Ixon ultra 897 camera

Embryos at stage 14–15 of development (11–12 h after egg laying) were dechorionated in 50% bleach for 2 min and rinsed with water. Embryos were aligned on an apple juice–agar pad, glued ventrolateral-side-down onto a coverslip using heptane glue, and covered with a 1:1 mix of halocarbon oil 27 and halocarbon oil 700 (Sigma-Aldrich). A Revolution XD spinning disk confocal (Andor) with a 60× oil-immersion lens (NA 1.35; Olympus) was used to image embryos. Images were acquired using an iXon Ultra 897 camera (Andor) and Metamorph (Molecular Devices) as the image acquisition software. Sixteen-bit Z-stacks were acquired at 0.5- to 0.75-μm steps every 3–30 s (7–11 slices per stack). Analysis used maximum intensity projections. The same linear contrast adjustment was applied to all the images in each experiment.

Ablations were done using a pulsed micropoint N₂ laser (Andor Technology) tuned to 365 nm in endo-DEcad::GFP embryos (Yu and Fernandez-Gonzalez, 2016 (link)). Embryos were mounted as described in the previous section for live imaging and then imaged using a Revolution XD spinning-disk confocal microscope equipped with an iXon Ultra 897 camera (Andor Technology) and a 1.5× coupling lens. All embryos were imaged with a 60×/1.35 NA oil immersion lens (Olympus) at 20–22°C. Images were acquired using Microscopy Automation and Image Analysis software (MetaMorph). For experiments involving ablation at the apical domain, five consecutive laser pulses of 60 µJ with a duration of 67 ms each were delivered to a single spot at the apical domain of late ingressing NBs or NICs. For ablation of cell boundaries around NBs, one pulse of 42 µJ and 67-ms duration was applied simultaneously at each junction. Two z stacks of 5–20 planes (0.35 µm each) were acquired before ablation, and after ablation every 1–3 s. In sham-irradiated controls, junctions were targeted with the laser completely attenuated, using the same number of pulses, to mimic the ablations performed in the corresponding experiments.