2 az plan fluor objective

Animals expressing cameleon YC2.60 were imaged with a ×2 AZ-Plan Fluor objective (Nikon) on a Nikon AZ100 microscope fitted with ORCA-Flash4.0 digital cameras (Hamamatsu). Excitation light was provided from an Intensilight C-HGFI (Nikon), through a 438/24 nm filter and an FF458DiO2 dichroic (Semrock). Emission light was split using a TwinCam dual camera adapter (Cairn Research) and passed through CFP (483/32 nm) and YFP (542/27) filters, and a DC/T510LPXRXTUf2 dichroic. We acquired movies using NIS-Elements (Nikon), with 100 ms exposure time.
To image neural activity in freely moving animals (Supplementary Fig. 4g), single young adults were transferred to peptone-free agar plates spotted with 4 µl of concentrated OP50 food in M9 buffer, and imaged at 2× zoom. For all other figures, 4–8 young adults were transferred to peptone-free agar plates, immobilized on a 2 µl patch of concentrated OP50 in M9 buffer using Dermabond adhesive, leaving the nose exposed, and imaged at 4× zoom.

Neural imaging was performed as previously described (Flynn et al., 2020 (link)), with a 2× AZ-Plan Fluor objective (Nikon) on a Nikon AZ100 microscope fitted with ORCA-Flash4.0 digital cameras (Hamamatsu). Excitation light was provided from an Intensilight C-HGFI (Nikon), through a 438/24 nm filter and an FF458DiO2 dichroic (Semrock). Emission light was split using a TwinCam dual-camera adapter (Cairn Research) bearing a filter cube containing a DC/T510LPXRXTUf2 dichroic and CFP (483/32 nm) and YFP (542/27) filters. We acquired movies using NIS-Elements (Nikon), with 100 ms or 500 ms exposure time. YFP/CFP ratios in URX were reported by YC2.60 driven from the gcy-37 promoter, in BAG by YC3.60 and TN-XL driven from the flp-17 promoter, in AFD by YC3.60 driven from the gcy-8 promoter.