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.

中文翻译：

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高通量仪器可在双光子激发下筛选荧光蛋白

双光子显微镜以及荧光蛋白和基于荧光蛋白的生物传感器是神经科学中常用的工具。为了扩大其实验范围，重要的是优化用于双光子激发的荧光蛋白。在单光子激发下荧光蛋白的定向进化是很普遍的，但是许多单光子特性与双光子特性并不相关。表达荧光蛋白突变体的简单系统是大肠杆菌琼脂平板上的菌落。双光子激发的小焦点体积使得在该系统中创建高通量屏幕成为传统点扫描方法的挑战。我们提出一种仪器和随附的软件，通过基于在单光子激发下捕获的菌落图选择性地扫描每个菌落来解决此难题。该仪器称为GIZMO，可在7小时内测量10,000个大肠杆菌菌落的双光子激发荧光。我们表明，GIZMO可用于在双光子激发下演化荧光蛋白。