V3 confocal spinning disk head 89 north

All images were acquired at room temperature on a Leica DMI8 with an xLIGHT V3 confocal spinning disk head (89 North) with a 63× Plan-Apochromat (1.4 NA) objective and an ORCAFusion GenIII sCMOS camera (Hamamatsu Photonics) controlled by microManager¹⁰¹ . RFPs were excited with a 555 nm laser, GFPs were excited with a 488 nm laser, and CFP was excited by a 445 nm laser. Z-stacks through the gonad were acquired with a step-size of 1µm unless otherwise noted. Worms were mounted on agar pads with 0.01 M sodium azide.

All images were acquired on a Leica DMI8 with an xLIGHT V3 confocal spinning disk head (89 North) with a ×63 Plan-Apochromat (1.4 NA) objective and an ORCAFusion GenIII sCMOS camera (Hamamatsu Photonics) controlled by microManager (Edelstein et al., 2010 (link)). RFPs were excited with a 555 nm laser, GFPs were excited with a 488 nm laser, and DAPI was excited with a 405 nm laser. Worms were mounted on agar pads with 0.01 M sodium azide (live) or in 75% glycerol (DAPI stained).

DAPI staining was done by modifying standard protocols (Francis and Nayack, 2000), with the cold methanol fixation done for a shorter time (2.5 min) and the concentration of DAPI higher at 1 μg/ml in 0.01% Tween in PBS in the dark for 5 min, washed once with 0.1% Tween in PBS. Samples were briefly stored at 4°C in 75% glycerol and imaged directly in glycerol solution on a Leica DMI8 with an xLIGHT V3 confocal spinning disk head (89 North) with a ×63 Plan-Apochromat (1.4 NA) objective and an ORCAFusion GenIII sCMOS camera (Hamamatsu Photonics) controlled by microManager (Edelstein et al., 2010). DAPI was excited with a 405 nm laser. Worms were mounted on agar pads in 75% glycerol.