The overall goal of this procedure is to show how to assemble and align a laser ablation system for studying axon regeneration in CL gans. This is accomplished by first mounting a laser gland Thompson polarizer half wave plate turning mirror, periscope rail and rail mirror to a breadboard. The next step of the procedure is to align the laser beam to the outgoing beam from the microscope condenser lens.
Then laser beam expander lens mounted, aligned, and positioned to center the laser beam and make it zero divergent. The final step of the procedure is to make the laser ablation spot congruent with the image focus by making fine adjustments. Ultimately, it is possible to precisely cut half micron axons with minimal collateral tissue damage using the diffraction limited laser beam when it is imperfect XY NC alignment.
The main advantage of this technique over existing methods like Femto second lasers, is that it is relatively inexpensive at 15, 000. It is relatively maintenance free and provides very precise axon cutting. Though this method can provide insight into sea elgan axon regeneration, it can also be applied to other systems such as flies, zebrafish mouse, peripheral nervous system, and the in vitro cells.
Basically any cell that is within 30 to 50 micrometers of the surface. Begin with the components positioned on the breadboard and bolt down the laser periscope post and elevated rail supports. This system uses a Class three B laser that can easily damage your eyes, so please take laser safe safety seriously.
Talk to somebody in your local physics department about how to work with free beam lasers safely. Always during the alignment process, set the laser at the lowest power. When you look through the oculars, make sure the laser is mechanically shuttered and the laser is turned off.
Now, position the microscope first, align it with the rail axis. Second, align it with the transmitted light beam from the microscope condenser lens. Third, align both ends of the rail to the condenser beam using an adjustable aperture or lens affixed to the rail.
Use a level and lab jacks to adjust the rail height before proceeding. Make certain that both ends of the rail are aligned. Now, make sure the laser beam is passing through the gland, Thompson polarizer and halfway plate, and is not hitting their edges rotating.
The halfway plate will adjust the laser's power with all the components secured to the breadboard. Turn on the laser, set the pulse frequency to 100 continuous mode and reduce the power to a minimum using the halfway plate. Use a post-it note or lens paper to visualize the laser beam.
Now to bring the laser beam up from the breadboard. Adjust and fix the kinematically mounted mirrors in sequence from the laser to bring the beam up to the mirror that is mounted on the end of the elevated rail. When finished, the laser beam should be aligned roughly to the center of the mirrors, and the mirrors should be oriented roughly 45 degrees to the laser beam axis before aligning the laser beam with the transmitted light beam of the microscope.
Remove or open any ND filters and apertures in the intermediate module. We now begin the most critical step to give precise cutting of axons without excessive collateral damage. We're going to align the laser precisely in the x, y, and Z axis at the lowest laser power to give the smallest ablation spot.
The laser and the transmitted light beams can be seen simultaneously against paper. Position the paper near the microscope port to begin. Now begin aligning the laser using the course adjustments on the mirror on top of the periscope and the mirror on the end of the rail.
Place a paper between the aligned condenser lens and the open objective turret. Continue using the fine adjustments on the rail mirror to align the laser beam to the center of the larger transmitted light beam. Now, fix the microscope in place with clamps.
Additional steps on assessing the alignment of the laser can be found in the written manuscript. Dual galilean beam expanders must be installed to expand the 300 micron laser beam 33 times to fill the back aperture of the 10 millimeter objective. Begin by mounting the four lenses onto carriers.
Then attach the lenses to the rail such that they're all at the same optical axis and exactly orthogonal to the laser beam. Turn on the laser and adjust it to minimum power and to continuous mode. Roughly adjust the beam alignment through each lens.
Use paper to see the beam. Now turn off the laser. Apply a safety shutter to the laser and remove the microscope from the laser beam's path.
Turn the laser back on and visualize it against a nearby wall. Use the fine adjustment of the lenses to expand the beam. Then adjust the last two kinematically mounted mirrors to align the beam into a circle of uniform brightness at the estimated position of the objective back aperture.
View the size and uniformity of the beam against paper. Then adjust the beam to zero convergence by noting how the beam size changes as the paper is moved farther from the lens. Turn off the laser and engage the mechanical shutter.
Slide the microscope back into the laser beam path using the fixed clamps to define the correct alignment. Replace the clamps on the free side of the microscope, but do not tighten them down. Now turn on the laser.
Remove the safety shutter and rotate the objective turret to an open position using paper on the stage and expanded and uniform laser beam centered on the transmitted light beam from the condenser should be visible. If not centered, center the beams by loosening the microscope clamps and carefully nudging the microscope. Now engage the safety shutter and set the laser to trigger mode.
Rotate the 60 times objective in place. Now image of the surface scratches on a target slide. Remove the safety shutter, trigger the ablation laser, and adjust the laser power for the minimum ablation spot.
The spot should be circular and within five to 10 microns of the image center, center, the ablation spot. Using the fine adjustments of the kinematically mounted rail mirror to keep the spot centered and the beam profile uniform, it'll be necessary to iteratively adjust the rail mounted mirror and top periscope mirror. Evaluate the Z focus of the laser by moving the focus up and down systematically in one micron steps trigger the ablation laser at each step.
If the beam is correctly expanded and aligned and with correctly adjusted convergence, then it should ablate maximally at the image focus and weak or not at all, one micron above or below focus. If the maximum ablation spot is above or below the image focus, then make the final adjustments to the Z focus of the expanded beam by moving lens L three of the Galilean telescope until the laser ablation is congruent with the image focus. Nematodes were mounted to a customized slide and axons were ablated using the constructed system Laser exo somy and time-lapse imaging of axon regeneration were very robust.
Using a motorized stage, it was possible to routinely and precisely cut and image five axons in five different animals. On a single slide. Imaging time for axons was the limiting factor.
About 10%of experiments produced high quality time-lapse movies. The remaining experiments all provided good data, but were aesthetically less appealing because of small jittering movements of the worm that generally began after five to eight hours of immobilization. While attempting this procedure, it's always important to accurately evaluate the alignment of the laser in the x, y and Z axis.
The most common problem people have trying to cut axon is collateral damage or the complete inability to cut axons, and this is almost always due to a misaligned laser. This laser ablation system and technique for visualizing axon regeneration in c Agains will allow you to routinely assay the effect of any genetic mutation on Axon regeneration. NCL Agains.
