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Proceedings Paper

A multidimensional screening method for the selection of two-photon enhanced fluorescent proteins
Author(s): Caleb Stoltzfus; Lauren Barnett; Aleksander Rebane; Thomas Hughes; Mikhail Drobizhev; Geoffrey Wicks; Alexandr Mikhailov
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Paper Abstract

Two-photon excitation of fluorescent proteins (FPs) is widely used in imaging whole organisms or living tissues. Many different FPs are now available but these proteins have only been optimized for their one-photon properties. We have developed a technique for screening entire libraries of E. coli colonies expressing FPs that utilizes multiple wavelengths of linear excitation as well as two-photon excitation. Single mutations in a particular protein that affect one or twophoton properties are easily identified, providing new views of structure/function relationships. An amplified femtosecond Ti:sapphire laser and a spectrally filtered lamp source are used to acquire the fluorescence signals of up to ~1000 E. coli colonies on a standard Petri dish. Automation of the analysis and acquisition of the fluorescent signals makes it feasible to rapidly screen tens of thousands of colonies. In a proof of principle experiment with the commonly used EGFP, we used two rounds of error prone PCR and selection to evolve new proteins with shifted absorption and increased two-photon cross sections at 790nm. This method of screening, coupled with careful measurements of photo bleaching dynamics and two-photon cross sections, should make it possible to optimize a wide variety of fluorescent proteins and biosensors for use in two-photon microscopes.

Paper Details

Date Published: 5 March 2014
PDF: 7 pages
Proc. SPIE 8956, Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications VI, 895611 (5 March 2014); doi: 10.1117/12.2039873
Show Author Affiliations
Caleb Stoltzfus, Montana State Univ. (United States)
Lauren Barnett, Montana State Univ. (United States)
Aleksander Rebane, Montana State Univ. (United States)
Thomas Hughes, Montana State Univ. (United States)
Mikhail Drobizhev, Montana State Univ. (United States)
Geoffrey Wicks, Montana State Univ. (United States)
Alexandr Mikhailov, Montana State Univ. (United States)


Published in SPIE Proceedings Vol. 8956:
Reporters, Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications VI
Samuel Achilefu; Ramesh Raghavachari, Editor(s)

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