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Fluorescent Proteins: Spectroscopic Studies, Microfluidic Analysis, and Generation of Improved Variants

Event Details

Event Dates: 

Monday, April 22, 2013 - 4:00pm

Seminar Location: 

  • Engineering Building Room ECCR 105

Speaker Name(s): 

Kevin Dean

Speaker Affiliation(s): 

University of Colorado Boulder
Seminar Type/Subject

Scientific Seminar Type: 

  • COSI Seminar

Event Details & 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 10-100x less photons than their small - molecule counterparts. Here, we present methodology that can be used to measure irreversible photobleaching, it’s 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 themicrofluidic photobleaching platform, this technology complements existing methods for fluorescent protein selection, thereby facilitating the development of next-generation RFPs for single-molecule research.