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A continuous wave diode laser module operating at 488 nm (Cobolt, MLD 488) was used as the excitation source. The laser intensity was tuned with a variable neutral density filter (Thorlabs, RSP1D). A circularly polarized excitation light was obtained using a Berek compensator (Newport, Model 5540). Samples were excited through an oil immersion objective (Olympus, TIRF UApo N, × 100, NA 1.49) by focusing the excitation light at the back aperture of the objective lens. Resulting fluorescence was separated from the excitation laser by a dichroic beam splitter (Semrock, FF506-Di03) followed by an emission filter (Semrock, FF01-609/181) and collected by a high-speed EMCCD camera (iXon Ultra 897, Andor Technology).

For screening cells morphology and checking cell localization in acute brain slices we used a widefield infrared illumination system. This consisted of an IR-LED source (M780L2, Thorlabs) installed at the rear port of a SliceScope Scientifica microscope, an orientable blocking element to create oblique illumination and a condenser focusing the light on the sample. IR light transmitted through the sample was collected with an IR antireflection coated water-immersion objective (Nikon NIR MRD07420 N40X/0.80W) and sent to an IR CCD (IR-1000, DAGE-MIT).
For a first control of ReaChR expression, we performed widefield fluorescence imaging with a system comprising 2 interchangeable LED sources (Thorlabs M470L2, for YFP and M565L3 for dTomato) filtered by 2 interchangeable bandwidth excitation filters (Semrock FF01-452/45 for YFP and F01-545/55-25 for dTomato) and coupled to a diffuser (DG10-1500, Thorlabs) and an achromatic lens (f = 30 mm, #LA1805 Thorlabs). Fluorescence was collected through a tube lens (f = 200 mm), separated from excitation light using a dichroic mirror (Semrock FF510-Di02 for YFP and FF580-FDi01 for dTomato) and detected by a CCD camera (Orca-05G, Hamamatsu) after passing through a visible bandwidth filter (Semrock FF01-609/181 for YFP and FF01-665/150-25 for dTomato).