Date of Award

Spring 1-1-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Amy E. Palmer

Second Advisor

Ralph Jimenez

Third Advisor

Arthur Pardi

Fourth Advisor

Thomas T. Perkins

Fifth Advisor

Rafael Piestun

Abstract

Fluorescent proteins (FPs) are powerful tools that permit real-time visualization of cellular processes. The utility of a given FP for a specific experiment depends strongly on its effective brightness, and propensity to undergo photodegradation and dark-state conversion. However, photobleaching of fluorescence is a complex phenomenon that depends on a variety of experimental and environmental factors. Under single-molecule conditions, FPs are particularly subject to photobleaching, emitting 10-100× less photons than their small-molecule counterparts. Here, we present methodology that can be used to measure irreversible photobleaching, its adaptation for high-throughput microfluidics-based directed evolution, and its use in the development of improved Red FPs (RFPs). Progress towards incorporation of frequency-domain fluorescence lifetime assays is also discussed. Given the quantitative and high-throughput nature of the microfluidic pho- tobleaching platform, this technology complements existing methods for fluorescent protein selection, thereby facilitating the development of next-generation RFPs for single-molecule research.

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