Csu x1 confocal scanner

Confocal fluorescence imaging was performed using NIS Elements imaging software on a Nikon confocal spinning disk system equipped with an Andor EMCCD camera, a CSU-X1 confocal scanner (Yokogawa), 405, 488, and 561 nm solid-state lasers, and 455/50, 525/26 and 605/70 nm emission filters. Transgenic animals expressing sfGFP₁₁ or split-wrmScarlet₁₁ were screened using a Leica M165 FC fluorescent stereomicroscope equipped with a Sola SE-V with GFP and mCherry filters.

Adult gravid hermaphrodite worms were dissected in a watch glass filled with Egg Salts medium (118 mM KCl, 3.4 mM MgCl₂, 3.4 mM CaCl₂, 5 mM HEPES, pH 7.4), and embryos were mounted on a fresh 2 % agarose pad and covered with an 18 mm x 18 mm coverslip (No. 1.5H, Marienfeld). Imaging was performed in a temperature-controlled room at 20°C using a Nikon Eclipse Ti microscope coupled to an Andor Revolution XD spinning disk confocal system, composed of an iXon Ultra 897 CCD camera (Andor Technology), a solid-state laser combiner (ALC-UVP 350i, Andor Technology), and a CSU-X1 confocal scanner (Yokogawa Electric Corporation), controlled by iQ3 software (Andor Technology). A 12 x 1 μm z-stack was acquired every 20 s using a 60x NA 1.4 or 100x NA 1.45 Plan-Apochromat objective.

Adult gravid hermaphrodite worms were dissected in a watch glass filled with Egg Salts medium (118 mM KCl, 3.4 mM MgCl₂, 3.4 mM CaCl₂ and 5 mM HEPES pH 7.4), and embryos were mounted on a fresh 2% agarose pad and covered with an 18 mm×18 mm coverslip (No. 1.5H, Marienfeld). Embryos co-expressing GFP::histone H2B and GFP::γ-tubulin for tracking of centrosomes and nuclei (Fig. 1F–H; Fig. 2C–G; Fig. 3G) were imaged on an Axio Observer microscope (Zeiss) equipped with an Orca Flash 4.0 camera (Hamamatsu), a Colibri.2 light source, and controlled by ZEN software (Zeiss). All other imaging was performed on a Nikon Eclipse Ti microscope coupled to an Andor Revolution XD spinning disk confocal system composed of an iXon Ultra 897 CCD camera (Andor Technology), a solid-state laser combiner (ALC-UVP 350i, Andor Technology), and a CSU-X1 confocal scanner (Yokogawa Electric Corporation), controlled by Andor IQ3 software (Andor Technology). All imaging was performed in temperature-controlled rooms kept at 20°C. Time-lapse sequences were processed and analyzed with Fiji software (Image J version 2.0.0-rc-56/1.51 h).

INS1 cells were cultured on gridded coverslips (MatTek P35G-1.5-14-C-GRID). Live-cell imaging was performed using Inverted Nikon Eclipse Ti microscope (Nikon Ti PFS Spinning Disk) equipped with x100 TIRF objective (NA = 1.49), with the PFS (perfect focus system), an EMCCD camera (Evolve, Photometrics) and Yokogawa CSU-X1 Confocal Scanner. GFP and m-Cherry were excited with a 488-nm and a 561-nm laser, respectively (100 mW). Live INS1 cells were imaged at 20 frames per second. 4D imaging was performed using consecutive xy(c)tz time-lapse acquisitions. Time series of 2D sections were analyzed using FIJI and IMARIS software. In the end of experiment, the cells were immediately fixed in PFA 2.5%, Glutaraldehyde 2.5% in sodium cacodylate 0.1 M for 20 min at room temperature. The samples were then post fixed in 1% osmium tetroxide in 0.1 M cacodylate buffer for 1 h at 4 °C, dehydrated through graded alcohol (50, 70, 90, and 100%), embedded in Epon 812 cut at 70 nm (Leica Ultracut UCT) and contrasted with uranyl acetate and lead citrate. Serial sections were examined using a Philips CM12 transmission electron microscope (CM12, Philips; FEI Electron Optics, Eindhoven, the Netherlands) operated at 80 kV and equipped with an Orius 1000 CCD camera (Gatan, Pleasanton, USA).