High throughput instrument to screen fluorescent proteins under two-photon excitation
| Autor: | Jacob Franklin, Mikhail Drobizhev, Daniel Flickinger, Nathan G. Clack, Jonathan P. King, Karel Svoboda, Vasily Goncharov, Thomas E. Hughes, Rosana S. Molina, Christopher McRaven |
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| Rok vydání: | 2020 |
| Předmět: |
0303 health sciences
Materials science Directed evolution 01 natural sciences Fluorescence Atomic and Molecular Physics and Optics Article 010309 optics 03 medical and health sciences Two-photon excitation microscopy Excited state 0103 physical sciences Microscopy Biophysics Throughput (business) Biosensor Excitation 030304 developmental biology Biotechnology |
| Zdroj: | Biomed Opt Express |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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