Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.04.283572v1?rss=1 Authors: Molina, R. S., King, J., Franklin, J., Clack, N., McRaven, C., Goncharov, V., Flickinger, D., Svoboda, K., Drobizhev, M., Hughes, T. E. 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. Copy rights belong to original authors. Visit the link for more info