title: Two-photon directed evolution of green fluorescent proteins.
abstract: Directed evolution has been used extensively to improve the properties of a variety of fluorescent proteins (FPs). Evolutionary strategies, however, have not yet been used to improve the two-photon absorption (2PA) properties of a fluorescent protein, properties that are important for two-photon imaging in living tissues, including the brain. Here we demonstrate a technique for quantitatively screening the two-photon excited fluorescence (2PEF) efficiency and 2PA cross section of tens of thousands of mutant FPs expressed in E. coli colonies. We use this procedure to move EGFP through three rounds of two-photon directed evolution leading to new variants showing up to a 50% enhancement in peak 2PA cross section and brightness within the near-IR tissue transparency wavelength range.
authors.affiliation: Physics Department, Montana State University, Bozeman MT. 59717.
authors.forename: Caleb R
authors.initials: CR
authors.lastname: Stoltzfus
authors.affiliation: Cell Biology Neuroscience Department, Montana State University, Bozeman MT. 59717.
authors.forename: Lauren M
authors.initials: LM
authors.lastname: Barnett
authors.affiliation: Physics Department, Montana State University, Bozeman MT. 59717.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Drobizhev
authors.affiliation: Physics Department, Montana State University, Bozeman MT. 59717.
authors.forename: Geoffrey
authors.initials: G
authors.lastname: Wicks
authors.affiliation: Physics Department, Montana State University, Bozeman MT. 59717.
authors.forename: Alexander
authors.initials: A
authors.lastname: Mikhaylov
authors.affiliation: Cell Biology Neuroscience Department, Montana State University, Bozeman MT. 59717.
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Hughes
authors.affiliation: 1] Physics Department, Montana State University, Bozeman MT. 59717 [2] National Institute of Chemical Physics and Biophysics, Tallinn, Estonia, 12618.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Rebane
mesh_terms: D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D017785:Photons; D019265:Spectroscopy, Near-Infrared
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins