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>

利害の衝突は宣言されません。

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><tbody><tr><td>Name of the reagent</td><td>Company</td><td>Catalogue number</td><td>Comments (optional)</td></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></td><td></td><td>(Thor offers comparable optics and hardware)</td></tr><tr><td>SUPREMA Optical Mount, 1.0-in diameter</td><td>Newport Corp.</td><td>SS100-F2KN</td><td>2</td></tr><tr><td>1&quot; diameter post, 1&quot; height</td><td>Newport Corp.</td><td>PS-1</td><td>1</td></tr><tr><td>1&quot; diameter post fork</td><td>Newport Corp.</td><td>PS-F</td><td>1</td></tr><tr><td>Achromatic Zero-Order Wavel Plate, &amp;frac12; Wave Retardation, 400-700nm</td><td>Newport Corp.</td><td>10RP52-1</td><td>1</td></tr><tr><td>Rotation Stage, 1&quot; Aperature</td><td>Newport Corp.</td><td>RSP-1T</td><td>1</td></tr><tr><td>1&quot; diameter post, 1&quot; height</td><td>Newport Corp.</td><td>PS-1</td><td>1</td></tr><tr><td>1&quot; diameter post fork</td><td>Newport Corp.</td><td>PS-F</td><td>1</td></tr><tr><td>Glan-Laser Calcite Polarizer, 430-700nm</td><td>Newport Corp.</td><td>10GL08AR.14</td><td>1</td></tr><tr><td>Polarizer Rotation Mount</td><td>Newport Corp.</td><td>RM25A</td><td>1</td></tr><tr><td>&amp;#188;&quot; spacer for 1&quot; diameter post</td><td>Newport Corp.</td><td>PS-0.25</td><td>1</td></tr><tr><td>&amp;frac12;&quot; height, 1&quot;diameter post</td><td>Newport Corp.</td><td>PS-0.5</td><td>1</td></tr><tr><td>Fork, 1&quot; diameter post</td><td>Newport Corp.</td><td>PS-F</td><td>1</td></tr><tr><td>Beam Dump</td><td>Newport Corp.</td><td>PL15</td><td>1</td></tr><tr><td>Microscope Objective Lens Mount</td><td>Newport Corp.</td><td>LH-OBJ1</td><td>1</td></tr><tr><td>1&quot; diameter post, 1&quot; height</td><td>Newport Corp.</td><td>PS-1</td><td>1</td></tr><tr><td>1&quot; diameter post fork</td><td>Newport Corp.</td><td>PS-F</td><td>1</td></tr><tr><td>High-Energy Nd:YAG Laser Mirror, 25.4 mm Diameter, 45&amp;#x00B0;, 532 nm</td><td>Newport Corp.</td><td>10Q20HE.2</td><td>4</td></tr><tr><td>SUPREMA Optical Mount, 1.0 inch diameter, clearance mounting hole</td><td>Newport Corp.</td><td>SN100C-F</td><td>2</td></tr><tr><td>High Precision Knob Adjustment Screw, 12.7mm travel, 100TPI</td><td>Newport Corp.</td><td>AJS100-0.5K</td><td>4</td></tr><tr><td>Thread adaptor, &amp;#188;-20 male, 8-32 female</td><td>Newport Corp.</td><td>SS-1-B</td><td>2</td></tr><tr><td>Rod Clamp for 1.5 inch diameter rod</td><td>Newport Corp.</td><td>340-RC</td><td>2</td></tr><tr><td>Rod, 14 inch (35.5 cm) tall</td><td>Newport Corp.</td><td>40</td><td>1</td></tr><tr><td>Rail carrier for X26, square 40mm length</td><td>Newport Corp.</td><td>CN26-40</td><td>4</td></tr><tr><td>Steel Rail, 384mm (15&quot;) length</td><td>Newport Corp.</td><td>X26-384</td><td>1</td></tr><tr><td>Rod Platform for 1.5 inch diameter rod</td><td>Newport Corp.</td><td>300-P</td><td>2</td></tr><tr><td>Rod, 14 inch (35.5 cm) tall</td><td>Newport Corp.</td><td>40</td><td>2</td></tr><tr><td>Plano-Concave Lens, 12.7mm diameter, -25mm EFL, 430-700nm</td><td>Newport Corp.</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 Corp.</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 Corp.</td><td>KPX085AR.14</td><td>1</td></tr><tr><td>Fixed Lens Mount, 0.5&quot; diameter, 1.0&quot; Axis height</td><td>Newport Corp.</td><td>LH-0.5</td><td>2</td></tr><tr><td>Fixed Lens Mount, 1.0&quot; diameter, 1.0&quot; Axis height</td><td>Newport Corp.</td><td>LH-1</td><td>2</td></tr><tr><td>Microspheres 0.1um</td><td>Polysciences, Inc.</td><td>00876</td><td></td></tr><tr><td>Agarose</td><td>Research Products International Corp.</td><td>A20090</td><td>EEO matters</td></tr><tr><td>Muscimol</td><td>Sigma-Aldrich</td><td>M1523</td><td></td></tr></tbody></table>

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

タイムラプス顕微鏡に続くレーザー軸索切断はCの軸索再生の表現型の高感度アッセイであるエレガンス1。このアッセイの主要な難しさは認知コスト（$ 25 - 100K）とレーザーアブレーションシステム2,3を実装するために必要な技術的専門知識です。しかし、控えめなコスト（&lt;$ 10K）の固体パルスレーザーは、ターゲットの軸索は、組織の表面に&quot;近い&quot;透明な製剤中のレーザーアブレーションのための堅牢な性能を提供することができます。システムの構築と配置は一日で達成することができます。アブレーションレーザーに焦点を当てたコンデンサーからの光が提供する光路は、便利な位置合わせガイドを提供します。削除されたすべての光学系との中間モジュールがアブレーションレーザーに専念しない光学素子は、レーザーアブレーションのセッション中に移動する必要はありませんことを保証することができます。中間モジュールのダイクロイックは同時イメージングとレーザーアブレーションすることができます。レーザービームtをセンタリングOは、焦点を絞った顕微鏡の集光レンズからの出射ビームは、システムの初期配置をガイドします。レンズの様々な条件に使用され、選択された対物レンズの背面に開口部を埋めるためにレーザービームを展開されています。最終的なアラインメントとテストは、フロント面のミラー化ガラスのスライドのターゲットで実行されます。レーザパワーは、最小サイズのアブレーションのスポット（&lt;1um）を与えるために調整されます。アブレーションのスポットは、イメージングのウィンドウで修正が十字カーソルに最後の運動学的にマウントされているミラーの微調整で中央に配置されます。軸索切断用レーザパワーは、ターゲットのスライド上の最小アブレーションスポット（これは、使用するターゲットと異なる場合があります）に必要なより高い約10倍になります。ワームは、アガロースパッド（またはマイクロ流体室4内）に取り付けてレーザー軸索切断とタイムラプスイメージングのために固定化することができる。アガロースパッドは簡単に電子レンジで溶かしたバランスのとれた食塩水で10％アガロースで作られています。溶融アガロースの液滴をガラススライド上に配置され、別の平坦化されパッドにスライドガラスは、厚さ約200ミクロン（隣接したスライド上に時間のテープの単層は、スペーサとして使用されます）。 &quot;シャーピー&quot;キャップは13mmの均一な直径の円形パッドを切り出すために使用されます。彼らは、その左側に横たわっているように、麻酔薬（1ULムシモール20mMの）とミクロ​​スフェア（クリス牙 - 円私信）（1UL 2.65パーセントポリスチレン水中0.1 UM）は指向3月5日ワームに続いてパッドの中央に追加されます。ガラスのカバースリップが適用され、その後ワセリンはカバーを密封し、試料の蒸発を防ぐために使用されます。

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.

の軸索再生を研究するために、低予算レーザー軸索切断システムの構築 C.エレガンス</em

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

Research

JoVE Journal

Bioengineering

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

15.4K ビュー.  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.

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

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

Cの生体レーザー軸索切断にエレガンス

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

神経科学

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

ライブゼブラフィッシュの胚における二光子軸索切断とタイムラプス共焦点イメージング

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

生物学

Laser Nanosurgery of Cerebellar Axons In Vivo

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to understand the mechanism that promotes neuronal regeneration, an in-depth analysis of the neuronal types that can remodel after injury is needed. Several studies showed that damaged climbing fibers are capable of regrowing also in adult animals1,2. The investigation of the time-lapse dynamics of degeneration and regeneration of these axons within their complex environment can be performed by time-lapse two-photon fluorescence (TPF) imaging in vivo3,4. This technique is here combined with laser surgery, which proved to be a highly selective tool to disrupt fluorescent structures in the intact mouse cortex5-9.
This protocol describes how to perform TPF time-lapse imaging and laser nanosurgery of single axonal branches in the cerebellum in vivo. Olivocerebellar neurons are labeled by anterograde tracing with a dextran-conjugated dye and then monitored by TPF imaging through a cranial window. The terminal portion of their axons are then dissected by irradiation with a Ti:Sapphire laser at high power. The degeneration and potential regrowth of the damaged neuron are monitored by TPF in vivo imaging during the days following the injury.

Laser Nanosurgery of Cerebellar Axons In Vivo

Two-photon imaging, coupled to laser nanodissection, are useful tools to study degenerative and regenerative processes in the central nervous system with subcellular resolution. This protocol shows how to label, image, and dissect single climbing fibers in the cerebellar cortex in vivo.

小脳軸索のレーザーNanosurgery<em&gt;インビボ</em

Only a few neuronal populations in the central nervous system (CNS) of adult mammals show local regrowth upon dissection of their axon. In order to ...

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various intrinsic and extrinsic inhibitory factors involved in the restriction of axon regeneration have been identified. However, simply removing these inhibitory cues is insufficient for successful regeneration, indicating the existence of additional regulatory machinery. Drosophila melanogaster, the fruit fly, shares evolutionarily conserved genes and signaling pathways with vertebrates, including humans. Combining the powerful genetic toolbox of flies with two-photon laser axotomy/dendriotomy, we describe here the Drosophila sensory neuron &#8211; dendritic arborization (da) neuron injury model as a platform for systematically screening for novel regeneration regulators. Briefly, this paradigm includes a) the preparation of larvae, b) lesion induction to dendrite(s) or axon(s) using a two-photon laser, c) live confocal imaging post-injury and d) data analysis. Our model enables highly reproducible injury of single labeled neurons, axons, and dendrites of well-defined neuronal subtypes, in both the peripheral and central nervous system.

A Drosophila In Vivo Injury Model for Studying Neuroregeneration in the Peripheral and Central Nervous System

Here, we present a protocol using the Drosophila sensory neuron - dendritic arborization (da) neuron injury model, which combines in vivo live imaging, two-photon laser axotomy/dendriotomy, and the powerful fly genetic toolbox, as a platform for screening potential promoters and inhibitors of neuroregeneration.

再生に関する末梢および中枢神経系を研究するため生体内でショウジョウバエ損傷モデル

The regrowth capacity of damaged neurons governs neuroregeneration and functional recovery after nervous system trauma. Over the past few decades, various ...

医学

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed orientation. Here we describe methods to label, image, and analyze the microtubule dynamics and growth during the development and regeneration of touch neurons in C. elegans. Using genetically encoded fluorescent reporters of microtubule tips, we imaged the axonal microtubules. The local changes in microtubule behavior that initiates axon regeneration following axotomy can be quantified using this protocol. This assay is adaptable to other neurons and genetic backgrounds to investigate the regulation of microtubule dynamics in various cellular processes.

In vivo Assessment of Microtubule Dynamics and Orientation in Caenorhabditis elegans Neurons

A protocol for imaging the dynamic microtubules in vivo using fluorescently labeled End binding protein has been presented. We described the methods to label, image, and analyze the dynamic microtubules in the Posterior lateral microtubule (PLM) neuron of C. elegans.

インビボカエノハブディティス・エレガンスニューロンにおける微小管動態と向きの評価

In neurons, microtubule orientation has been a key assessor to identify axons that have plus-end out microtubules and dendrites that generally have mixed ...

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca2+ (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Methods for Skin Wounding and Assays for Wound Responses in C. elegans

The adult C. elegans skin is a tractable model for studies of epithelial wound responses, including wound closure, scar formation, and innate immunity.

創傷応答のためのスキン傷つけ処理のための方法およびアッセイ<em&gt; C。エレガンス</em

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound ...

<meta charset="utf8"></head><body>PDMS金型を用いた動物配向制御用チャネリング寒天パッドの作製

Fine Adjustment of Caenorhabditis elegans Orientation on Channeled Agar Pads for Imaging Neuroregeneration

Caenorhabditis elegans is a model organism widely used for studying biological processes. Its transparency and small size make it ideal for imaging tissues, cells, and subcellular structures. Traditional flat agar pads for imaging C. elegans limit control over the animal's orientation, restricting views primarily to lateral perspectives. This limitation complicates the visualization of dorsal-ventral structures and reduces image clarity, especially in older animals with increased pigmentation and larger diameters. To overcome these challenges, we developed channeled agarose pads that allow precise control of animal orientation. These channels enable researchers to rotate and fix C. elegans in specified positions, facilitating the simultaneous imaging of multiple structures and improving image resolution by bringing target cells closer to the microscope objective. This is particularly useful for imaging regenerated neuronal fibers after surgery, which may grow in directions difficult to capture with traditional flat agar pads. This method is accessible, as fabricating channeled agar pads requires the same time and materials as flat pads, making it a practical option for most laboratories.

<meta charset="utf8"></head><body>イメージング神経再生のためのチャネル寒天パッド上の 線虫 の配向の微調整

Here, we present a protocol for fabricating channeled agar pads using PDMS molds created from vinyl records. The channels enable users to finely orient Caenorhabditis elegans to improve imaging contrast and facilitate the comparison of structures. These capabilities are particularly useful in neuroregeneration studies.

イメージング神経再生のためのチャネル寒天パッド上の 線虫 の配向の微調整

Caenorhabditis elegans is a model organism widely used for studying biological processes. Its transparency and small size make it ideal for imaging ...

C. elegans Maintenance

Ceanorhabditis elegans has been, and is still, used to great success as a model organism for studying a variety of developmental, genetic, molecular and even physical phenomena. In order to use C. elegans to its full potential, proper care and attention to the basic maintenance of this powerful organism is essential.
In this video you will learn the basic housing and feeding requirements of C. elegans, how to correctly handle and manipulate worms using a worm pick and how to freeze and recover important worm stocks. Towards the end of the video we will visit a few applications of modifying the housing, feeding and manipulation of these important animals.

Maintenance of Caenorhabditis elegans

C. elegansの基礎研究のためのメンテナンスについて

Ceanorhabditis elegans has been, and is still, used to great success as a model organism for studying a variety of developmental, genetic, molecular and ...

の軸索再生を研究するために、低予算レーザー軸索切断システムの構築 C.エレガンス

要約

さらに動画を探す

この動画の章

Cの生体レーザー軸索切断にエレガンス

ライブゼブラフィッシュの胚における二光子軸索切断とタイムラプス共焦点イメージング

小脳軸索のレーザーNanosurgery

再生に関する末梢および中枢神経系を研究するため生体内でショウジョウバエ損傷モデル

インビボカエノハブディティス・エレガンスニューロンにおける微小管動態と向きの評価

創傷応答のためのスキン傷つけ処理のための方法およびアッセイ

イメージング神経再生のためのチャネル寒天パッド上の線虫の配向の微調整

イメージング神経再生のためのチャネル寒天パッド上の線虫の配向の微調整

イメージング神経再生のためのチャネル寒天パッド上の線虫の配向の微調整

C. elegansの基礎研究のためのメンテナンスについて

の軸索再生を研究するために、低予算レーザー軸索切断システムの構築 C.エレガンス

要約

さらに動画を探す

この動画の章

Cの生体レーザー軸索切断にエレガンス

ライブゼブラフィッシュの胚における二光子軸索切断とタイムラプス共焦点イメージング

小脳軸索のレーザーNanosurgery

再生に関する末梢および中枢神経系を研究するため生体内でショウジョウバエ損傷モデル

インビボカエノハブディティス・エレガンスニューロンにおける微小管動態と向きの評価

創傷応答のためのスキン傷つけ処理のための方法およびアッセイ

イメージング神経再生のためのチャネル寒天パッド上の 線虫 の配向の微調整

イメージング神経再生のためのチャネル寒天パッド上の 線虫 の配向の微調整

イメージング神経再生のためのチャネル寒天パッド上の 線虫 の配向の微調整

C. elegansの基礎研究のためのメンテナンスについて

イメージング神経再生のためのチャネル寒天パッド上の線虫の配向の微調整

イメージング神経再生のためのチャネル寒天パッド上の線虫の配向の微調整

イメージング神経再生のためのチャネル寒天パッド上の線虫の配向の微調整