Flickinger Daniel

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  • Article
    High throughput instrument to screen fluorescent proteins under two-photon excitation
    (Optical Society of America, 2020-12-01) Molina, Rosana S. ; King, Jonathan ; Franklin, Jacob ; Clack, Nathan ; McRaven, Christopher ; Goncharov, Vasily ; Flickinger, Daniel ; Svoboda, Karel ; Drobizhev, Mikhail ; Hughes, Thomas E.
    Two-photon microscopy together with fluorescent proteins and fluorescent protein-based biosensors are commonly used tools in neuroscience. To enhance their experimental scope, it is important to optimize fluorescent proteins for two-photon excitation. Directed evolution of fluorescent proteins under one-photon excitation is common, but many one-photon properties do not correlate with two-photon properties. A simple system for expressing fluorescent protein mutants is E. coli colonies on an agar plate. The small focal volume of two-photon excitation makes creating a high throughput screen in this system a challenge for a conventional point-scanning approach. We present an instrument and accompanying software that solves this challenge by selectively scanning each colony based on a colony map captured under one-photon excitation. This instrument, called the GIZMO, can measure the two-photon excited fluorescence of 10,000 E. coli colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.