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Neuroscience JoVE (Open Access) Citable · DOI

Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans

DOI: 10.3791/3331-v
What you'll learn
  • ✓ Build a low-cost laser ablation system compatible with existing microscopes
  • ✓ Align and focus a laser beam for precise axon transection in C. elegans
  • ✓ Perform laser axotomy and acquire time-lapse imaging data for regeneration analysis
Protocol

Laser axotomy followed by time-lapse imaging is a sensitive way to assay the effects of mutations in C. elegans on axon regeneration. A high quality, but inexpensive, laser ablation system can be easily added to most microscopes. Time lapse imaging over 15 hours requires careful immobilization of the worm.

Difficulty
advanced
Total time
~15 hours per experiment (including 15-hour time-lapse imaging); system construction ~2-3 hours
Model organism
Caenorhabditis elegans

Steps

1
Construct laser ablation system components

Assemble optical and mechanical components into a functional laser axotomy apparatus. Design and integrate system to attach to standard microscope setup.

▶ 01:39
2
Align laser beam to target axis

Perform precise alignment of laser beam through optical path to ensure accurate targeting of axons during ablation.

▶ 03:42
3
Expand and focus laser beam output

Adjust beam expansion optics and focal length to achieve optimal spot size and intensity for axon transection without excessive collateral damage.

▶ 04:54
4
Perform axotomies and image regeneration

Execute laser axotomy on immobilized C. elegans neurons and acquire 15-hour time-lapse imaging sequence to document axon regeneration response.

▶ 08:29
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