Metamorph software package

Epifluorescence still images were collected using a mercury lamp as an illumination source with appropriate sets of filters on a Zeiss Axiovert 200M (Plan Apochromat 100×/1.4 numerical aperture [NA] objective lens; Carl Zeiss, Gottingen, Germany) microscope equipped with CoolSnap camera (Photometrics, Tucson, AZ) and Uniblitz shutter driver (Photonics, Rochester, NY) under the control of a MetaMorph software package (Universal Imaging, West Chester, PA).
Scanning confocal microscopy was performed on a LSM510 microscope (Carl Zeiss) equipped with Achroplan 100×/1.25 NA objective lens, a 488-nm argon laser (for GFP excitation), and a 543-nm HeNe laser (for mCherry excitation). Time-lapse fluorescence microscopy images were generated on a Zeiss Axiovert 200M microscope equipped with UltraView RS-3 confocal system: CSU21 confocal optical scanner and 12-bit digital cooled Hamamatsu Orca-ER camera (OPELCO, Sterling, VA). The z-stack maximum projection images were obtained by MetaMorph built-in module. Imaging was performed on S. pombe cells placed in sealed growth chambers containing 2% agarose yeast extract supplement medium.

For live imaging, embryos were collected for 15 min to 1 hr, depending on the stage to be imaged, dechorionated with 4.2% sodium hypochlorite for 2 minutes, washed with dH₂O, mounted as a monolayer of embryos in a drop of halocarbon oil (Series 56) on the outer surface of the Greiner Lumox culture dishes (Sigma-Aldrich Cat# Z376744), and covered with a coverslip before imaging. All movies were captured using a 40x Planapochromat objective (NA 1.5) on a Nikon Eclipse TE2000-E inverted microscope with a Perkin-Elmer spinning disk and a Hamamatsu Orca-EM CCD camera. Movies were processed using the Metamorph software package (Universal Imaging, Downington, Pennsylvania).

For live imaging, embryos were collected for 15 min to 1 hour, depending on the stage to be imaged, dechorionated with 4.2% sodium hypochlorite for 2 min, washed with dH₂O, mounted as a monolayer of embryos in a drop of halocarbon oil (series 56) on the outer surface of the Greiner Lumox culture dishes (Sigma-Aldrich, catalog no. Z376744), and covered with a coverslip before imaging. All movies were captured using a 40× Planapochromat objective [numerical aperture (NA) 1.5] on a Nikon Eclipse TE2000-E inverted microscope with a Perkin-Elmer spinning disk and a Hamamatsu Orca-EM CCD camera. Movies were processed using the Metamorph software package (Universal Imaging, Downington, Pennsylvania).