moerner, W. E. (William Esco), 1953- | |
| Thesis advisor | Moerner, W. E. (William Esco), 1953- | |
| Thesis advisor | Pande, Vijay | |
| Thesis advisor | Zare, Richard N | |
| Advisor | Pande, Vijay | |
| Advisor | Zare, Richard N |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Samuel Joseph Lord. |
|---|---|
| Note | Submitted to the Department of Chemistry. |
| Thesis | Ph. D. Stanford University 2010 |
| Location | electronic resource |
Access conditions
- Copyright
- © 2010 by Samuel Joseph Lord
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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