Ultraview spinning disc confocal microscope

L4 worms were anesthetized in 3 mM tetramisole (Sigma- Aldrich) and mounted on 5% agarose pads. Time-lapse images were acquired in Olympus IX83 with Perkin Elmer Ultraview Spinning Disc confocal microscope and a Hamamatsu EMCCD camera or the Olympus Fluoview FV1000 confocal laser scanning microscope. Dual color simultaneous imaging was performed at 3 frames per second (fps), dual color sequential imaging was done at 1.3 fps, and single fluorophore imaging for analysis of vesicle length was done at 5 fps. All movies were 3 minutes long, and the region of imaging in the PLM comprised the first 60–100 μm of the neuronal process immediately outside the cell body, with the cell body in the frame of imaging. Live imaging of EBP-2::GFP to assess microtubule polarity was carried out using an Olympus IX73 Epifluorescence microscope with an Andor EMCCD camera at 3 fps. UNC-104::GFP was imaged on Olympus Spin SR10 (SoRA, 50 μm disk) fitted with Teledyne Photometrics sCMOS camera.

L4 or 1-day adult worms were immobilized using 30 mM sodium azide and mounted on 2–5% agarose pads. Images were acquired on an Olympus IX73 Epifluorescence microscope with an Andor EMCCD camera or the Olympus Fluoview FV1000 confocal laser scanning microscope or Olympus IX83 with Perkin Elmer Ultraview Spinning Disc confocal microscope fitted with a Hamamatsu EMCCD camera. Since AP-3 localization is sensitive to levels of ATP [73 (link)], static imaging of APB-3::GFP was performed using 5 mM Tetramisole. APB-3::GFP was imaged on Olympus Spin SR10 (SoRA, 50 μm disk) fitted with Teledyne Photometrics sCMOS camera.