Neuronal microsurgery with an Yb-doped fiber femtosecond laser

Abstract

Laser microsurgery allows the user to ablate cell bodies or disconnect nerve fibers by using a laser microbeam focused through a microscope. This technique was pioneered in C. elegans where it led to exciting discoveries in the fields of development and neurobiology. All neurons studied so far in C. elegans can regenerate and regrow axons and dendrites after injury, allowing studies of the molecular and cellular basis of neuroregeneration. In this chapter, we describe how to assemble and operate a platform for Yb-doped fiber laser microsurgery. The novel laser setup described here is a more robust, lower cost, and user-friendly alternative to other femtosecond-pulsed laser systems.

1. Introduction

Ablation of cells or cell parts such as nerve fibers with a laser microbeam has proven to be a useful tool to address questions of necessity and regeneration in multiple fields. Laser microsurgery has been widely used in C. elegans since it was first described by John White[1] in 1980 and was instrumental in multiple discoveries such as details of cell-cell interactions[1], the elucidation of the touch sensitivity circuit[2], and the role of GABA in C. elegans nervous system[3]^. More recently, in 2004, Yanik[4] showed that some C. elegans axons are capable of regenerating, which established C. elegans as a model for regeneration studies. Further, the technique can also help identify specific behaviors associated with injury in addition to providing insight about factors that affect regeneration [5, 6].

Laser microsurgery platforms have evolved significantly since the 1980s, most modern laser ablation platforms are one of two kinds, either nanosecond or femtosecond lasers[7]. Nanosecond lasers are more suited for cell ablation experiments while femtosecond lasers can be used for both cell ablation and axotomy as they can be adjusted to generate large areas of damage or to be precise and dissect subcellular structures as the laser beam can be focused onto a point in the sample with minimal damage to surrounding tissues[8]. This difference is mainly due to the million-fold decrease in pulse width increasing the required energy per pulse by the same ratio. While femtosecond laser pulses are typically in the order of tens of nanojoules (nJ), nanosecond pulses are usually in the order of tens of millijoules (mJ). Because excess energy diffuses away from the injury spot, nanosecond-long pulses generate more damage surrounding the injury[6].

Most commonly used femtosecond lasers in the field are Ti:Sapphire lasers which produce near-infrared (NIR) pulses with energies up to 50 nJ, a center wavelength of approximately 800 nm, pulse duration of 100–200 femtoseconds, and repetition rates of 80 MHz[9]. However, Ti:Sapphire lasers need constant maintenance, are very susceptible to room environmental conditions such as high humidity and tend to have a very high cost. In this chapter we describe how to assemble an operate a laser microsurgery platform using a Yb-dope fiber laser. The advantages of this type of laser are higher possible power, lower maintenance, smaller footprint, and air cooling [10].

The specific Yb-fiber system described here (BlueCut, Menlo Systems GmbH, Germany) which generates ~400 fs pulses in the infrared (1030 nm) includes an internal pulse picker which simplifies set up and lowers the overall cost compared to Ti:Sapphire systems that require an external pulse picker. Further, Yb-fiber system can ablate with user-defined repetition rate of single shot to 50 MHz and pulse energies of nJ to μJ. We have recently demonstrated that our novel setup shows comparable microsurgery results to those obtained with the Ti: Sapphire systems[10] without its disadvantages.

2. Materials and Reagents

2.1. Microscope assembly

1. Yb-fiber system (BlueCut, Menlo Systems GmbH, Germany)

2. 4 circular mirrors: 2 for alignment and 2 for periscope

3. Optical rails and base

4. 10X Achromatic Galilean Beam Expander, AR Coated: 650–1050 nm (can be replaced by a Galilean pair and optical rails)

5. Microscope (e.g. RAMM configured by Applied Scientific Instrumentation)

6. High NA Objective with high IR transmission (e.g. Olympus UAPO 40×, 1.35 NA)

7. Immersion Oil

8. 750 nm long-pass dichroic Mirror

9. Dichroic mirror cube holder

10. sCMOS Camera (e.g. Flash4.0, Hamamatsu)

11. IR cut-off filter (that prevents 1030 nm from hitting camera, e.g. Chroma ET750sp-2p8)

12. LED light source (e.g. LED 120, X-cite)

13. 4×8 passive optical table

14. Power and Energy Meter Console with S170C Microscope Slide Power Sensor

15. NIR detector card (VRC4, Thorlabs)

16. Alignment plate

17. Laser safety goggles for 1030 nm

18. Beam blockers

2.2. Laser Microsurgery

1. Immobilization Solution: Pluronic F127 36%, Tetramisole 1μM (or similar anesthetic). See note 1

2. #1 Rectangular Microscopy Coverslips

3. Dissection Microscope

4. 60 mm petri dishes for strain maintenance

5. NGM-Agar [11] 3 g of NaCl, 2.5 g of peptone,20 g of agar,1 mL of cholesterol (5 mg/mL in ethanol), 1 mL of 1 M CaCl2, 1 mL of 1 M MgSO4, and 25 mL of 1 M (pH 6.0) KPO4 to prepare 1 L

6. OP-50–1 bacteria (GCG)

7. Transgenic C. elegans strain (e.g. NW1229 pan-neuronal GFP expression, CGC)

3. Methods

3.0. Laser Safety

Lasers are dangerous and should always be operated with caution. This laser is a class 4 laser which means it is hazardous for eye exposure and that it can burn through skin and some materials at close range and should be handled with extreme care. Always use the minimal energy and number of pulses that produce the required lesion. Before using the laser, it is very important to receive laser safety training. The laser manufacturer can provide further guidance.

In addition, beam blockers should always be in place even when the laser is off and only removed when performing experiments. When the laser is on, safety goggles should be worn at all times and the user’s hands should be free of any reflective surfaces such as jewelry or watches. “Laser in use” signs should be displayed on the door outside the room where the laser is setup to prevent people without appropriate protection from coming in.

3.1. Microscope and laser setup (Fig 1A).

Figure 1. Laser setup and alignment.

A) Laser setup. BlueCut laser source (a) is fastened to an optical table. The beam is directed in free space to aligning mirrors (b), a pair of periscope mirrors (c and d), and a beam expander (e), to fill the back focal aperture (f) of the microscope (g) and onto the dichroic mirror (h). B) Black Slide test. The laser damage spot should be circular and the focus of the microscope is adjusted in order to get defined and crisp edges. A circular region of interest is selected with the white lesion in its center. Scale bar = 5 μm

1. Set up optical table and secure the laser system so that the beam is aligned with the center of the table.

2. Secure all connection between the laser console, the AOM seed, and the computer, and make sure that no cables are in the path of the laser beam.

3. Set up the microscope, the LED fluorescent light source and the camera at the other end of the table. See Note 2

4. Install the IR cut-off filter to avoid damage to the camera.

5. Assemble the periscope and align it to direct the laser through the beam expander into the back focal plane of the microscope at the side of the filter cube.

6. Turn the laser on at a very low power setting and in continuous mode and using the NIR Detector Card to observe the path of le laser beam secure the small circular mirrors to the table so that their configuration allows to direct the beam to the middle of the lower periscope mirror. See Note 3

7. Adjust the angle of the lower periscope mirror to center the laser beam on the upper periscope mirror.

8. Set up the dichroic mirror in the dichroic mirror cube holder. See Note 4

9. Attach the dichroic mirror to the top of the microscope’s fluorescent filter turret.

10. Using the optical rails from the periscope attach the beam expander so that the beam expander optics are close to the back focal opening of the microscope.

11. Turn the laser in a low power setting again and using the NIR card adjust the iris on the beam expander so that the entire back focal aperture of the microscope is illuminated.

12. Proceed with alignment procedures.

3.2. Laser Alignment

3.2.1. Alignment process

Alignment is probably the most dangerous process when working with a laser as the user is adjusting, moving mirrors and introducing objects in the laser beam path. Please read section 3.0 before starting alignment. See Notes 5 and 6

1. Turn on the laser in continuous mode at a low power setting and using the NIR card adjust the circular mirrors to hit the center of each mirror, starting from the one closest to the laser and up to the upper periscope mirror.

2. Use the alignment knobs and the alignment plate, placed on the rails between the periscope mirror and the beam expander, to adjust the mirrors further so that the laser beam is exactly at the center of the optical frame before the beam expander. Move the beam with the lower mirror when the plate is farthest from the microscope, and with the upper mirror when it is closer to the microscope.

3. You might need to repeat steps 1–2 several times as adjusting one mirror might alter were the laser hits the others.

4. Adjust the beam expander so that the back focal aperture of the microscope is fully illuminated. The expanded beam should be as collimated as possible. You can test this by removing the microscope and verify that the shape and size of the expanded beam does not change over some distance.

5. Proceed to fine alignment using the “Black slide test” (See 3.2.2) with the laser to pulse mode.

6. After optimal alignment has been reached, measure laser power with the power meter and record the number. Measuring power before every experiment is a good practice and can also help determine if the laser is in need of alignment.

3.2.2. Fine alignment test: “Black ink slide”

1. Cover a #1 rectangular coverslip in black ink using a black permanent marker and allow the ink to dry for a few minutes.

2. Place the coverslip under the microscope with the ink side facing away from the objective and focus on it. Use immersion oil for the high NA objective and use the microscope in well-Köhlered brightfield mode. See Note 7

3. Starting with a low power setting fire the laser and keep increasing power until a white spot is visible on the slide.

4. Keep doing this and adjust the microscope focus and the setting of the beam expander until the white spot is the smallest possible size, looks circular and has defined and crisp edges (Fib 1B).

5. To be able to aim the laser for microsurgery, use ImageJ to draw a circular Region of Interest (ROI), with the laser spot in the center of the circle and save the ROI so it is easily available (Fig 1B). See Note 8

3.3. Laser Microsurgery

1. On the day before performing the laser microsurgery, pick animals of the desired age and transfer them on to a new, seeded NGM plate. See Note 9

2. Before starting the experiment turn the laser on, make sure everything is working appropriately, perform the black slide test, and open the saved ROI and adjust it if needed (See section 3.2.2).

3. Fill a bucket with ice and place an empty tip-box lid on top of the ice making sure the flat surface is level.

4. Mix Pluronic F127 36% solution with enough concentrated Tetramisole 1 mM solution to dilute to 1μM Tetramisole in a 1.5 ml tube and keep the tube in ice. See Note 10.

5. Place 1 rectangular coverslip on top of the tip box and wait a minute until the surface of the coverslip is cold, add 25 μL of the Pluronic + Tetramisole mixture to the coverslip surface. See Note 11

6. With a pick transfer the animals onto the drop of Pluronic + Tetramisole mixture and gently press another coverslip on top until the liquid between the coverslips is evenly spread. See Note 12

7. Remove the coverslip from the ice and wait until the liquid mixture become solid or the glass does not feel cold to the touch anymore.

8. Using a dissection microscope make sure that the animals are present between the coverslips and that the animals are healthy. See Note 13

9. Place the coverslip in the microscope and secure it as if it were a slide. See Note 14

10. Using the microscope software and adjusting the focus find the target neuron or cell. See Note 15.

11. Once you locate the target cell, select the injury spot by placing it in the middle of the ROI.

12. Put on the laser safety goggles, remove the beam blocker from the laser opening and press the pulse key to fire the laser. The injury should be immediately visible. If the injury is not visible, increase laser power slightly and try again. See Note 16.

13. Take before and after images of the injury site (Figure 2A).

14. After all the desired cells have been injured remove the coverslip from under the microscope and move it back on the ice.

15. After 5 minutes on the ice slowly separate both coverslips being careful not to break them. Once both sides are separated, place them under the dissection microscope to find the animals.

16. With a pick carefully remove the animals from the Pluronic + Tetramisole mixture which should have the consistency of a gel and place them into individual NGM plates.

17. After 24 hours or the desired time for your experiment, repeat steps 2–9 to mount the animals between two coverslips again and image the injury site (Figure 2A).

Figure 2. C. elegans neurites regrow following microsurgery with a Yb-doped fiber laser.

A) C. elegans axotomy. Commissures of GABAergic (D type) motoneurons injured in immobilized animals were found again on the next day to assess regeneration. Arrows indicate the sites of axotomy (red arrow) and regenerating branch (green arrow). B) Example of measuring the sizes of the injury size and outgrowth. Size of injury is measured between the two retracting branches. Outgrowth is measured from the site of injury to the tip of the regenerated branch. Scale= 10 μm

3.4. Data Analysis

1. The specific assay and statistical test depends on your scientific question For example, you can score the number of neurons that regenerated or cells that survived by looking at images taken 24 or 48 hours after injury. In that case, a Fisher’s Exact test is appropriate.

2. For continuous values, such as extent of neurite regeneration, measure the initial injury size as well as outgrowth with a software such as FIJI (is just ImageJ v.1.53c [12, 13]). (Fig 2B) See Note 17. In this case, we suggest to visualize the data in shared-control Gardner–Altman plots, and to calculate the p-value by two-sided permutation t-test (Estimation Statistics;[14]), or to compare among treatment groups with an ANOVA test.

4. Notes

1. Mix Pluronic F127 to 36% in distilled water by gradually adding the powder to a solution kept at 4°C and stirred continuously. This can take a few hours. Do not let the solution warm up because it will solidify. Aliquot and keep at 4°C. Prepare a concentrated Tetramisole 1 mM solution and use the Pluronic F127 36% solution to dilute to 1 μM

2. We prefer a custom-made microscope without eyepieces for the obvious safety advantage. The suggested platform can be set up with any epifluorescence microscope that can accommodate a second dichroic mirror in its light path.

3. The laser beam should always hit the center of every mirror.

4. The dichroic mounting adapter directs the laser beam into the objective lens without interfering with the normal optical paths of the microscope.

5. For safety and efficiency reasons, we recommend that two people align the laser.

6. It is not reasonable to try and comprehensively cover laser alignment in this chapter. Your institution might have laboratories that are proficient in laser alignment or you could ask the vendor for advice.

7. Scoring a line or a grid on the ink with a sharp object might help with focusing.

8. The location of the ROI might need to be adjusted or at least checked with a black ink slide before every experiment.

9. For adult axotomy pick L4s the day before, it is easier to perform axotomy when the animal has few or no eggs.

10. It is very important to make and keep the Pluronic F127 36% solution at 4°C or below as it will become solid at Room temperature.

11. Try to avoid bubbles.

12. Four to five animals per coverslip works best.

13. You can use a thin permanent marker to draw a circle around each animal to make it easier to find under the microscope. You will use immersion oil so mark the coverslip that will be away from the objective and draw large circles.

14. If needed, you can place the coverslips on a glass slide. Mounting the animals between two coverslips allows optical access from either side of the animal more easily. We routinely lesion commissure neurites on the far side of the animal but they can end up closer to one coverslip or the other.

15. If epifluorescence is necessary to find the target cell, keep the shutter open as little as possible to avoid bleaching.

16. Always begin with a low laser power and slowly increase it until the injury is visible.

17. In case there is more than one branch in the regenerated neurite, sum the values.