S159 500

To create the imaging chambers, 150 mm petri dishes were plasma treated (Harrick Plasma, PDC-001) for 2 min at high power to create a hydrophilic surface. Ten circular templates with 20 mm diameter were traced in each dish with a permanent marker to pattern a hydrophobic mask. Each region was first loaded with a single planarian in 800 μl of Instant Ocean water to ensure complete wetting and then reduced to ~350 μl. This flattened the top surface of the droplet and reduced reflections during imaging. After loading, the dish was gently filled with ~50 mL of silicone oil (Fisher Scientific S159-500) until the top of all droplets were covered. The oil phase both reduced evaporation and increased surface tension at the droplet boundary to prevent planarians from escaping. Planarians remained viable and active for at least ~7 d at 20 °C under these conditions. All animals were starved at least 4 d prior to imaging and all imaging sessions lasted no longer than 7 d. For imaging regeneration time courses, animals were loaded at 8 hpa and rinsed before loading to reduce the accumulation of material ejected from the wound site within the droplet. To cover the complete span of regeneration, a second cohort of animals was imaged starting 3 dpa for ~7 days. Activity responses at matching regeneration time were merged from both datasets and used for all analyses.