Micropoint laser system

Photobleaching and laser ablations were performed using 514 or 551 nm ns-pulsed laser light and a galvo-controlled MicroPoint Laser System (Andor, Oxford Instruments) operated through MetaMorph or Micro-Manager. Single z-planes were chosen to pick the clearest k-fiber visible from plus- to minus-end, parallel to the coverslip, that was long enough to ablate. Non-ablated unfocused k-fibers in the same imaging plane were not necessarily parallel to the coverslip, so their full length was not always captured in the single z-plane due to tilt. Photobleaching was performed by firing the laser at the lowest possible power to make a visible bleach mark (~20% of total power), whereas ablations were performed at the lowest possible power to fully cut a k-fiber (~60% of total power). K-fiber ablations were verified by observing complete depolymerization of newly created plus-ends, relaxation of interkinetochore distance, or poleward transport of k-fiber stubs (control only). When firing the laser, 1–3 areas around the region of interest were targeted and hit with 5–20 pulses each. Ablations were imaged using one z-plane every 12 s to assay short-term dynamics, then switching to every 1 min after approximately 10 min following ablation to avoid phototoxicity.

Laser ablations were performed on a Nikon A1 confocal microscope equipped with an Andor MicroPoint laser system consisting of a pulsed 440 nm nitrogen laser. We adjusted a variable neutral density filter to attenuate the output laser to limit damage to targeted cells, as assessed by confocal imaging. The observation of cell collapse was used to confirm successful ablation (Fig 2C and 2F).

Adult day 3 and day 4 hermaphrodites were mounted in S-basal containing 10 mM levamisole on a 2% agarose pad under a cover slip. GFP-expressing cholinergic neurons and commissures were monitored with a fluorescence microscope using a Nikon 100X, 1.4 NA lens. Selected commissural axons were cut using a MicroPoint Laser System (Andor Technology). After surgery, animals were placed on a fresh OP50-1 spotted NGA plate, and then remounted for quantitative light microscopy imaging approximately 24-28 hours post-surgery. A minimum of 10 individuals (with 1 axotomized commissure per worm) were observed for most experiments and repeated 3 times.