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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.

SUBMITTER: Stoltzfus CR

PROVIDER: S-EPMC4491718 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Two-photon directed evolution of green fluorescent proteins.

Stoltzfus Caleb R CR Barnett Lauren M LM Drobizhev Mikhail M Wicks Geoffrey G Mikhaylov Alexander A Hughes Thomas E TE Rebane Aleksander A

Scientific reports 20150706

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 thousan ...[more]

PMID: 26145791

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