Embryos were imaged using a Zeiss LSM 900 confocal microscope. Plan-Apochromat 40x/1.4 N.A. oil immersion objective was used. Images were acquired with the following settings: 512 × 512 pixels, 16-bit depth, 18 z-slices separated by 0.6 μm, ~16.8 s/frame time-resolution. The fluorescence of GFP, mCherry, and eBFP2 was excited using 488-, 561-, and 405-nm lasers, respectively. Excitation power was measured and calibrated using X-Cite XR2100/XP750 Optical Power Measurement System (EXCELITAS Technologies) to keep the same experimental setting for each set of experiments. Image acquisition was started before the end of nc13 and ended after the onset of gastrulation at nc14. During imaging, data acquisition was occasionally stopped for a few seconds to correct the z-position. Obtained data were concatenated and cropped into 430 × 512 pixels (sna shadow enhancer), 430 × 430 pixels (Ubx BRE), 300 × 512 pixels (rho NEE), or 300 × 350 pixels (hairy enhancer) to remove nuclei outside of the expression domain. One hundred eighty timeframes starting from the entry into nc14 as defined by the progression of prior anaphase were used for subsequent image analysis. The temperature was kept between 22.0 to 23.0 °C during imaging.
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