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

互联网2013-11-13

1194

实验材料

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

实验步骤

1. Construction of a laser ablation system

Wear laser safety goggles and use good laser safety practices during the initial alignment. Never look through the oculars when the laser is on.

1) Arrange the components on the breadboard as shown in Fig. 1 (see also example² ). Bolt down the laser (with a riser plate if needed), periscope post, and the elevated rail supports.

2) Position your microscope to align with the rail axis. Align with the transmitted light beam from the microscope condenser lens. Align the rail height (use a level) and position with an adjustable aperture or a lens affixed to the rail. The aperture or lens must be aligned at both ends of the rail to the condenser beam. Lab jacks can help with the rail height adjustment.

3) Align the Glan-Thompson polarizer and half-wave plate to the laser beam. You do not need the laser on to do this alignment. The beam just needs to go through both without hitting their edges. Rotate the polarizer to provide a convenient orientation and position for the beam dump (fig.1). You will rotate the half wave plate to adjust laser power. Secure the components to the breadboard.

4) Turn on the laser, set the pulse frequency to 100, continuous mode, and reduce power to a minimum using the half-wave plate. Use a Post-It note or lens paper to visualize the laser beam.

5) 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. The laser beam should be aligned roughly to the center of the mirrors. The mirrors should be oriented roughly 45 degrees to the laser beam axis (fig.1).

6) Coarsely adjust the kinematically mounted mirror on top of the periscope and on the end of rail to align the laser beam to the center of the transmitted light beam from the microscope. Both the laser beam and the transmitted light beam from the microscope can be simultaneously visualized by inserting paper in the beam path. Make sure any ND filters and apertures in the intermediate module are out or fully open.

7) Finely align the laser beam to the center of the transmitted light beam with the fine adjustments on the top periscope and rail mirrors. Hold the paper close to the rail mirror and align the laser beam to the center of the transmitted light beam with the top periscope mirror. Hold the paper near the microscope port and align the laser beam to the center of the transmitted light beam with the rail mirror adjustments (fig.2).

8) Position a Post-It note in the beam path between the aligned condenser lens and the open objective turret. You should see the transmitted light beam with the smaller laser beam near the center. Use the fine adjustments on the rail mirror to center the laser beam. Fix the microscope in place with clamps.

Steps 1.9-1.12 are optional, but it is easy and useful to assess alignment and laser ablation before adding the beam expanding lenses.

9) Turn off the laser and engage the mechanical safety shutter. Rotate the 50% dichroic out of the beam path. Even though no direct laser light will pass to the oculars, the reflected light can be quite intense. Mount the target slide for the fine alignment and image with the 60X oil objective. Focus on scratches on the slide. Put in the ND4 and ND8 filter in intermediate module.

10) Image the target slide with your CLSM, Spinning disk, or CCD camera system. Rotate the 50% dichroic into the laser path. From this point, you should never look through oculars while the ablation laser is on. Turn on the ablation laser and set the mode to triggered, frequency to 100, and pulse number to 10. Make sure the power is still set to minimum with the half-wave plate and then open the mechanical safety shutter. You can simultaneously image and use the ablation laser.

11) While imaging the target slide, trigger the ablation laser. Check the target slide for an ablation spot 1-10 um in diameter. Sequentially remove ND filters until an ablation spot is seen. Adjust the ablation spot to the center of the image with the rail mirror. You will want to add back a ND filter to have room to finely increase laser power with the half-wave plate to give a <1 um ablation spot. Image at 0.2 um/pixel or less and finely adjust the ablation spot to the image center (position 256,256 with 512X512 image).

12) Check the depth of focus and axis of the laser beam. Focus up and down 1-2 um and check the ablation position and ablation spot size. If the laser beam is axially aligned the ablation spot will not move. The unconditioned laser beam will distribute power over several um (about 3-5 um from top to bottom of focus).

2. Add lenses to expand the laser beam to fill the objective back aperture and adjust convergence to control focus

1) This system uses dual Galilean beam expanders to expand the laser beam to fill the back aperture of the objective (10 mm). Mount the 4 lenses on to carriers and attach them to the rail (L1f1 L2f2 and L3f1' L4f2'). Adjust the positions so that all lenses are at the same optical axis and exactly orthogonal to the laser beam (fig. 2).

2) Turn on the laser and adjust it to minimum power and continuous mode. Roughly adjust the beam alignment through each lens using paper to see the laser beam and transmitted light beam from microscope condenser. Turn off and shutter the laser.

3) Remove the clamps from one side of the microscope and slide the microscope out of the laser beam path. Turn on the laser and remove safety shutter.

4) The fine alignment of the lenses and the laser beam can be performed by viewing the expanded laser beam on a nearby wall. The beam should expand and contract symmetrically when the lenses are moved. Adjust the last two kinematically mounted mirrors to align the beam (fig.3).

5) The aligned and expanded beam profile should be circular and of uniform brightness (fig.3). The size and uniformity of the beam can be viewed with a Post-It note at the estimated position of the objective back aperture.

6) The beam should be adjusted to zero convergence for infinity optical systems. Convergence can be estimated by noting how the beam size changes as you move a Post-It note farther from lenses. Turn off the laser and engage the mechanical shutter.

7) 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 microscope, but do not tighten down.

8) Turn on the laser and remove the safety shutter. Rotate the objective turret to an open position. Place a Post-It note on the stage. You should see the expanded and uniform laser beam centered on the transmitted light beam from the condenser (fig.4). You may need to center it by loosening the microscope clamps and carefully nudging the microscope.

9) Engage the safety shutter, and set the laser to trigger mode. Rotate the 60X objective in place and image the surface scratches on the target slide.

10) Remove the safety shutter, trigger the ablation laser and adjust the laser power for the minimum ablation spot. The ablation spot should be circular and within 5-10 um of the image center.

11) Center the ablation spot with fine adjustments of kinematically mounted rail mirror. You will need to iteratively adjust the rail mounted and top periscope mirror to both center the ablation spot and maintain a uniform beam profile.

12) Evaluate the Z focus of the laser by moving the focus systematically up and down in 1 um steps and triggering the ablation laser. The expanded, aligned, and convergence adjusted beam should ablate maximally at the image focus and weakly or not at all 1 um above or below focus (fig 5).

13) Adjust the Z focus of the expanded beam by moving lens L3 of the Galilean telescope (fig.2).

3. Laser axotomy and time-lapse microscopy of axon regeneration

1) Microwave 10% Agarose (RPI Molecular Biology Grade EEO 0.1 A20090) in balanced saline till fully melted. Set on a hot plate to keep it melted during use for mounting.

2) 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).

3) A "Sharpie" marker cap is used to cut out a uniformed diameter circular pad of 13mm.

4) 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 (fig.6).

5) The ablation laser is aligned to crosshairs, as described above, at the beginning of each session. Engage the laser safety shutter.

6) Remove the laser alignment target and image a mounted adult worm with a suitable fluorescent marker in the target neurons. Image axons at a similar magnification (0.2 um/pixel or higher magnification) and position under the crosshairs (fig.7).

7) Open the laser safety shutter and trigger the ablation laser while imaging. Evaluate the axon after triggering the laser and slowly increase laser power until the axon is cut. You will need about 10X more laser power to cut axons than to form an ablation spot on the alignment target slide (about 1uJ/ ns pulse). We typically cut with 100 pulses at 2.5 kHz and power set to about 0.27mW (average power measured at specimen with Coherent FieldMaxII power meter and laser set to continuous at 2500 Hz). The ablation laser power setting will be consistent for cutting axons in future experiments (fig.7).

8) A successfully cut axon will show a break of about 0.5-1 um without a loss of brightness in the two cut ends (fig.7). A large loss of brightness or a large gap (2-10 um) often signifies extensive damage beyond the axon by a cavitation bubble. The proximal and distal axons will separate and form retraction bulbs over the next 30 minutes (fig. 8 and Movie).

9) Equilibrate your mounted worms with your microscope stage for 30 minutes before starting your time lapse recording. This will minimize drift due to temperature differences and agarose contraction.

10) ) Time-lapse imaging parameters are set up using the imaging software associated with your system. We typically image 10-20 Z steps (1um/step) at 0.1-0.2 um/pixel using gain, pinhole, scan rate, and imaging laser power settings that minimize exposure while giving the desired spatial resolution. Sampling intervals are typically 1-5 minutes over 15 hours depending on the desired temporal resolution (fig. 8 and Movie).

11) Time-lapse image data are converted into movies using NIS Elements or ImageJ.

4. Representative Results:

Laser axotomy using this system is a reliable and routine. The results shown in figure 7 are typical. Time-lapse imaging of axon regeneration is very robust using this protocol. We routinely cut and image up to 5 axons in 5 different worms on a single slide using a motorized stage. The only limitation is the time it takes to collect the images of each axon, e.g., if it takes 20 seconds to collect a stack for an axon then at most you can sample 9 axons (9 stacks) if you are sampling every 180 seconds. The example shown in Figures 8 and 9 is a good representative result. About 10% of experiments give results of this quality. The remaining experiments provide good data on the regeneration phenotype, but are esthetically unappealing because of small jittering movements of the worm that generally begin after 5-8 hours of immobilization.

1. Hammarlund, M., Nix, P., Hauth, L., Jorgensen, E.M., & Bastiani, M. Axon regeneration requires a conserved MAP kinase pathway. Science. 323, 802-806 (2009).

2. Steinmeyer, J. D. et al . Construction of a femtosecond laser microsurgery system. Nature protocols. 5, 395-407, doi:10.1038/nprot.2010.4 (2010).

3. Wu, Z. et al . Caenorhabditis elegans neuronal regeneration is influenced by life stage, ephrin signaling, and synaptic branching. Proc Natl Acad Sci. U. S. A. 104, 15132-15137 (2007).

4. Gilleland, C.L., Rohde, C.B., Zeng, F., & Yanik, M.F. Microfluidic immobilization of physiologically active Caenorhabditis elegans. Nature protocols. 5, 1888-1902, doi:10.1038/nprot.2010.143 (2010).

5. Raabe, I., Vogel, S. K., Peychl, J., & Tolic-Norrelykke, I. M. Intracellular nanosurgery and cell enucleation using a picosecond laser. J. Microsc. 234, 1-8, doi:JMI3142 [pii] 10.1111/j.1365-818.2009.03142.x (2009).

6. Hutson, M. S., & Ma, X. Plasma and cavitation dynamics during pulsed laser microsurgery in vivo . Physical review letters. 99, 158104 (2007).

7. Venugopalan, V., Guerra, A., 3rd, Nahen, K., & Vogel, A. Role of laser-induced plasma formation in pulsed cellular microsurgery and micromanipulation. Physical review letters. 88, 078103 (2002).

8. Bourgeois, F., & Ben-Yakar, A. Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans . Opt Express. 16, 5963 (2008).

9. O'Brien, G.S., Rieger, S., Martin, S.M., Cavanaugh, A.M., Portera-Cailliau, C., & Sagasti, A. Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos. J. Vis. Exp. (24), e1129, DOI: 10.3791/1129 (2009).

10. Tsai, P.S., et al . Plasma-mediated ablation: an optical tool for submicrometer surgery on neuronal and vascular systems. Curr. Opin. Biotechnol. 20, 90-99 (2009).

11. Chung, S. H., Clark, D. A., Gabel, C. V., Mazur, E., & Samuel, A. D. The role of the AFD neuron in C. elegans thermotaxis analyzed using femtosecond laser ablation. BMC Neurosci. 7, 30 (2006).

12. Shen, N. et al . Ablation of cytoskeletal filaments and mitochondria in live cells using a femtosecond laser nanoscissor. Mech. Chem. Biosyst. 2, 17-25 (2005).

13. Rao, G.N., Kulkarni, S.S., Koushika, S.P., & Rau, K.R. In vivo nanosecond laser axotomy: cavitation dynamics and vesicle transport. Opt. Express. 16, 9884-9894 (2008).

14. Rohde, C.B. & Yanik, M.F. Subcellular in vivo time-lapse imaging and optical manipulation of Caenorhabditis elegans in standard multiwell plates. Nat. Commun. 2, 271, doi:10.1038/ncomms1266 (2011).

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