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1 Publications

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    Svoboda Lab
    12/01/20 | High throughput instrument to screen fluorescent proteins under two-photon excitation.
    Molina RS, King J, Franklin J, Clack N, McRaven C, Goncharov V, Flickinger D, Svoboda K, Drobizhev M, Hughes TE
    Biomedical Optics Express. 2020 Dec 01;11(12):7192-7203. doi: 10.1364/BOE.409353

    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 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 colonies in 7 hours. We show that the GIZMO can be used to evolve a fluorescent protein under two-photon excitation.

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