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bioRxiv · 10.1101/2020.09.04.283572

High throughput instrument to screen fluorescent proteins under two-photon excitation

Molina, R. S.·King, J.·Franklin, J.·Clack, N.·McRaven, C.·Goncharov, V.·Flickinger, D.·Svoboda, K.·Drobizhev, M.·Hughes, T. E.
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Abstract

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.

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Molina, R. S., King, J., Franklin, J., Clack, N., McRaven, C., Goncharov, V., Flickinger, D., Svoboda, K., Drobizhev, M., Hughes, T. E.. 2020-09-04. High throughput instrument to screen fluorescent proteins under two-photon excitation. https://doi.org/10.1101/2020.09.04.283572

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