Unless otherwise indicated, all images were generated using an AxioCam HRc mounted on a Zeiss Axioplan 2 imaging microscope with the software AxioVision version 4.8; with an Axio Imager.Z1 equipped with an AxioCamMR3; or with a confocal microscope OLYMPUS BX61Wi with the software OLYMPUS FLUOVIEW version 3.1. Microscopic images were processed using Adobe Photoshop and Illustrator software.
For live-cell imaging of semi-in vivo fertilization, the following microscopy settings were used as described previously (Hamamura et al., 2014 (link); Gooh et al., 2015 (link)). Confocal images were acquired using an inverted microscope IX-83 (Olympus) equipped with a disk-scan confocal system (CSU-W1; Yokogawa Electric). A silicone oil immersion objective lens, UPLSAPO60XS (Olympus), mounted on a Piezo z-drive (P-721; Physik Instrumente) was used. Time-lapse and z-stack images were acquired every 5-10 min in seven planes (3 μm intervals). The exposure time of 488 nm laser was 250-300 ms for eGFP, and of 561 nm laser was 50-200 ms for mRFP and tdTomato. Images were processed by Metamorph version 7.8.4.0 (Universal Imaging) to display maximum-intensity projection images and to add pseudo-colors. The images and movies were edited by MacBiophotonics ImageJ software (http://www.macbiophotonics.ca/ ).
For live-cell imaging of semi-in vivo fertilization, the following microscopy settings were used as described previously (Hamamura et al., 2014 (link); Gooh et al., 2015 (link)). Confocal images were acquired using an inverted microscope IX-83 (Olympus) equipped with a disk-scan confocal system (CSU-W1; Yokogawa Electric). A silicone oil immersion objective lens, UPLSAPO60XS (Olympus), mounted on a Piezo z-drive (P-721; Physik Instrumente) was used. Time-lapse and z-stack images were acquired every 5-10 min in seven planes (3 μm intervals). The exposure time of 488 nm laser was 250-300 ms for eGFP, and of 561 nm laser was 50-200 ms for mRFP and tdTomato. Images were processed by Metamorph version 7.8.4.0 (Universal Imaging) to display maximum-intensity projection images and to add pseudo-colors. The images and movies were edited by MacBiophotonics ImageJ software (
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