The overall goal of this procedure is to precisely cut the neurons of CL agains with a laser in vivo. This is accomplished by assembling the taxotomy system and precisely focusing and aligning and adjusting the power of the laser. When worms are immobilized on a microscope slide, their neurons can be cut with precise spatial and temporal control.
The extent to which severed neurons regenerate is then measured by scoring for growth cones and or by tracing neurite lengths. The main advantage of this technique over existing methods like the femtosecond laser, is that the micro point pulse laser is a cost-effective system that is easy to set up and maintain and is readily adaptable for a wide variety of applications, Though we typically apply this method to study regeneration of the GABA motor neurons. The system can also be used to ablate other cell types such as skin muscle, or specific neuronal synapsis.
This method requires a micro point laser system. The laser mounts to the epi fluorescence port of a compound microscope, and includes a single dichroic that matches the fluorophore in the cells to be cut a pulse generator and a foot pedal to operate the laser. At a minimum, the microscope should be outfitted with a 100 times 1.4 NA oil objective and a four times objective.
Have a motorized stage with joystick operation and be mounted to an air table for illumination. A light supply with light guide and a shutter mechanism is attached to the micro point laser. The shutter mechanism is useful to reduce the illumination intensity independently of the laser.
A camera which is connected to a computer requires a frame rate of at least 16 hertz and must operate well in reduced light. The computer requires imaging software such as Nikon elements to view the neurons, drive the camera, and manipulate the motorized stage. When fully assembled, the user can focus the microscope with one hand, move the stage with the other, activate the laser with the foot pedal, and have a clear view of the onscreen image.
Cutting neurons in an organism the size of a grain of sand can be challenging. To accomplish this, the laser must be tightly focused in all three dimensions. Begin by focusing the Z plane.
Push in the neutral density filter ND 16 to attenuate the laser while focusing for finer focusing, pushing both the ND 16 and ND four fluorescent filters. Next, set the micro point laser to one pulse with the switch turn to select, and then move the filter wheel to the appropriate long pass barrier filter. Now place a mirrored slide on the stage and focus on pinholes within it using the four times objective and transmitted light.
Once focused, add a drop of immersion oil to the slide and refocus on the pinholes with the 100 times objective. Then use the optical path switching lever to open the camera shutter and open the laser shutter. If getting accurate, focus is difficult, reduce the transmitted light intensity so the edges of the pinhole are crisp.
To test the focus, use the foot pedal to fire the laser. If the Z plane is properly focused, a small hole should show up in the mirror. Now focus the microscope slightly above the plane of the mirrored slide and shoot the laser.Again.
This should make an even smaller hole than the first shot or leave no mark at all. The same results should be seen while focusing below the mirrored slide. However, if a larger hole is produced, then refocus the laser with the focus ring on the ablation head.
Finally, verify that the hole is still properly aligned with the cross hairs in the eyepiece. With consistent use, the Z plane focus rarely needs to be adjusted. To focus the laser in the XY plane, make another hole in the mirror, and then access the software in Nikon elements.
Initiate the live capture mode in the acquire menu. In the measure toolbar, select ROI editor, select the double cross tool and drag the cross hairs to align them with the center of the new hole. Click save ROI and then exit editor to resume live capture.
To manipulate the stage with the mouse, activate the mouse XY setting. Now the laser is focused. Reset the neutral density filters to their original position and set the laser pulses from one to 10 for future use.
First, prepare a solution of 3%molten aros in M nine solution and quat approximately five milliliters into a 50 milliliter tube. Place the tube in a 55 to 65 degrees Celsius water bath and store the remainder for future use. Now use two slides covered in labeling.
Tape a pair of clean slides and a drop of agarose to make a pad where the worms can be placed. After 30 seconds, the aros will have congealed pipette three to five microliters of 0.05 to 0.1 micrometer polystyrene beads onto the pad within 10 minutes and quickly arrange five to 10 transgenic worms onto the beads with a platinum pick, so their labeled neurons are easily viewed. To avoid damaging the worms carefully set a standard cover slip onto the worms.
Sliding it around. The worm slide is now prepared for performing taxonomies. Using the eyepiece, locate the worms under four times power refocus with the 100 times objective, turn off the transmitted light, turn on the fluorescence and switch the optical path to the camera.
Use the mouse to center the neuron on the cross hairs. Now fire the laser with the foot pedal. The right amount of laser power will sever the neuron in one or two pulses.
Too much laser power will cause peripheral damage or blast a hole in the worm to adjust the laser's power, move the attenuator plate. If the laser becomes weak, the attenuator plate position can be adjusted to keep cutting. However, this is likely an indication that the Kumar in four 40 in the laser's D cell needs to be replaced when performed correctly.
Laser taxotomy produces a small break in the neuron. In certain cases, a small scar may form around the site of injury. Practically between one and three neurons are cut per animal, but there is no theoretical limit.
When finished, recover the worms by removing the cover slip in a direct upward movement, being careful that the worms remain on the agro rose pad. Do not slide the cover slip off. Use needle nose forceps to cut out the pad around the worms.
Place the pad on a freshly seeded NGM plate containing OP 50 and free the worms with 10 microliters of sterile M nine. Between eight and 48 hours after the ex otomy regeneration can be assessed to do this, remount the healthy worms on three to five microliters of polystyrene beads and visualize their neurons in severed neurons. A neuronal stump distal to the cut site will remain Both the distal and proximal stumps initially retract after being cut, but regeneration is expected from the proximal stump.
Regeneration can be scored for the formation of a growth cone. Alternatively, neurite length can be traced and compared typically between 60 to 70%of GABA neurons form growth combs within 24 hours of being cut. If the laser isn't cutting properly, it is likely because either the laser is outta focus or because the dye in the dye cell needs to be replaced.
Also, if you find that the worms are moving around, it is probably because the agros pad is too moist. We find that pads need to be left for approximately 30 seconds before worms are placed on them to circumvent this problem. In addition, you can try using smaller size microbeads to immobilize the worms.
Finally, if you find that the axons have a beaded appearance, it's likely because the agros pad is too dry. After watching this video, you should have a good understanding of how to set up the micro point laser system. Mount worms for taxotomy, cut individual neurons and assess subsequent regeneration.
