CO2-immobilization of C. elegans embryos aids in imaging events that occur after twitching begins.

To construct a durable and reusable chamber for imaging, this chamber was designed using AUTODESK Tinkercad (https://www.tinkercad.com/). The chamber comprises two parts: a bottom part with a square chamber that transforms into a bottom coverslip chamber connected to an exit and entry tubing, surrounded by a moat to house an O-ring; and a top part that seals the chamber’s O-ring. The device was printed on a DLP 3D printer Photon Ultra (Anycubic, Inc.) using a UV curing SLA resin. The printing process involved 3 seconds of on and 1 second of off exposure with a z-step of 50 µm and an XY pixel size of 80 µm. After printing, the two parts were cleaned with 100% isopropanol, and a final cure was performed using a UV blue-light LED for 15 minutes. The device was then dried in an oven for 30 minutes. The top and bottom imaging windows were added and sealed using UV clear resin and 22x40mm #1.5 coverslips. A nitrile rubber O-ring (32x28x2 mm) was added to the chamber’s moat to create a seal. Both the top and bottom parts were lined up with N52 5x2 mm round magnets. For immobilization, CO₂ was allowed to flow through an ozone water humidifier and into the chamber via tubing for 5-10 minutes at a rate of 60-80 cc/min until immobilization. The chamber was then sealed by pinching the tubing at the entry and exit valve. After the imaging was complete, ambient air was inserted into the chamber using an aquarium air pump through a flow derivation system.