Evolve 512 emccd camera

For bright field (BF) and fluorescence microscopy 2 µl of an exponentially growing culture sample were placed on a microscope slide coated with a thin agarose layer (1%) made using the growth medium. The slide was incubated at 30°C during the images acquisition. The images were acquired with an Evolve 512 EMCCD camera attached to an Axio Observe spinning disk from Zeiss and recorded every 30 seconds with step size of 0.4 µm in the Z-axis (3 images were acquired for each channel). The BF image 3 is subtracted from the BF image 1 to obtain the phase image.

For all live imaging shown, embryos at the two-to-four-cell stage were dissected from adult hermaphrodites and mounted onto 3% agarose pads, covered with number 1.5 cover slip, and sealed with Vaseline. Embryos were then incubated at 23°C for 240 min. Imaging was done in a temperature-controlled room set to 23°C on a Laser Spinning Disk Confocal Microscope with a Yokogawa scan head, on a Zeiss AxioImager Z1m Microscope using the Plan-Apo 63X/1.4NA or Plan-Apo 40X/1.3NA oil lenses. Images were captured on a Photometrics Evolve 512 EMCCD Camera using MetaMorph software, and analyzed using ImageJ. Some images (Fig. 1) were done on a Zeiss Axioskop 2 microscope, using Plan-Apo 40X/1.3NA oil lens, and captured with a Roper camera. Controls and mutants were imaged within 3 days of each other with the same imaging conditions. All measurements were performed on raw data. For fluorescent measurements, background intensity was subtracted by using a box or line of the same size and measuring average intensity in the same focal plane, near the embryo. Actin intensity measurements, protrusion and retraction analysis was performed as described previously (Wallace et al., 2018 (link)) on embryos imaged at 2-min intervals for at least 120 min beginning at 240 min after the two-to-four-cell stage.