This research was supported by the National Science Foundation, the McKnight Endowment Fund for Neuroscience, the Christopher and Dana Reeve Foundation and Amerisure Charitable Foundation.

<ol>
	<li>Hammarlund, M., Nix, P., Hauth, L., Jorgensen, E. M., Bastiani, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axon+regeneration+requires+a+conserved+MAP+kinase+pathway.">Axon regeneration requires a conserved MAP kinase pathway.</a> Science. 323, 802-806 (2009).</li><li>Steinmeyer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+femtosecond+laser+microsurgery+system.">Construction of a femtosecond laser microsurgery system.</a> Nature protocols. 5, 395-407 (2010).</li><li>Wu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caenorhabditis+elegans+neuronal+regeneration+is+influenced+by+life+stage,+ephrin+signaling,+and+synaptic+branching.">Caenorhabditis elegans neuronal regeneration is influenced by life stage, ephrin signaling, and synaptic branching.</a> Proc Natl Acad. 104, 15132-15137 (2007).</li><li>Gilleland, C. L., Rohde, C. B., Zeng, F., Yanik, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+immobilization+of+physiologically+active+Caenorhabditis+elegans.">Microfluidic immobilization of physiologically active Caenorhabditis elegans.</a> Nature protocols. 5, 1888-1902 (2010).</li><li>Raabe, I., Vogel, S. K., Peychl, J., Tolic-Norrelykke, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+nanosurgery+and+cell+enucleation+using+a+picosecond+laser.">Intracellular nanosurgery and cell enucleation using a picosecond laser.</a> J. Microsc. 234, 1-8 (2009).</li><li>Hutson, M. S., Ma, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+and+cavitation+dynamics+during+pulsed+laser+microsurgery+in+vivo.">Plasma and cavitation dynamics during pulsed laser microsurgery in vivo.</a> Physical review letters. 99, 158104-158104 (2007).</li><li>Venugopalan, V., Guerra, A., Nahen, K., Vogel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+laser-induced+plasma+formation+in+pulsed+cellular+microsurgery+and+micromanipulation.">Role of laser-induced plasma formation in pulsed cellular microsurgery and micromanipulation.</a> Physical review letters. 88, 078103-078103 (2002).</li><li>Bourgeois, F., Ben-Yakar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Femtosecond+laser+nanoaxotomy+properties+and+their+effect+on+axonal+recovery+in+C.+elegans.">Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans.</a> Opt Express. 16, 5963-5963 (2008).</li><li>O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A. M., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J. Vis. Exp. (24), e1129-e1129 (2009).</li><li>Tsai, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma-mediated+ablation:+an+optical+tool+for+submicrometer+surgery+on+neuronal+and+vascular+systems.">Plasma-mediated ablation: an optical tool for submicrometer surgery on neuronal and vascular systems.</a> Curr. Opin. Biotechnol. 20, 90-99 (2009).</li><li>Chung, S. H., Clark, D. A., Gabel, C. V., Mazur, E., Samuel, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+AFD+neuron+in+C.+elegans+thermotaxis+analyzed+using+femtosecond+laser+ablation.">The role of the AFD neuron in C. elegans thermotaxis analyzed using femtosecond laser ablation.</a> BMC Neurosci. 7, 30-30 (2006).</li><li>Shen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ablation+of+cytoskeletal+filaments+and+mitochondria+in+live+cells+using+a+femtosecond+laser+nanoscissor.">Ablation of cytoskeletal filaments and mitochondria in live cells using a femtosecond laser nanoscissor.</a> Mech. Chem. Biosyst. 2, 17-25 (2005).</li><li>Rao, G. N., Kulkarni, S. S., Koushika, S. P., Rau, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+nanosecond+laser+axotomy:+cavitation+dynamics+and+vesicle+transport.">In vivo nanosecond laser axotomy: cavitation dynamics and vesicle transport.</a> Opt. Express. 16, 9884-9894 (2008).</li><li>Rohde, C. B., Yanik, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subcellular+in+vivo+time-lapse+imaging+and+optical+manipulation+of+Caenorhabditis+elegans+in+standard+multiwell+plates.">Subcellular in vivo time-lapse imaging and optical manipulation of Caenorhabditis elegans in standard multiwell plates.</a> Nat. Commun. 2, 271-271 (2011).</li></ol>

No conflicts of interest declared.

There are several good discussions of laser microsurgery with different laser systems3,5-11. Femtosecond IR lasers are the "gold standard" for subcellular laser ablation12 and convenient if associated with an imaging facility, but they are often too costly for individual users. If you require a femtosecond IR laser for imaging your sample because of depth of imaging then you will probably need one for laser axotomy. Transparent tissues with target axons within 30-50 um of the surface are probably feasible with blue and green pulse lasers in the 20 uJ/pulse range targeted through a high na immersion lens. There will be more collateral damage with the nano and pico second lasers compared with the femtosecond laser, especially as the depth of the target axon increases. C. elegans is transparent and all axons are within 20-30 um of the surface. We routinely cut motor axons that are within 5 um of the surface. We have also easily cut axons within the nerve ring that are about 20 um from the cuticle surface. We found the limit for cutting axons to be about 30-50 um through the diameter of an adult worm. It is likely that the laser ablation system described here would work well with many different preparations that fit the criteria of transparency and depth of target axons. Still, it is a bit surprising, given the theoretical advantages of the femtosecond IR lasers, that nano and picosecond 355 nm and 532 nm lasers perform so well for laser axotomy in C. elegans6,13. We see no differences in axon regeneration in response to laser axotomy with nanosecond 440 nm, nanosecond 532 nm, and femtosecond IR lasers.
Solid state 355 nm UV lasers are about the same cost as the 532 nm diode pumped passively Q-Switched solid state laser, but require either higher powers or optics that efficiently pass these shorter wavelengths. Most optics are designed to perform well with visible 400-700 nm light. 355 nm lasers would offer some advantages6 such as reduced plasma threshold, smaller spot size, and a long pass dichroic would efficiently direct 100% of the ablation laser to the target and allow simultaneous imaging of GFP without loss of sample signal. Blue 440 nm lasers would retain the advantages of working with a visible light laser (good performance with standard optics and safety). Unfortunately, the cost of a DPP Q-switched solid state 440 nm laser is 3 times the cost of a 355nm or 532 nm laser of similar power at this time. If we were designing a new system for C. elegans , where target depth is minimal, we would opt for a UV transparent lens (cost about $3,000 for a 40X 1.3na oil lens with 50-70% transmittance at 355nm) and a 355 nm laser producing 5-10 uJ/pulse at 1-20 kHz.
Axon ablation is thought to be due to plasma formation. Shorter pulses will produce plasma thresholds at lower power and the plasma volumes will be smaller6. The goal is to produce the smallest and shortest-lived plasma by adjusting the pulse energy to the lowest power. Larger long-lived plasmas will generate cavitation bubbles that damage surrounding cells. We have generally found that ablation using about 50-100 pulses at the highest frequency (i.e. 2.5kHz) gives the best results. This suggests that damage can be gradually integrated over a series of pulses to better control the extent of damage. The laser ablation system described here is very forgiving if the beam is aligned and expanded accurately. We start cutting axons in adult worms at 10% laser power (average of 0.3 mW measured at 2500 Hz and estimated 1 uJ/pulse) and can cut with limited localized damage through 14% power. You can observe larger areas of damage from 15-39% laser power, but only above 40% power (about 1 mW average measured at 2500 Hz) do the worms explode due to large cavitation bubbles and damage to the worm's cuticle.
The Steinmeyer et al. 20102 paper is an excellent resource for constructing a laser ablation system. Leon Avery also has a nice practical description of a laser ablation system based on an inexpensive N2 gas 337 nm laser and he provides some great practical advice (elegans.swmed.edu/Worm_labs/Avery/). When designing your own system you should first talk with your microscope representative to determine how to target the ablation laser to the microscope objective. Your microscope should be mounted on a vibration isolation table (Newport, TMI, Thor). A breadboard top is optimal, but a solid steel top can still be used with magnetic positioners. A dedicated intermediate module on an upright scope is nice because all optical elements can be left in place. However, it may be less expensive and more convenient to use a camera port (inverted) or a "combiner" attached to an epi-fluorescent port. You need to know the size of the back aperture and the transmittance (at the ablation laser wavelength) of the objective lens you intend to use. Once you decide on a laser (e.g., Crylas, TEEM, Crystal, or CRC) you should contact Thor or Newport for help with selecting the correct optical elements, hardware and safety equipment. We decided on a dual Galilean beam expander to save space, but a simpler Keplerian expander is an option (f2/f1=expansion factor). A custom beam expander will be the least expensive, but Newport, Thor, and several of the laser manufacturers offer excellent quality commercial beam expanders. Some laser manufacturers also offer manual and electronically controlled attenuators that may be cost effective and space saving, but not as versatile as the Glan-Thompson polarizer and half wave plate. You will also need to decide how to control the laser. You can design a LabView based controller, use a commercial TTL generator, or software provided by the laser company. Discuss the options with the specific laser manufacturer.
We have provided a list of the hardware we have used only as a general guide. The system described here cost about $15,000 when added to our existing imaging system. Your local Physics department will often have someone willing to provide a practical introduction to safely working with free beam lasers.
Alternative methods for immobilization and time lapse imaging of C. elegans have been recently described.14
Troubleshooting
The most difficult and critical step is alignment of the centered expanded beam to the optical axis of the microscope. Once you have the beam centered and expanded through the Galilean lenses you should work hard to get it aligned to within 5 um of the microscope's optical axis BEFORE using the steering mirrors for the final alignment to the center. Excessive use of the steering mirrors to align the beam to the microscope will move it off axis through the beam expander. USING EXTREME CAUTION you can attenuate the laser beam with the appropriate ND filter and directly view the laser beam profile on a reflective target (front surface mirror). You can gently nudge the microscope to precisely center the beam in the 60X objective field of view (if it can't be centered in the up/down range you need to adjust the rail height). The beam profile can be used to precisely align the microscope optical axis to give a centered beam that is circular and "flares" symmetrically. An asymmetric flare is an indication that the microscope axis is not perfectly aligned (or the rail is not level). Finally, you should adjust L3 and see an even and symmetrical expansion and contraction of the beam. Focus the microscope precisely on the target surface and then adjust L3 to focus the laser beam to the smallest spot. You should now find that the laser beam is within 5 um of the center when imaged via LSCM or CCD system and the ablation spot is within 1um of Z focus.
If you find you are "blowing up" worms or consistently generating large cavitation bubbles to cut axons your problem is most likely the fine alignment of the ablation laser. Make sure the minimum (&lt;500nm) ablation spot is localized to the Z focus (adjust convergence with L3 lens) and to the XY fiduciary spot (adjust with last kinematically mounted mirror).
Targeting axons closer to the surface will require less laser power. Similarly, smaller animals (L1 and L2) will require less laser power for axotomy compared to targeting the same axons in adults.
If your wild type axons do not regenerate or the axons bleach you should try reducing imaging laser power or decreasing the sampling rate. If your worms die or become sickly try reducing the percent Agarose in 1% steps. Make sure you are not moving the coverslip after mounting worms as this will often cause them to die when using high percentage agarose and microspheres.
If your worms burst or die during a time-lapse session it is due to: 1. Percentage agarose is too high. 2. Damage to cuticle by ablation laser. 3. Movement of coverslip after mounting. If damage to the cuticle is at fault it will only affect mounted worms that have been targeted with the ablation laser.
If your worm moves too much during a time-lapse session try increasing the percentage agarose in 0.5% steps. Healthy axons in healthy worms are always "active" and display a consistent level of fluorescence. If you see a sudden decrease in fluorescence or activity the worm is dying or dead. Multiple beaded or fragmented axons are a sure sign of a dying or dead worm.
If you have trouble keeping your axons in focus during the entire time lapse session there may be several different problems. First check your stage drift by imaging an inert sample on a glass slide over 10 hrs. A few microns drift may be due to thermal instability, but greater than 5 um is probably due to mechanical issues with your microscope. Problems with mounting are more common. Let your mounted worms equilibrate with your microscope stage for 30 minutes before starting your time lapse. Check that your Vaseline seal did not develop leaks during the course of your time lapse session.

<table border="1">
	<tbody>
		<tr>
			<th>
				Name of the reagent</th>
			<th>
				Company</th>
			<th>
				Catalogue number</th>
			<th>
				Comments (optional)</th>
		</tr>
		<tr>
			<td>535 nm Laser</td>
			<td>Crylas</td>
			<td>FDSS532Q3</td>
			<td>(TEEM, Crystal, and CRC also offer comparable lasers)</td>
		</tr>
		<tr>
			<td>All optics and hardware</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>(Thor offers comparable optics and hardware)</td>
		</tr>
		<tr>
			<td>SUPREMA Optical Mount, 1.0-in diameter</td>
			<td>Newport</td>
			<td>SS100-F2KN</td>
			<td>2</td>
		</tr>
		<tr>
			<td>1" diameter post, 1" height</td>
			<td>Newport</td>
			<td>PS-1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1" diameter post fork</td>
			<td>Newport</td>
			<td>PS-F</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Achromatic Zero-Order Wavel Plate, &frac12; Wave Retardation, 400-700nm</td>
			<td>Newport</td>
			<td>10RP52-1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Rotation Stage, 1" Aperature</td>
			<td>Newport</td>
			<td>RSP-1T</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1" diameter post, 1" height</td>
			<td>Newport</td>
			<td>PS-1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1" diameter post fork</td>
			<td>Newport</td>
			<td>PS-F</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Glan-Laser Calcite Polarizer, 430-700nm</td>
			<td>Newport</td>
			<td>10GL08AR.14</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Polarizer Rotation Mount</td>
			<td>Newport</td>
			<td>RM25A</td>
			<td>1</td>
		</tr>
		<tr>
			<td>&frac14;" spacer for 1" diameter post</td>
			<td>Newport</td>
			<td>PS-0.25</td>
			<td>1</td>
		</tr>
		<tr>
			<td>&frac12;" height, 1"diameter post</td>
			<td>Newport</td>
			<td>PS-0.5</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Fork, 1" diameter post</td>
			<td>Newport</td>
			<td>PS-F</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Beam Dump</td>
			<td>Newport</td>
			<td>PL15</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Microscope Objective Lens Mount</td>
			<td>Newport</td>
			<td>LH-OBJ1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1" diameter post, 1" height</td>
			<td>Newport</td>
			<td>PS-1</td>
			<td>1</td>
		</tr>
		<tr>
			<td>1" diameter post fork</td>
			<td>Newport</td>
			<td>PS-F</td>
			<td>1</td>
		</tr>
		<tr>
			<td>High-Energy Nd:YAG Laser Mirror, 25.4 mm Diameter, 45&deg;, 532 nm</td>
			<td>Newport</td>
			<td>10Q20HE.2</td>
			<td>4</td>
		</tr>
		<tr>
			<td>SUPREMA Optical Mount, 1.0 inch diameter, clearance mounting hole</td>
			<td>Newport</td>
			<td>SN100C-F</td>
			<td>2</td>
		</tr>
		<tr>
			<td>High Precision Knob Adjustment Screw, 12.7mm travel, 100TPI</td>
			<td>Newport</td>
			<td>AJS100-0.5K</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Thread adaptor, &frac14;-20 male, 8-32 female</td>
			<td>Newport</td>
			<td>SS-1-B</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Rod Clamp for 1.5 inch diameter rod</td>
			<td>Newport</td>
			<td>340-RC</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Rod, 14 inch (35.5 cm) tall</td>
			<td>Newport</td>
			<td>40</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Rail carrier for X26, square 40mm length</td>
			<td>Newport</td>
			<td>CN26-40</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Steel Rail, 384mm (15") length</td>
			<td>Newport</td>
			<td>X26-384</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Rod Platform for 1.5 inch diameter rod</td>
			<td>Newport</td>
			<td>300-P</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Rod, 14 inch (35.5 cm) tall</td>
			<td>Newport</td>
			<td>40</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Plano-Concave Lens, 12.7mm diameter, -25mm EFL, 430-700nm</td>
			<td>Newport</td>
			<td>KPC025AR.14</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Plano-Convex Lens, 25.4mm diameter, 50.2mm EFL, 430-700nm</td>
			<td>Newport</td>
			<td>KPX082AR.14</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Plano-Convex Lens, 25.4mm diameter, 62.9mm EFL, 430-700nm</td>
			<td>Newport</td>
			<td>KPX085AR.14</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Fixed Lens Mount, 0.5" diameter, 1.0" Axis height</td>
			<td>Newport</td>
			<td>LH-0.5</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Fixed Lens Mount, 1.0" diameter, 1.0" Axis height</td>
			<td>Newport</td>
			<td>LH-1</td>
			<td>2</td>
		</tr>
		<tr>
			<td>Microspheres 0.1um</td>
			<td>Polysciences</td>
			<td>00876</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>RPI</td>
			<td>A20090</td>
			<td>EEO matters</td>
		</tr>
		<tr>
			<td>Muscimol</td>
			<td>Sigma</td>
			<td>M1523</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

constructing a low-budget laser axotomy system to study axon regeneration in c. elegans

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1. The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system2,3. However, solid-state pulse lasers of modest costs (&lt;$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (&lt;1um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers4). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13mm. Anesthetic (1ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.

Watch this Scientific Journal Video about Constructing a Low-budget Laser Axotomy System to Study Axon Regeneration in C. elegans at JoVE.com

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

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.

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

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans1.  The main difficulty of this assay ...

constructing-low-budget-laser-axotomy-system-to-study-axon

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15.3K vistas.  University of Utah. 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.