Ti u inverted microscope

For live imaging of L3 and young adult hermaphrodites, animals were sedated with 10 mM Levamisole M9 and mounted on 5% agarose. To perform imaging on embryos, gravid adults were dissected in M9 and mounted on 5% agarose. Two main microscopes were used. First, a wide-field Zeis Axioplan2 upright microscope equipped with 25x – 0.8 NA, 63x – 1.4 NA, and 100x – 1.4 NA objectives, and an Axiocam MRm CCD monochrome camera. Zeiss filter sets used were set 34 for DAPI, set 13 for GFP, and set 31 for mCherry. The microscope and camera were controlled by Zeiss Axiovision 4.x software. Second, an Andor spinning disc platform controlled by MetaMorph software and consisting of a Nikon Ti-U inverted microscope with 60x – 1.4 NA and 100x – 1.4 NA oil objectives, a Yokogawa CSU-X1 spinning disk unit, 488 nm and 561 nm lasers, Semrock 512 – 23 + 630 – 91, 525 – 30, and 617 – 73 emission filters, and an Andor iXON DU-885 camera. Images in Figure 6 were captured using a Zeiss AxioImager, 40x – 1.3 NA objective, and Hammamatsu Orca-R2 camera. Images of fixed embryos were deconvolved using AxioVision software, and are shown as maximum intensity projections of 3–5 adjacent planes spaced 0.3 μm apart. Images were cropped, rotated, and levels were adjusted in ImageJ and Adobe Photoshop.