Graduate Thesis Or Dissertation

 

Fluorescent Proteins: Spectroscopic Studies, Micro uidic Analysis, and Generation of Improved Variants Public Deposited

https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/gf06g277n
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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  • 2013-01-01
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  • 2019-11-13
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