Axio imager

Fluorescence confocal microscopy was performed using a Zeiss Axio Imager with a Yokogawa spinning-disc confocal scanner. Embryo images were taken using Slidebook v6.0 software (Intelligent Imaging Innovations) using a 63x objective. Embryos were staged by DAPI-stained nuclei in optical Z-sections and multiple Z-sections were taken to include germ cells. For In vitro condensation reactions, images are single planes taken using a 40x objective unless otherwise indicated. For fluorescence super-resolution microscopy, images were acquired using ZEISS LSM 880-AiryScan (Carl Zeiss) equipped with a 63X objective. Images were processed using ZEN imaging software (Carl Zeiss). Equally normalized images were exported via either Slidebook v6.0 or ZEN, and contrasts of images were equally adjusted between control and experimental sets. For in vitro fluorescence recovery after photobleaching experiments, images were acquired using Zeiss LSM 800 GaAsp. Images are single confocal planes imaged using a 63x objective every 3 s during a recovery phase of 300 s. All image analyses were conducted using the Fiji image-processing package (http://fiji.sc/Fiji).

Fluorescence microscopy was performed using a Zeiss Axio Imager with a Yokogawa spinning-disc confocal scanner. Images were taken using Slidebook v6.0 software (Intelligent Imaging Innovations) using either a 63× or 40× objective. Equally normalized images were exported by Slidebook v6.0, and contrasts of images were equally adjusted between control and experimental sets using ImageJ. For live imaging, embryos were dissected from adult hermaphrodites in M9 solution, mounted onto 2% agarose pads and imaged at 20°C. We noticed that par-1(T983A) embryos were sensitive to compression under the glass coverslip, which gave variable results. To eliminate this variability, we only examined embryos that were compressed by the coverslip during or after the pronuclear migration stage (cell fate determinants within these zygotes are polarized while inside the hermaphrodite uterus).

Fluorescence microscopy was performed using a Zeiss Axio Imager with a Yokogawa spinning-disc confocal scanner. Images were taken and stored using Slidebook v6.0 software (Intelligent Imaging Innovations) using a 40x or 63x objective. Embryos were staged by DAPI-stained nuclei in optical Z-sections and multiple Z-sections were taken to include germ cells marked by α-PGL-1 (K76) staining. For images of embryonic PGCs, a single Z-section was extracted at a plane with the widest area of DAPI staining for nuclear signal of LIN-15B, MES-3, and MES-4. For MES-2-GFP, the Z-section was determined based on widest area of GFP signal. Equally normalized images were first taken by Slidebook v6.0, and contrasts of images were equally adjusted between control and experimental sets using Image J.

Images were acquired using a Zeiss Axio Imager fitted with a Yokogawa spinning disc confocal scanner with Slidebook software (Intelligent Imaging Innovations, Denver, CO) using a 63× objective (embryos) or 40× objective (in utero movies). Embryos were dissected from gravid mothers and mounted on 3% agarose pads in M9 solution at room temperature.
For single time point images, 10 z planes with a z step size of 1 μm, spanning 9 μm, were acquired. Exposure time was 100 ms per plane per color with one exposure acquired in each color before proceeding to the next plane. Granule counts were performed with Slidebook using Mask Segmentation tools and manually verified for each embryo.
For time-lapse movies of embryos, z-stacks (8 z planes, step size 1 μm) were acquired every 8 s. Exposure time was 100 ms per plane per color with one exposure acquired in each color before proceeding to the next plane.
For in utero movies, young adult mothers were anesthetized in 0.3 mM levamisole for 15 min prior to mounting on 3% agarose pads. Z-stacks (11 z planes with a z step size of 1 μm) were acquired every 30 s. Exposure time was 100 ms per plane per color with one exposure acquired in each color before proceeding to the next plane.

Fluorescence microscopy was performed using a Zeiss Axio Imager with a Yokogawa spinning-disc confocal scanner. Images were taken and stored using Slidebook v 6.0 software (Intelligent Imaging Innovations) using a 63x objective. For live imaging, embryos were dissected from adult hermaphrodites in M9 salt solution and mounted onto 3% agarose pads. All embryo images are z stack maximum projections using a z step size of 1 μm, spanning the entire width of the embryo.