Imaging Striatal Dopamine Release Using a Non-Genetically Encoded Near-Infrared Fluorescent Catecholamine Nanosensor

Abraham G. Beyene; Kristen Delevich; Jackson Travis Del Bonis-O’Donnell; David J. Piekarski; Wan Chen Lin; A. Wren Thomas; Sarah J. Yang; Polina Kosillo; Darwin Yang; Linda Wilbrecht; Markita P. Landry

Ex vivo slice imaging was performed with a modified upright epifluorescent microscope (Olympus, Sutter Instruments) mounted onto a motorized stage. Nanosensor excitation was supplied by a 785 nm, CW, DPSS laser with adjustable output power to a maximum of 300 mW and a near TEM00, top hat beam profile (OptoEngine LLC). The beam was expanded using a Keplerian beam expander comprised of two plano-convex lenses (Thorlabs, f=25 mm, f=75 mm, AR coating B) to a final beam diameter of approximately 1 cm. The beam was passed through a custom fluorescence filter cube (excitation: 800 nm shortpass (FESH0800), dichroic: 900 longpass (DMLP990R), emission: 900 longpass (FELH0900), Thorlabs) to a 60X Apo objective (1.0 NA, 2.8 mm WD, water dipping, high NIR transmission, Nikon CFI Apo 60XW NIR). Emission photons collected from the sample were passed through the filter cube and were focused onto a two-dimensional InGaAs array detector (500-600 nm: 40% quantum efficiency (QE); 1000-1500 nm: >85% QE; Ninox 640, Raptor Photonics) and recorded using Micro-Manager Open Source Microscopy Software. 46 Laser power was adjusted to maximize collected photons and fill the pixel bit depth on the detector but did not exceed 70 mW at the objective back focal plane. YFP fluorescence was imaged by switching the filter cube (FESH 0899 for excitation, FELH 0900 for emission, Thorlabs) and using a mercury-vapor lamp (Olympus) for excitation.