Date of Award

8-2011

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Physics

Advisor

Samuel T. Hess

Second Committee Member

Charles T. Hess

Third Committee Member

Michael D. Mason

Abstract

Applicability to living specimens and genetically encodable tagging has made fluorescence microscopy both powerful and versatile in life sciences, yet image resolution of fluorescence far-field microscopy has been limited by diffraction. Superseding this limitation, recently discovered super-resolution microscopic techniques can now resolve structures down to a few nanometers, at least ten fold better compared to conventional methods. The localization based super-resolution microscopic method fluorescence photoactivation localization microscopy (FPALM) utilizes genetically encodable photoactivatable fluorescent proteins (PAFPs) to label specimens that facilitate maintaining spatially sparse subsets of fluorescent molecules during imaging. Even though the image of a fluorescent single molecule itself is diffraction limited, it can be localized with precision better than the diffraction based resolution. This precision is primarily limited by the number of photons collected. A sequence of images sampling either all or most of the specimen's tagged PAFPs are subsequently localized and rendered as high density maps revealing structures of interest at tens of nanometer resolution. Rapidly evolving, localization based super-resolution microscopic methods are now capable of imaging live specimens, multiple species, and single molecule anisotropics, and have been extended to three dimensions. This thesis primarily discusses methodology developed to couple the superresolution capability of FPALM to measure single molecule anisotropy, three dimensional orientations (Chapter 2) and simultaneous imaging of multiple (three) PAFPs (Chapter 3) and used to supplement the understanding of the organization and interaction of the cell plasma membrane with cytoskeletal and viral proteins. A method based on single molecule counting statistics and a first order linear kinetic model are presented to estimate the photophysical property that quantifies the nature of photoactivation (photoactivation yield) of a PAFP (Chapter 4). Photoactivation yields were estimated for three extensively used PAFPs. Chapter 5 discusses a scheme able to compensate for sample stage drift both in axial and radial directions. The concluding Chapter 6 discusses future directions.

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