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


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.

SUBMITTER: Molina RS 

PROVIDER: S-EPMC7747914 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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

Molina Rosana S RS   King Jonathan J   Franklin Jacob J   Clack Nathan N   McRaven Christopher C   Goncharov Vasily V   Flickinger Daniel D   Svoboda Karel K   Drobizhev Mikhail M   Hughes Thomas E TE  

Biomedical optics express 20201117 12


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 <i>E. coli</i> colonies on a  ...[more]

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