Prime 95b

Confocal imaging of fluorescently-tagged RIMB-1::Skylan-S were collected from L4 animals anesthetized with 10 mM levamisole in M9 buffer. using a Zeiss Axio Observer Z1 microscope stage outfitted with a Plan-Apochromat 63× 1.4 NA objective and a Yokogawa CSU-W1 spinning disk confocal scanner attached to a Prime 95B camera. Maximum intensity projections of z-stack images were cropped and straightened around the dorsal nerve cord (DNC) using ImageJ/Fiji, and were analyzed using a Matlab script designed to identify puncta in individual TIFF files based on local means thresholding coupled with watershed segmentation. (Details in SI Appendix, Methods).

For static imaging, worms were immobilized using 1 mM levamisole solution and placed on 4% agarose pads, and then imaged using an Olympus IX83 fluorescence microscope equipped with a spinning-disk confocal scanner (Yokogawa CSU-W1), an sCMOS camera (Prime 95B), and a 60x oil Apochromat objective (NA: 1.49). Z stack images were processed by the projection of maximum intensity except for Figs 2A and S5A.
Time-lapse imaging was performed as previously described with some modifications [60 (link)]. Briefly, 2 μl of 1 mM levamisole solution was added into the center of the glass bottom of a microwell dish, then about 20 worms were transferred into the drop of levamisole solution. Next, a 4% agarose pad was gently added onto the animals. All time-lapse movies were taken using the spinning-disk confocal microscope, and Z stack images were processed by projection of maximum intensity except for S10 and S11 Figs, in which single-layer images were shown.
For Stimulated Emission Depletion Microscopy (STED) imaging, worms were immobilized using 1 mM levamisole solution and placed on 4% agarose pads. A Leica TCS SP8 STED fluorescence microscope equipped with 592/660/775 nm lasers, and a HC PL APO CS2 100×/1.40 oil objective was used for imaging. Z stack images were processed by the projection of maximum intensity.