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

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.

Monkey-MIMMS: Towards Automated Cellular Resolution Large- Scale Two-Photon Microscopy In The Awake Macaque Monkey.

The size and curvature of the macaque brain present challenges for two photon laser scanning microscopy (2P-LSM). General access to the cortex requires 5-axis positioning over a range of motion wider than existing designs offer. In addition, movement artifacts due to physiological pulsations and bodily movement present particular challenges. We present a microscope and implant platform that allows for repeatable, motorized positioning and stable imaging at any point on the dorsal convexity of macaque cortex. While testing the system to image neurons expressing fluorescent proteins in an awake macaque, motion artifacts were limited to several microns.

Supercontinuum Fourier transform spectrometry with balanced detection on a single photodiode.

We demonstrate a method of combining a supercontinuum light source with a commercial Fourier transform spectrometer, using a novel approach to dual-beam balanced detection, implemented with phase-sensitive detection on a single light detector. A 40 dB reduction in the relative intensity noise is achieved for broadband light, analogous to conventional balanced detection methods using two matched photodetectors. Unlike conventional balanced detection, however, this method exploits the time structure of the broadband source to interleave signal and reference pulse trains in the time domain, recording the broadband differential signal at the fundamental pulse repetition frequency of the supercontinuum. The method is capable of real-time correction for instability in the supercontinuum spectral structure over a broad range of wavelengths and is compatible with commercially designed spectrometers. A proof-of-principle experimental setup is demonstrated for weak absorption in the 1500-1600 nm region.