Epidermal Laser Wounding: Inducing Localized Damage to Examine Repair Processes in C. elegans

Overview

This video introduces a method of laser wounding, which is used to cause very precise injuries in the worm epidermis.  The sample protocol is from a study of wound response and repair.

Protocol

This protocol is an excerpt from Xu and Chisholm, Methods for Skin Wounding and Assays for Wound Responses in C. elegans, J. Vis. Exp. (2014).

1. Laser Wounding

NOTE: Use femtosecond laser irradiation (800 nm) to perform more precise wounding.

1. Prepare agar pads on a glass slide with melted 2% agar. Ensure the pads are similar to the agar pads used for live imaging of C. elegans.
2. Transfer 10 young adult worms onto the agarose pad with worm pick, and add a 2 µl drop of 12 mM Levamisole solution. Cover the animals and the liquid with a cover slip. Wait 1-2 min for the worms to paralyze.
3. Place the glass slide onto a spinning disk confocal microscope. Move the stage to find the worms and focus on the lateral anterior or posterior syncytial epidermis with a 100X objective (NA 1.4-1.46).
4. Set the power of the femtosecond laser to 140 mW (measured before the objective). Use the femtosecond laser at a repetition rate of 80 MHz.
   NOTE: Two pulses of 200 msec each (separated by 20 msec) are sufficient for wounding in our hands.
5. Focus on the apical surface of the epidermal cell and wound the epidermis. Observe local disruption of the cytoplasm (bubbling), or local bleaching of any fluorescent marker used.
   NOTE: Follow appropriate laser safety procedures when using the femtosecond laser. Line or point scans using femtosecond lasers such as those in two photon microscopes should provide sufficient power for laser wounding.
   NOTE: While we do not have direct experience of using other lasers, in principle wounding should be possible using conventional UV lasers (as used in C. elegans cell ablations). Optionally, use a Fluorescence Recovery After Photobleaching (FRAP) module on spinning disk confocal to wound the epidermis (N. Pujol, personal communication).