Large Animal 2P Microscope Design

The 2P microscope for imaging swine was designed with four key specifications: 1) ability to accommodate large animals with considerable flexibility, 2) intensity and fluorescence lifetime imaging without switching detection hardware and scan path, 3) externally triggered high speed volumetric scanning, and 4) low-cost construction from off-the-shelf parts. The overall design of the microscope was based heavily on the previously published open source TIMAHC (Two-photon Imaging that is Modular, Adaptable, High-performance and Cost-effective) design^{14 (link)} with exceptions detailed below. All 2P imaging was performed using excitation light from a MaiTai HP laser (Spectra Physics, Milpitas, CA) 16× , 0.8 N.A. objective (Nikon Instruments, Tokyo, Japan). Emission light was filtered through 440/80 (cyan fluorescent protein and the chloride-sensitive organic dye), 525/50 (YFP and GFP), or 605/70 (TurboRFP and tdTomato, Chroma Technology, Bellows Falls, VT).