Laser ablation

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Laser ablation, more exactly the ablation of single cells by aiming a laser beam at them, is mainly used for studying vulva development and neurology.

This section describes how to use a setup with a manually (or "pedially") pulse-triggered VSL-337ND-S Nitrogen laser and a Zeiss Axioskop with a live camera as it is installed in the Sommer lab.

The most important point about doing an ablation experiment is to know the cell lineage you want to follow. Another issue is timing: for example, "standard" vulva precursor cell ablations should always be done within 1 hour after hatching.


ablating cells

  • Melt 4ml 5% Noble Agar and add 20ul of a 1M SodiumAzide solution (toxic!)
  • Put around 30-100 "late" eggs on a OP50 plate, taking care not to ransfer any larvae.
  • Check regularly. Carefully pick 1-5 newly hatched larvae onto an agar pad with M9 (see Nomarski microscopy)
  • Use the 100x objective and remove the polarization filter above the objective revolver. The shutter on the right side has to be in "medium" position.
  • The pulse-trigger should be set at a "medium" pulse rate (around "15" in our machine , this is NOT 15Hz !)
  • Focus at the cover slip (the place where many e. coli hang around) and give it a shot. The cover slip should show a little crack.
  • Mark the exact location of the impact on the monitor. It should be checked regularly.
  • Focus at the centre of your cell's nucleus and give a brief pulse. An exact time cannot given here, since it largely depends on the power of your laser, which decreases over time (see below). Instead, you have to obeserve the cell during shooting. Stop the beam as soon as you see a little black spot in the nucleus.



troubleshooting

If you cannot produce a crack in the cover slide:

  • Often it is just a matter of proper illumination to see the beam.
  • Check if the microscope shutter is in the correct position and the upper polarization filter is removed.
  • Check the pulse trigger - you should hear its "tiggi-tiggi-tiggi"-noise.
  • Move the attenuation shutter in the laser path one or two positions further to the left and try again (the attenuation shutter is the gray slide that you find at the tube coming from the dye cell of the laser)
  • If this shutter is in left-most position, it might be necessary to replace the coumarin dye: open the screw of the dye cell (little black box at the end of the tube) and remove liquid with a pipette. Wash several times with water-free methanol. Never use water to clean the cell! Fill with new coumarin dye.
  • If this doesn't help, there is a secret second attenuation shutter directly at the exit port of the laser. Push it to the right a little bit and try again.
  • If you tried all this and could not solve the problem, you are in deep shit. Do not make any plans for the rest of the day.
  • It might be necessary to re-adjust the laser. Get the "special screwdriver" (little black thing with red handle). It fits to two screws on top of the laser tube, which can both be moved in two dimensions by pushing and twisting. The "hidden" screw next to the dye cell is for focussing the beam, the other one just changes the x/y coordinates of the beam. In addition, there is a moveable ring which can be used to focus in Z-axis. Place a piece of yellow paper(e.g."post-it" notes) onto the microscope stage, shut down the microscope- and the room-lights and try to see the laser beam on the paper when firing through the 10x objective; all shutters should be maximally open. If you cannot find the beam, SLOWLY move and twist the hidden screw next to the dye cell. Once you found the beam, insert a microscope slide (with agar pad) and focus on the cover slide. Try to find the beam first by playing with the x/y screw. Try to remember its initial position and go back to that if you did not find the beam. Next try the "hidden screw". Move it VERY slowly, and look out for the beam. Once you found it, move it to the centre with the x/y screw and try to focus it with the "hidden screw" until you can make a crack. Fine-tune it with the Z-ring. Stepwise close the attenuation shutters and further improve the focus. First close the shutter at the laser itself, then the one in the tube. Finally try a worm. You might want to drink a beer now, and you earned one indeed.
  • If you cannot find any beam, carefully remove the optic fibre at the exit port of the laser. DO NOT look into the opening while the laser is operating! Place a piece of white paper around 10cm away from the opening, attenuator should be wide open. If there is no beam, probably the Nitrogen cell needs to be exchanged( happens every 2-4 years). Contact "Laseranalytik Starna Gmbh"
  • If the laser itself is ok, check the fibre: Put it in the exit port and hold the other end against white paper.
  • You can do the same thing with the entire tube: Use the "special screwdriver" and remove it from the microscope.

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