Date of Award

12-2015

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Physics

Advisor

Samuel T. Hess

Second Committee Member

Michael Mason

Third Committee Member

R. Dean Astumian

Additional Committee Members

George Bernhardt

David Batuski

Abstract

Due to the diffraction limit of light, the resolution of fluorescence light microscopy is limited to ~250 nm. Super resolution techniques such as Fluorescence Photoactivation Localization Microscopy (FPALM) can circumvent this limit and improve the final image resolution by roughly a factor of ten. Photoactivatable fluorescent molecules are stochastically converted from a fluorescent dark state to a bright state and imaged until they photobleach. Only a sparse subset of molecules emits light at any given time, and the cycle of emission and bleaching is repeated through time as data are simultaneously acquired. The resultant single molecules are mathematically localized in order to find their spatial positions to a much higher precision than allowed by the diffraction limit. Super resolved images are generated from the localized molecule positions; typically resulting in a final image resolution on the scale of tens of nanometers.

While localization microscopy can image nanoscale cellular details, the ability to distinguish multiple fluorescent species simultaneously is invaluable in addressing a number of biological questions. Previously published multispecies schemes have divided the detected fluorescence into two distinct spectral channels, often with the ratio between channels used for species identification. However, such ratiometric methods have been limited in the number of species that can be detected simultaneously, and are unable to obtain the emission spectra of the imaged species, thus limiting their ability to distinguish multiple species. We present a localization microscopy method which detects the emission spectrum of each localized single molecule. In this scheme, a prism in the detection path spatially disperses the fluorescence signal according to the emission spectrum of each single molecule, which is recorded in one of two detection channels. The emission spectrum of each single molecule can be used for fluorescent species identification, theoretically allowing a major increase in the number of different species that can be simultaneously imaged in a sample. This technique enables an exciting new family of super-resolution imaging experiments which can report spectral emission changes due to pH, hydrophobicity, redox state, ion concentrations, temperature, or other factors, while also recording precise nanometer molecular locations.

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