Our purpose was to provide an updated, easy-to-follow guide on the fabrication and testing of epimysial electromyography electrodes. To that end, we provide instructions for material sourcing and a detailed walkthrough of the fabrication and testing process.
Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to measure EMG signals in preclinical models. Although classical resources exist describing the principles of epimysial electrode fabrication, there is a sparsity of illustrative information translating electrode theory to practice. To remedy this, we provide an updated, easy-to-follow guide on fabricating and testing a low-cost epimysial electrode.
Electrodes were made by folding and inserting two platinum-iridium foils into a precut silicone base to form the contact surfaces. Next, coated stainless steel wires were welded to each contact surface to form the electrode leads. Lastly, a silicone mixture was used to seal the electrode. Ex vivo testing was conducted to compare our custom-fabricated electrode to an industry standard electrode in a saline bath, where high levels of signal agreement (sine [intraclass correlation – ICC= 0.993], square [ICC = 0.995], triangle [ICC = 0.958]), and temporal-synchrony (sine [r = 0.987], square [r = 0.990], triangle [r= 0.931]) were found across all waveforms. Low levels of electrode impedance were also quantified via electrochemical impedance spectroscopy.
An in vivo performance assessment was also conducted where the vastus lateralis muscle of a rat was surgically instrumented with the custom-fabricated electrode and signaling was acquired during uphill and downhill walking. As expected, peak EMG activity was significantly lower during downhill walking (0.008 ± 0.005 mV) than uphill (0.031 ± 0.180 mV, p = 0.005), supporting the validity of the device. The reliability and biocompatibility of the device were also supported by consistent signaling during level walking at 14 days and 56 days post implantation (0.01 ± 0.007 mV, 0.012 ± 0.007 mV respectively; p > 0.05) and the absence of histological inflammation. Collectively, we provide an updated workflow for the fabrication and testing of low-cost epimysial electrodes.
Electromyography (EMG) is a powerful tool for studying the electrical activity of muscle. EMG recordings can be especially useful in preclinical animal models to assess the effectiveness of interventions to treat neuromuscular dysfunction. In these models, implantable biocompatible electrodes are commonly used to assess the neurophysiological interface between motor neurons and muscle fibers. These implantable electrodes can provide localized measurements of muscle excitation and can be diverse in terms of their configuration, shape, and material, with the optimal design ultimately dictated by the location and intended use.
Despite their suitability for assessing muscle excitation in preclinical models, the use of epimysial electrodes can be limited by cost. As a result, many investigators use custom-fabricated epimysial electrodes that are produced in-house. Although resources exist detailing the fundamental considerations of electrode fabrication, testing, and use1,2, there is a need for an updated instructional guide detailing the sourcing, fabrication, and validation of epimysial electrodes using modern methods. Informed by the foundational works of Loeb and Gans3 and others in electrode theory, we present modern instructions on the sourcing and fabrication of low-cost epimysial electrodes and test their performance in a series of ex vivo and in vivo experiments. The aim is to offer a user-friendly guide for others in the scientific community to source, fabricate, and test in-house low-cost epimysial electrodes for animal use, enabling the broader quantification of muscle excitation in preclinical models.
In this protocol, we provide an instructional guide to the sourcing, fabrication, and testing of epimysial electrodes for animal use in the modern electrophysiology laboratory. Electrode parameters chosen for fabrication, such as the shape, dimensions, contact surface area, interelectrode distance, lead length, etc., were selected to suit our experimental needs and were comparable to a commercially available industry standard epimysial electrode (see the Table of Materials). We encourage other groups to modify these parameters to suit their needs in addition to selecting a reliable industry standard electrode that matches their use case.
In an effort to give readers a relatively quick sense of electrode performance, we also provide an example of an ex vivo testing protocol with the option of measuring electrode impedance. Additionally, we give an example assessment of electrode performance in vivo. The ex vivo experiment compared the custom-fabricated electrode to an industry standard in a saline bath to mimic stable physiological conditions. Impedance was also assessed ex vivo via electrochemical impedance spectroscopy (EIS). The in vivo experiment consisted of the surgical implantation of the custom-fabricated electrode into the vastus lateralis (VL) muscle of a 16-week-old female Long Evans rat (HsdBlu: LE, Envigo) to measure the EMG signal during conditions known to elicit a high or low signal (uphill, downhill walking). To assess the reliability of the custom-fabricated electrode, EMG signaling was acquired during level walking following full surgical recovery and prior to sacrifice (14 days and 56 days post implantation, respectively). Hematoxylin-eosin (H&E) staining was conducted on the instrumented muscle to assess the biocompatibility of the custom-fabricated electrode.
The in vivo procedure was conducted under the approval of the Institutional Animal Care & Use Committee at the University of Michigan (IACUC approval #PRO00010765) and in accordance with the National Institutes of Health guidelines on the care and use of laboratory animals.
1. Electrode sourcing and fabrication
NOTE: Figure 1 provides a high-level summary of all key fabrication steps with a QR link that provides additional visual instructions.
Figure 1. Steps for electrode fabrication. (A) Perforate silicone base. (B) Form U-shaped foils using the folding jig. (C) Insert U-shaped foils into perforated silicone base. (D) Silicone base contains 12 foils to form 6 bipolar electrodes. (E) Apply surgical tape to the base to secure foils during removal from the cutting jig. (F) Remove silicone base from the cutting jig. (G) Denude PFA-coated stainless-steel wire and weld to the upright foil arm using a Micro TIG welder. (H) Align denuded wires, apply silicone-toluene sealant, and let set. (I) Divide the silicone base into individual electrodes and clean in an ultrasonic bath. Please click here to view a larger version of this figure.
2. Ex vivo testing
Figure 2: Ex vivo testing: Saline bath containing the custom-fabricated electrode, industry standard electrodes, two stimulating electrodes, and a ground source. Signal agreement and temporal synchrony were assessed by delivering sine, square, and triangle waves into the saline bath from the signal generator and recording the waveforms detected by the respective electrodes using a data acquisition platform. NOTE: Electrochemical impedance spectroscopy is not pictured. Please click here to view a larger version of this figure.
3. In vivo testing
NOTE: The in vivo testing procedure describes our experimental use case. It is recommended that custom-fabricated epimysial electrodes are tested in vivo in a way that matches the user's intended experimental conditions.
4. Biocompatibility testing
5. Suggested statistical analyses
Ex vivo performance
ICCs revealed high levels of agreement between the custom-fabricated and industry standard electrodes across all waveforms (sine [ICC = 0.993], square [ICC = 0.995], triangle [ICC = 0.958]; p < .001). Bland-Altman plots also revealed a high degree of signal agreement between electrodes. Bland Altman plots and Pearson correlations are summarized in Figure 3 with strong positive correlations between the custom-fabricated and industry standard electrodes. Pearson correlations revealed high levels of temporal synchrony between the custom-fabricated and industry standard electrode across all waveforms (sine [r = 0.987], square [r = 0.990], triangle [r = 0.931]).
Figure 3: Ex vivo testing results. Raw data from custom and industry standard electrodes for a repeated (A) sine wave, (D) square wave, and (G) triangle wave at 0.1 V and 5 Hz. (B,E,H) Correlation between voltage measurements for the custom and MicroProbe electrodes for 8,000 samples of the respective waveforms. (C,F,I) Bland Altman plot assessing the percent difference between the custom and MicroProbe electrode across average values of the respective waveforms. Please click here to view a larger version of this figure.
A representative impedance spectrum for a custom-fabricated epimysial electrode is shown in Figure 4. Although impedance measurements were collected from 10 Hz to 31 kHz, results are reported at 1 kHz, which is a relevant frequency for EMG acquisition. The industry standard (reference electrode) had an impedance of 2 kΩ. For comparison, the mean impedance of 10 custom-fabricated epimysial electrodes was 3.61 ± 7.95 kΩ and 1.63 ± 1.59 kΩ at 1 kHz for contact surfaces 1 and 2, respectively.
Figure 4: Impedance. Bode magnitude plot depicting the impedance value in Ohms (Ω) at ~ 1 kHz for a representative custom-fabricated epimysial electrode. Channels 1 and 2 pertain to contact surfaces 1 and 2 respectively. Please click here to view a larger version of this figure.
In vivo performance (Supplemental Video S1)
The custom-fabricated electrode effectively captured the physiological fluctuations in VL EMG activity induced during various treadmill walking conditions. Notably, the mean peak EMG activity was found to be significantly lower during downhill walking (0.008 ± 0.005 mV) than uphill (0.031 ± 0.180 mV, p = 0.005), in agreement with previous findings10. Furthermore, a paired t-test revealed that the peak EMG amplitude values at 14 days (0.01 ± 0.007 mV) did not exhibit a significant difference from those at 56 days (0.012 ± 0.007 mV, p > 0.05), supporting the long-term reliability of the device (Figure 5). Significantly different EMG magnitude values under the same recording conditions may be indicative of electrode damage or failure.
Figure 5: In vivo electrode reliability. Tukey boxplot of peak EMG amplitude (mV) of the vastus lateralis during level treadmill walking (16 m/min) at 14 days (T14) and 56 days (T56) following instrumentation. Abbreviations: EMG = electromyography; ns = not significant. Please click here to view a larger version of this figure.
Biocompatibility
H&E revealed no evidence of inflammation in the control muscle (Figure 6, left panel) relative to the instrumented muscle (Figure 6, right panel). This included no clear evidence of immune cell infiltration, internal myonuclear accumulation, fibrogenesis, and/or sarcolemma fragmentation between cross-sections extracted from the control or instrumented muscle.
Figure 6: Biocompatibility. Histological comparison of control (left) and instrumented (right) vastus lateralis muscles stained with hematoxylin and eosin (40x). Scale bars = 100 µm. Please click here to view a larger version of this figure.
Supplemental File 1: Catch_tray.gcode. 3D printing template of the catch tray, designed as part of the folding jig to catch the folded foils from the folding jig. Please click here to download this File.
Supplemental File 2: Cutting_jig.gcode. 3D printing template of the cutting jig, which contains evenly spaced slots as a guide to consistently perforate the silicone base. The cutting jig is configured for the placement of twenty foils to form ten bipolar epimysial electrodes. However, we currently use the cutting jig to produce a batch of six bipolar electrodes as we suggest leaving a space between electrodes. Please click here to download this File.
Supplemental File 3: Folding_jig.gcode. 3D printing template of the folding jig, which allows for easier folding of the platinum-iridium foils to form the desired U-shape. Folding of the foils can also be performed manually if needed. Please click here to download this File.
Supplemental Video S1: In vivo testing. EMG signaling of the VL during decline walking (-16°) on a motorized treadmill at 16m/min using the custom-fabricated epimysial EMG electrode. Please click here to download this File.
Our objective was to streamline the EMG fabrication process, enabling broader adoption and implementation of epimysial electrode designs, thus promoting accessibility, and advancing neuromuscular research. To this end, we present a user-friendly guide for sourcing, fabricating, and testing low-cost epimysial electrodes in-house. In hopes of supporting other research groups, we also provide supplemental 3D printing templates to facilitate the production of in-house epimysial electrodes for their research endeavors.
Researchers considering the adoption of these fabrication techniques readers should consider the following: 1) the electrode parameters specified in this workflow (shape, dimensions, contact surface area, interelectrode distance, lead length, etc.) were selected to suit our experimental model and will need to be modified for different use cases, 2) the welding of the lead wires to the contact foils is a crucial step that requires practice for successful execution; adjusting the pulse width and weld energy during welding can enhance success, 3) it is essential to apply tension to the lead wires and assess the interface's performance post welding to test the lead-to-contact surface connection, 4) the final fabrication step involves coating and sealing the electrode with silicone, a simple yet crucial process for optimal electrode performance. The silicone coating should be thin and relatively smooth to prevent irritation to dermal tissue during implantation, but an excessively thin coating can impair electrode performance. Researchers are advised to modify the silicone-toluene ratio according to their specifications to achieve a robust yet thin and smooth layer of silicone sealant.
When assessing electrode performance, others should note the following: 1) we have provided an example of an in vivo assessment, but we encourage the reader to use an in vivo testing procedure that mimics the intended use case of their custom-fabricated epimysial electrode, 2) our in vivo testing protocol also did not spatially permit for the simultaneous implantation of the custom-fabricated and industry standard electrodes. As such, signal agreement between electrodes could not be measured in vivo and, therefore, was not evaluated, 3) additionally, our in vivo testing configuration did not permit the assessment of electrode impedance following implantation, 4) it is important to consider that our data were tested ex vivo at relatively low levels of voltage (<1 V) due to our experimental needs. Thus, researchers who consider onboarding these electrodes may wish to test their utility at higher voltages. These are considerations that others may take into account when testing their own epimysial electrodes.
Prior work has highlighted the potential of multi-channel and high-density recording electrodes to more accurately decode motor neuron activity17,18,19,20. We encourage those interested in developing and using multichannel epimysial electrodes to utilize and expand upon the instructional resources we provide here. In addition to serving as recording electrodes, epimysial electrodes are commonly used as stimulating electrodes in neurostimulation21,22,23 and prostheses control experiments24,25. For those interested in using epimysial electrodes for these purposes, we encourage them to build upon our work and test the stimulating capability of their own custom-fabricated epimyisal electrodes.
Overall, the quantification of the electrical activity of muscle is fundamental to understanding the underlying mechanisms of numerous pathologies and the effectiveness of potential treatments. This updated workflow represents a straightforward guide to producing in-house, cost-effective epimysial electrodes that can be used to reliably quantify this important metric of musculoskeletal health. We hope that this methodology can serve as a template for other groups to fabricate and test their own custom-fabricated epimysial electrodes.
The authors have nothing to disclose.
This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant R01AR081235 (to L. K. Lepley). The authors thank the following individuals for their contribution to the fabrication and testing of our biocompatible electrode: Joel Pingel, Grant Gueller, Akhil Ramesh, Joe Letner, Jacky Tian, and Ross Brancati.
Electrode Materials | |||
Quantity & price per electrode | |||
Contact surface | Prince and Izant PT90/IR10 1.25 mm x 5 mm foil | Catalog #1040055 | 2 per electrode $7.50 per foil $15.00 per electrode |
PFA coated stainless-steel electrode lead wire | A-M Systems Multi-Stranded PFA-Coated Stainless Steel Wire 50.8 µm strand diameter | Catalog #793500 | Dependent on desired lead length (e.g., 9 inch lead wires x2) $128 per 25 ft spool $5.12 per foot $0.42 per inch (x18) $7.68 per electrode |
Folding jig | 3D printed (see .gcode file) |
NA | NA |
Sealant for electrode body | Nusil Med-1137 liquid silicone | Catalog #MED-1137 | 1 gram $344.66 per 2 oz. (59.15 mL) $5.83 per electrode |
Silicone base | Implantech Alliedsil Silicone Sheeting-Reinforced, Long Term Implantable (8” x 6”) .007 thick | Catalog #701-07 | 10mm x 5mm sheet $225.00 per 8 x 6 inch $0.36 per electrode (10 mm x 5 mm) |
Thinner for sealant mixture | Toluene 99.5% ACS Reagent 500mL or Xylene ACS 99.5% | Catalog #179418-500 ML | 0.75 mL $25.53 per 500 mL $0.38 per electrode |
Template for perforating silicone base | Cutting jig – 3D printed (see CAD file) |
NA | NA |
Custom-fabricated electrode: $29.25 | |||
Industry standard electrode (EP105 EMG Patch Electrode, 2 contacts, single-sided, 7mm x 4mm, MicroProbe for Life Science): $305.00 | |||
Additional Fabrication Materials | |||
Quantity & price per electrode | |||
3D printing software | Solidworks (Solidworks, 2022) | ||
Micro-Tig welder | Micro-Tig Welder (CD1000SPM, Single Pulse Research and Light Production Resistance Spot Welder, Sunstone) | SKU 301010 | $3,500 |
Ultrasonic bath | Ultrasonic bath (CPX Series Ultrasonic Bath, Fisherbrand). | 15-337-403 | NA |
Ex Vivo Testing Materials | |||
Quantity & price per electrode | |||
Data acquisition platform and software | DigitalLynx 4sX Base Cheetah version 6.0 (Neuralynx Inc.) | NA | EMG acquisition hardware and software |
Electrode interface board (EIB) | EIB, EIB16-QC, Neuralynx Inc. | 31-0603-0007 | NA |
Signal generator | 5 MHz Function Generator, B&K Precision | 4005DDS220V | $387.46 |
Potentiostat | PGSTAT1 potentiostat (EcoChemie, Utrecht, Netherlands) | NA | NA |
Stainless steel screw | Fine Science Tools | 19010-00 | $98 |
Ex Vivo Testing Materials | |||
Quantity & price per electrode | |||
Rodent treadmill | Exer 3/6 Open Treadmill, Columbus Instruments | NA | NA |
Dental cement | Excel Formula® Pourable Dental Material, St. George Technology Inc. | #24211 | $125.60 |
Light microscope | Keyence BZ-X800, Keyence Corporation, Osaka, Japan | NA | NA |
Motion capture system | Optitrack Color Camera, Optitrack, NaturalPoint Inc. | NA | NA |
Peak detection algorithm | “SciPy.signal.find_peaks – SciPy v1.8.1 Manual”, 2022 | NA | NA |
Python software | Python Software Foundation. Python Language Reference, version 3.9. Available at http://www.python.org | NA | NA |
Rat | HsdBlu: LE, Envigo | 140 | NA |
Statistical sotware | GraphPad Prism version 10.0.0 (GraphPad Software, Boston, Massachusetts USA) | NA | NA |