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 (continuous-wave diode-pumped solid-state) laser with adjustable output power to a maximum of 300 mW and a near TEM00 top hat beam profile (Opto Engine LLC). The beam was expanded using a Keplerian beam expander composed of two plano-convex lenses (f = 25 and 75 mm; AR coating B, Thorlabs) 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), and emission: 900 longpass (FELH0900); Thorlabs] to a 60× Apo objective (numerical aperture, 1.0; working distance, 2.8 mm; water dipping; high nIR transmission; Nikon CFI Apo 60XW nIR). Emission photons collected from the sample were passed through the filter cube, were focused onto a two-dimensional InGaAs array detector [500 to 600 nm: 40% quantum efficiency (QE); 1000 to 1500 nm: >85% QE; Ninox 640, Raptor Photonics], and were recorded using the Micro-Manager Open Source Microscopy Software (53). Laser power was adjusted to maximize collected photons and to fill the pixel bit depth on the detector but did not exceed 70 mW at the objective back focal plane. Yellow fluorescent protein (YFP) was imaged by switching the filter cube (U-N41017XL, Olympus) and by using a mercury-vapor lamp (Olympus) for excitation.

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