Engineering new capabilities into optical microscopes : toward measuring the three-dimensional position and orientation of single molecules in living cells
Abstract/Contents
- Abstract
- The labeling of specific biological structures with single fluorescent molecules has ushered in a new era of imaging technology: super-resolution optical microscopy with resolution far beyond the diffraction limit down to some tens of nm. With the features of these exquisite tools in mind, this Dissertation discusses optical strategies for measuring the three-dimensional (3D) position and orientation of single molecules with nanoscale precision and several super-resolution imaging studies of structures in living cells. The concepts of single-molecule imaging, super-resolution microscopy, the engineering of optical point spread functions (PSFs), and quantitative analysis of single-molecule fluorescence images are introduced. The various computational methods and experimental apparatuses developed during the course of my graduate work are also discussed. Next, a new engineered point spread function, called the Corkscrew PSF, is shown for 3D imaging of point-like emitters. This PSF has been demonstrated to measure the location of nanoscale objects with 2-6 nm precision in 3D throughout a 3.2-micrometer depth range. Characterization and application of the Double-Helix (DH) PSF for super-resolution imaging of structures within mammalian and bacterial cells is discussed. The DH-PSF enables 3D single-molecule imaging within living cells with precisions of tens of nanometers throughout a ~2-micrometer depth range. Finally, the impact of single-molecule emission patterns and molecular orientation on optical imaging is treated, with particular emphasis on multiple strategies for improving the accuracy of super-resolution imaging. The DH microscope is shown to be well-suited for accurately and simultaneously measuring the 3D position and orientation of single molecules.
Description
| Type of resource | text |
|---|---|
| Form | electronic; electronic resource; remote |
| Extent | 1 online resource. |
| Publication date | 2015 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Associated with | Lew, Matthew D | |
|---|---|---|
| Associated with | Stanford University, Department of Electrical Engineering. | |
| Primary advisor | Moerner, W. E. (William Esco), 1953- | |
| Thesis advisor | Moerner, W. E. (William Esco), 1953- | |
| Thesis advisor | Fan, Jonathan Albert | |
| Thesis advisor | Solgaard, Olav | |
| Advisor | Fan, Jonathan Albert | |
| Advisor | Solgaard, Olav |
Subjects
| Genre | Theses |
|---|
Bibliographic information
| Statement of responsibility | Matthew D. Lew. |
|---|---|
| Note | Submitted to the Department of Electrical Engineering. |
| Thesis | Thesis (Ph.D.)--Stanford University, 2015. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2014 by Matthew Duk-Ying Lew
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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