Ff458dio2 dichroic

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.

Five to ten young adult transgenic animals (<24 hr old) expressing the YC2.60 (URX) or the YC3.60 (AQR and PQR) Ca²⁺ sensors were glued to agarose pads (2% in M9 buffer, 1 mM CaCl2) using Dermabond tissue adhesive, with their body immersed in OP50 washed off from a seeded plate using M9. The animals were quickly covered with a PDMS microfluidic chamber and 7% O₂ pumped into the chamber for 2 min before imaging, to allow animals to adjust to the new conditions. Neural activity was recorded for 6 min with switches in O₂ concentration every 2 min. Imaging was on an AZ100 microscope (Nikon) equipped with a TwinCam adaptor (Cairn Research), two ORCAFlash4.0 V2 digital cameras (Hamamatsu), and an AZ Plan Fluor 2x objective with 2x zoom. Recordings were at 2 frame-per-second (fps) with a 500ms exposure time. Excitation light from a C-HGFI Intensilight lamp (Nikon) was passed through a 438/24 nm filter and an FF458-DiO₂ dichroic (Semrock). Emitted light was passed to a DC/T510LPXRXTUf2 dichroic filter in the TwinCam adaptor cube and then through 483/32 nm (CFP) or 542/27 nm (YFP) filters before collection on the cameras.

To image young adults, we picked L4 animals expressing the YC2.60 Ca²⁺ sensor 24 hr before imaging. On the day of the assay, 5 – 10 day 1 adult animals were glued to agarose pads (2% in M9 buffer, 1 mM CaCl₂), using Dermabond tissue adhesive, with their body immersed in OP50 washed off from a seeded plate using M9 buffer. The animals were quickly covered with a PDMS microfluidic chamber and 7% O₂ was pumped into the chamber for 2 min before we began imaging, to allow animals to adjust to the new conditions. Neural activity was recorded for 9 min with switches in O₂ concentration every 2 min.
Imaging was performed on an AZ100 microscope (Nikon) bearing a TwinCam adaptor (Cairn Research, UK), two ORCAFlash4.0 V2 digital cameras (Hamamatsu, Japan), and an AZ Plan Fluor 2× lens with 2× zoom. Recordings were at 2Hz. Excitation light from a Lambda LS xenon arc lamp (Sutter) was passed through a 438/24 nm filter and an FF458DiO2 dichroic (Semrock). Emitted light was passed to a DC/T510LPXRXTUf2 dichroic filter in the TwinCam adaptor cube and then filtered using a 483/32 nm filter (CFP), or 542/27 nm filter (YFP) before collection on the cameras. Recordings were analyzed using Neuron Analyzer, a customwritten Matlab program available at https://github.com/neuronanalyser/neuronanalyser.