Imaging Embryo Development Dynamics

Embryos were prepared as described previously (Kong et al., 2019 (link)). Briefly, the staged embryos were collected and dechorionated with 50% hypochlorite bleach for 90 s, aligned on an agar block, and attached on the coverslips by homemade glue covered with halocarbon oil. Cross-sectional images were recorded from the dorsal side for E-CadGFP (488 nm excitation) on a spinning disc microscope with a 40x or ×100 oil objective (Carl Zeiss, ×40/oil, NA1.2, 100x/oil, NA1.4) with an emCCD camera (Carl Zeiss, AxioCam MRm). The apical plane of the embryo was acquired with axial sections of each 0.5 µm and a frame rate of 0.2/min or 1/min. Images in Figure 6A were acquired on a laser scanning confocal microscopy (Carl Zeiss, ZEISS LSM 980 with Airyscan 2) with a ×63 oil objective (Carl Zeiss, ×63/oil, NA1.4), and the apical plane of the embryo was acquired with axial sections of each 0.5 µm. Images in Figure 7A were acquired on a laser scanning confocal microscopy (Carl Zeiss, ZEISS LSM 980 with Airyscan 2) with a ×63 oil objective (Carl Zeiss, ×63/oil, NA1.4), and the apical plane of the embryo was acquired with axial sections of each 1 µm and a frame rate of 0.2/min.