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.
Highlights
The 1990s saw both the cloning of the green fluorescent protein [1] and the first application of two-photon microscopy to neuroscience [2]
The instrument should be set up to screen Fluorescent proteins (FPs)-expressing E. coli colonies. This makes it simple to recover the plasmid encoding a particular FP, and creating a larger library is as straightforward as transforming more E. coli
We have designed and implemented an instrument that can measure the two-photon excited fluorescence of 10,000 E. coli colonies expressing a library of FP mutants in 7 hours
Summary
The 1990s saw both the cloning of the green fluorescent protein [1] and the first application of two-photon microscopy to neuroscience [2]. Fluorescent proteins (FPs) were promising as genetically encoded probes, and two-photon microscopy made it possible to image fluorescence deep in living tissue, featuring confined excitation and decreased scattering due to the nearinfrared excitation light Today, their combination is ubiquitous in neuroscience. FPs have been engineered to sense Ca2+ and other indicators of brain activity [3], and two-photon microscopes have been engineered for faster imaging rates and larger fields of view to image these deep in the brains of behaving mice [4,5,6,7,8] Their combination can make an even greater impact if FPs are optimized for two-photon excitation. While there have been extensive efforts to improve FP-based probes, these efforts have been almost exclusively under one-photon excitation
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