Csu x1 spinning disk confocal system

Worms of desired stage/age were anesthetized in 0.5 mg/mL tetramisole (Sigma, T1512) diluted in M9 and mounted to 2% agarose pads. For imaging of the mitochondrial TOMM-20 reporter in the muscle, images were taken using a Zeiss Imager.M2 microscope. Apotome optical sectioning was used to acquire fluorescence and one picture with best focus was chosen for each worm for quantification (as described in Weir et al., 2017 (link)). For imaging of mitochondria in neurons and in intestine, images were taken in the Sabri Ulker imaging lab using a Yokogawa CSU-X1 spinning disk confocal system (Andor Technology, South Windsor, CT, USA) combined with a Nikon Ti-E inverted microscope (Nikon Instruments, Melville, NY, USA). Images were taken using a 100x/1.45 oil Plan Apo objective lens, Zyla cMOS (Zyla 4.2 Plus USB3) camera and 488 nm Laser for GFP. Optical slice thickness was 0.2 µm. NIS elements software was used for acquisition parameters, shutters, filter positions and focus control. For images shown of the intestine, images were taken as a z stack and each plane was then threaded together by concatenation of the stack. These concatenated stacks were then rendered into 3d by the 3d viewer function of FIJI.

RAGA-1::AID^Somatic animals were grown on NGM Carb plates until day 1 of adulthood (3 days after hatch). On day 1, some animals were taken to be imaged before auxin treatment. Of the remaining animals, half of the animals were transferred to control plates and half were transferred to 0.15 mM auxin plates. Animals were then imaged at 2.5 hours or 6 hours to monitor RAGA-1::AID::EmGFP degradation.
For imaging, worms were anesthetized in 0.5 mg/ml tetramisole in 1X M9 buffer on empty NGM plates and mounted on thin 2% agarose pads on glass slides with 0.05 mm Polybead microspheres (Polysciences) for immobilization. A No. 1.5 cover glass was gently placed on top of worms and sealed with clear nail polish. Images were performed on a Yokogawa CSU-X1 spinning disk confocal system (Andor Technology, SouthWindsor, CT) with a Nikon Ti-E inverted microscope (Nikon Instruments,Melville, NY), using a Plan-Apochromat 100x/1.45 objective lens. Images were acquired using a Zyla cMOS camera and NIS elements software was used for acquisition parameters, shutters, filter positions and focus control.