Electrode-Fabrication

Detailed step-by-step instructions for fabrication of Stereotrodes & EMG electrodes

https://kl-turner.github.io/Electrode-Fabrication/

Materials

Equipment

Other: ruler, razor blades, standard office scissors, any kind of tape, wire/solder for test equipment, NaCl, beaker, Parafilm, coffee, etc etc.

Instructions

The following instructions outline the step-by-step process for fabricating tungsten stereotrodes and seven-strand stainless steel EMG electrodes.

• [A] Unravel a small amount of the desired wire. I typically use ~ 1 inch (25 mm) as it makes handling the wire significantly easier.

• [B] Gather as many pieces of wire as you would like, the number of wires (N) is equal to N/2 electrodes. I typically make 20+ wire + pins before assembling the electrodes. You want to your wires to be as straight as possible throughout the entire process, so discard any wire at the end of the spool that is irreparably bent.

• [C] Use a pair of fine-tip forceps to strip ~ 3 mm of the Teflon coating off one end of each wire. It does not matter which side. You want to strip off just enough coating so that the bare end fits fully into the gold pin, but does not extend significally past the end of the opening. Careful when stripping stranded wire, as you do not want to separate the strands.

[A] Cutting wire	[B] 2 wires per electrode	[C] Stripping off coating

• [D] Shake the bottle of colloidal silver (CS) very well. The CS is electrically conductive, allowing current to pass from the wire to the pin. It starts off as a liquid, but quickly dries leaving the conductive particles. Gather up your stripped wires, some gold pins, and a crimper tool. It’s nice to work in a well-ventilated area under a fume extractor swing-arm, ventilation table, or fume hood.

• [E] Hold a gold pin with the forceps and apply a small amount of CS to the opening. It helps to use the brush to lightly work the CS liquid into the pin hole.

• [F] Insert the stripped end of the wire into the gold pin opening. I do this by holding the wire vertically and placing the gold pin upside down on top of the wire.

• [G] Set the forceps down and pick up the crimping tool. Still holding the wire vertically with the gold pin balancing on top, insert the gold pin into the crimping tool and crimp the pin 2-3 times. When finished, gently tug the wire to ensure that it is secure. If the wire comes out, toss the pin and try again with a new one. If the wire is secure but the gold pin is severely cracked open or fragile, pull out the wire and re-do it. The last thing you want is a fragile gold pin breaking in half after you’ve already completed a successful surgery and have the animal is ready to be imaged. Speaking from experience.

• [H] Repeat the process until you’ve collected at least twice as many completed wire + pins as the number of electrodes you desire. As mentioned before, I typically make dozens of these at once. Often times I make several dozen on slow days and keep them in a labeled container for later assembly. Be sure to label the container(s), as the stainless steal and tungesten wires look nearly identical unless under a microscope.

[D] CS and crimpers	[E] Apply CS to gold pin	[F] Insert wire into pin	[G] Crimp gold pin	[H] Repeat as necessary

The remaining steps are easier when done under a microscope, but can be done without one.

Using a microscope makes fabrication significantly easier

• [I] Cut the polyimide (PI) tubing into the appropriate lengths. For each tungsten stereotrode, I cut two pieces - one at 1 mm in length, and the other at 3 mm in length. For each EMG electrode, I cut two pieces - both 3 mm in length. Careful when cutting the tubing, as it tends to go flying after cut with a razor blade.

• [J],[K] While cutting the PI tubing, I find it extremely useful to adhere the small pieces to a piece of tape. This not only prevents the small, static-clinging pieces from sticking to your forceps and gloves, it stabilizes them for easy wire-insertion.

• [L] Use the forceps to insert two tungsten wires first into a single 1 mm piece of PI tubing. This can be extremely frustrating to do by eye, but is incredibly easy when using a microscope with just a small amount of magnification. The fit of the two wires inside the tubing will be tight. For EMG electrodes, insert one single wire into a single piece of 3 mm tubing. The fit for these wires will be very loose, but they serve an important purpose discussed later.

• [M] Lightly lift the tape and slide the PI tubing further down the wires so that it doesn’t slip off. For the tungsten stereotrodes, insert the tips into a second piece of 3 mm tubing. After doing this, remove the wires/tubing from the tape.

[I] Measure PI tubing	[J] Cut tubing to length	[K] Apply pieces to tape	[L] Insert wires into tubing	[M] Insert into second tubing for stereotrodes

• [N] Cut equal pieces (to the # of wires) of heat-shrink tubing of approximately equal length. Anywhere between 3-5 mm is fine. I shoot for around 4 mm, but don’t actually measure these when cutting as you learn rather quickly what is too short/long by eye. I cut several pieces at once into a container, and then fish out two of approximately equal length for each individual electrode. The tubing serves to physically separate the two gold pins from shorting the circuit.

• [O] Gather your parts, as the electrodes are ready to assemble. EMG wires will be separate, stereotrode wires will be together.

• [P] Slide a piece of heat-shrink tubing onto each wire. For the EMG wires, this can be done before or after putting the pins into the headers. For the stereotrode wires, you need to put the tubing on first from the pin-side. You made need to slide the pieces of PI tubing back towards the top of the wires to separate the two wires. Be sure to not bend the tungsten wires.

• [Q] Insert the gold pins into headers. Use a pair of larger (not fine) forceps to do this, as it may require a little bit of gentle force. For this example, I have just a single pair of headers per electrode. When I make mine, I typically make 5 electrodes at once and have several electrodes spaced along a single 24-pin header. After the pins are inserted, use a pair of forceps to slide the pieces of heat-shrink tubing to the desired position (See photo). You want the tubing to slightly extend past the top of the gold pin.

• [R] Use a heat gun to shrink the tubing. Do not overheat the tubing, it only needs to be heated to the point that it shrinks to its final OD. If you do overheat the electrode, you risk melting and deforming the Teflon coating and the PI tubing. This makes moving the PI tubing during the finishing steps difficult to impossible.

[N] Cut shrink tubing	[O] Parts ready to assemble	[P] Tubing on pins	[Q] Pins into headers	[R] Heat shrink tubing

• [S] To finish up the EMG electrodes, align the electrode vertically in the helping hands. Slide the two pieces of PI tubing down as far as they will comfortably go. Ideally, they go all the way past the heat-shrunk tubing and stop at the end of the crimped gold pin. Do not worry if the two pieces are slightly uneven in length. The purpose of these long pieces of PI tubing on the single EMG wires is provide a little bit of reinforcement against the torsional forces on the wire connection during electrode implantation. The stainless steel wires are very flexible, and are typically bent/curved into the muscles during implantation. This PI tubing helps prevent the base of the wire connection from flexing too much and breaking. Unlike the EMG wires, the tungsten stereotrode wires are never bent, and are already inherently very stiff.

• [T] Apply a small amount of cyanoacrylate glue to the vertical EMG electrode. You want to get the glue inside the PI tubing and down inside the heat-shrunk tubing. Remember that this tubing is very loose without the glue, so in order for it to serve its purpose, this needs to be let to dry for 30-60 minutes. Careful not to apply too much glue that it drips down the side of the electrode. If the glue drops down and gets inside the pin/header connection, you will unlikely be able to remove the electrode from the header without destroying it.

• [U] After the “bottom” of the electrode is dry, flip it over and apply a light, even layer of glue over the entire electrode. You want to get glue on the top of the heat-shrunk tubing (it is not electrically conductive and will not short the pins), all along the sides, and can allow a small amount to drip back down to the portion already glued. Use a Kim-wipe or paper towel to dab away any excess amount at the bottom. The glue serves to secure the two gold pins together, which until now are really only being held together by the actual header, which will be removed upon data acquision.

• [V] For the tungsten stereotrodes, there is a bit of a finicky step that makes a world of difference during electrode implantation and data quality. This step can be easy or an absolute pain depending on the electrode, but is nonetheless incredibly important. You want the stereotrode wires to remain parallel to each other as you slide the smaller 1 mm piece of PI tubing towards the base. Naturally, the wires will want to cross each other the further down the electrode you slide the tubing. The tubing is not meant to go all the way down (See photo), but should be as far down as possible so that the height of the electrode off the mouse’s head is not excessive. The easiest way to do this step is under the microscope with a pair of dedicated “glue” forceps. The longer piece of PI tubing is meant to be kept towards the top of the wire and serves to help keep them together/aligned. Use the forceps to gently squeeze the wires together, but do not puncture the Teflon coating or you risk shorting. If the wires persistently cross each other as you slide down the PI tubing down, use the glue up and down along the length of the top of the wire where the 3 mm PI tubing is. Make sure the wires are straight before doing this, as there is no going back afterwards. The glue will seap inside the PI tubing and after 30-60 seconds create modest adhesion to keep the wires straight. Do not get any on the smaller piece yet. After a brief wait, now gently slide the forceps up and down along the drying glue (re-apply more as necessary) and slide the smaller piece of PI tubing down with glue along your forceps. The wires should be less resistant to crossing at this point due to the glue on the top PI tubing, but will still require patience. Every time the wires cross, move the tubing back up, gently squeeze the wires together to straighten them, and slide the tubing back down. Over minute(s) as the glue dries, you should reach a point where the tubing is getting more difficult to slide up/down, but the wires should be better staying together. This is tedious, however, if you can keep the wires straight it will drastically improve your results. If you let the wires cross, they almost always separate from each other by several millimeters, either immediately or during trimming. While the (crossed) wires may appear to be glued together straight, the outward forces from the crossing causes the wire tips to separate as soon as the wires are trimmed to the shorter length and the long piece of PI tubing is removed. In my experience, 25% of electrodes require no extra work and are perfectly straight. 50% require a small amount of work little-to-no extra glue to get the wires straight. 25% are a 4-5 minute pain, but maybe one electrode in ten (total) just being tossed at this point due to the inability to prevent the wires from crossing. The benefits of having perfectly straight, parallel electrodes are reduced damage to the brain during implantation and improved localized data due to the tips being closer together. If the tips are separating during implantation, they typically end up several millimeters apart once in their final position.

• [W] If you made it this far and have survived (or given up) keeping the wires from crossing, flip the electrode over and apply the thin layer of glue identical to the EMG electrode outlined in [U]. For the stereotrodes, I prefer to allow a decent amount of glue to drop down to the bottom creating a sort of upside-down triangle from the heat-shrunk tubing down to the small piece of PI tubing. When it dries, this extra glue provides a noticeable amount of stability to the base of the electrode wires that helps prevent them from bending.

[S] Align EMG veritcally	[T] Glue PI tubing on top	[U] Flip and glue rest of EMG	[V] Straighten stereotrode wires	[W] Flip and glue rest of stereotrode

Allow the electrodes to dry overnight. After they are dry, the wires can be trimmed to the desired length.

Testing Electrode Impredence

Before using the electrode(s) during surgery, it is imperitive to check that each wire has a good electrical connection and that they are not shorted together. We can quickly do this by testing the impedence of each wire. I prefer to not trim the electrode wires to the desired length until the day of the surgery, testing the electrodes before starting.

Remove the necessary adapter wires from the Drew Lab Thorlabs box next to the meter, and plug them in to the MicroProbes impedance tester.

MicroProbes impendance tester

Make sure the REFERENCE pin is connected to the bath of 3M NaCl, and the ELECTRODE pin is connected to your desired electrode. Turn the meter on, and it should automatically jump to the right (max impedence). Depending on application, you can adjust the RANGE, but typically 500kΩ is enough for most of our applications. Attach the electrode the header of the adapter cable. If your electrode is already in headers, you can simply connect the headers to the headers on the adapter. It does not significantly change the impedance.

Testing each wire	Testing for shorts	Meter reading

Test the wire impedance by dipping the electrode tip into the NaCl beaker. The meter reading should move to between 70 and 250 kΩ (@1 kHz) for the tungsten, and around 20-150 kΩ for the stainless steel. After testing one side, flip the headers around and test the other wire. If you do not get a good reading from either wire, do not use the electrode. To check for shorts between the two wires, remove the copper clamp from the NaCl beaker and attach it to the exposed header pin. This meter should not move from max - as we do not want current to pass between the two electrodes. If you do get a reading, make sure that there’s no wet NaCl shorting the stereotrode tips, as a small amount of the salt water solution will (temporarily) complete the circuit. If the tips are dry and still read an impedance, do not use the electrode.

If you did not remove the headers when testing the electrode: remove it once before surgery to ensure that no glue got inside the header, as this will make it nearly impossible to remove for data acquisition. Keeping the header on to test the impedence is fine, but it should be removed when recording data. The header to pin connection is tight (which is what is used to connet to the differential amplifier), but the head to header connection is not, and will lead to bad data especially during running/movement events regardless of how stable the stereotax is. Keep the header on during surgical implantation, remove it during imaging, but replace it back on when finished imaging before freeing the mouse from the head-bar holder.

When finished, turn off the meter. Re-cover the NaCl beaker with Parafilm. Put the ELECTRODE connectors/wires into the Drew Lab box. The REFERENCE connector and copper clamp can stay out and connected to the NaCl beaker.

For detailed imformation on signal amplification and acquisition, see https://github.com/KL-Turner/LabVIEW-DAQ

For data analysis, check recent publications at https://sites.esm.psu.edu/~pjd17/Drew_Lab/Publications.html