Slicescope pro 6000

Two microscopes were used to visualize cell morphology. The in vitro microscope, a Scientifica SliceScope Pro 6000, used 980 nm illumination from a SpectraPhysics MaiTai Laser steered by a galvo scanner for 2P excitation and IR visualization. Software was SciScan version 1.5 by Scientifica in LabVIEW by National Instruments. Dyes for two-photon visualization were Alexa Fluor 488 and 568 Hydrazide from Invitrogen (A10436, A10437), the latter of which was found to be not gap junction permeable and was used for single-cell image isolation. Microscopy was continued on fixed retinas, which were stained with antibodies and fluorescent dyes for immunohistochemistry. The fixed-tissue microscope was a Nikon A1R confocal microscope with a 1.0 NA 40x oil immersion objective at the Northwestern Center for Advanced Microscopy. In Fig 1a–c and 7a, neurons were traced using Simple Neurite Tracer⁴⁷ in ImageJ/Fiji. Stratification analysis in Fig. 1c used ChAT layers to computationally flatten traced neural morphology. In Fig. 4 individual image channels from confocal microscopy were contrast adjusted and despeckled with a 3×3 median filter to improve clarity, then max-intensity projected through 7 μm of depth.