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.

 Fabricating Epimysial EMG Electrodes for Preclinical Rodent Models 

<ol><li>Grandjean, P. A., Mortimer, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recruitment+properties+of+monopolar+and+bipolar+epimysial+electrodes%5BTitle%5D)+AND+%22Ann.+Biomed.+Eng%22%5BJournal%5D)">Recruitment properties of monopolar and bipolar epimysial electrodes.</a> Ann. Biomed. Eng. 14 (1), 53-66 (1986).</li>
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<li>Loeb, G. E., Gans, C. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aLoeb%2C+GE+%22Electromyography+for+Experimentalists%22">Electromyography for Experimentalists</a>. , University of Chicago Press. Chicago, IL. (1986).</li>
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<li>Impedance measurement of bipolar stimulation electrodes immersed in medium. Sarolic, A., Skalic, I., Deftu, A., Sapunar, D. 2018 EMF-Med 1st World Conf. Biomed. Appl. Electromagn. Fields EMF-Med, , 1-2 (2018).</li>
<li>Pritchett-Corning, K. R., Mulder, G. B., Luo, Y., White, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Principles+of+rodent+surgery+for+the+New+Surgeon%5BTitle%5D)+AND+%22J.+Vis.+Exp.%3A+JoVE%22%5BJournal%5D)">Principles of rodent surgery for the New Surgeon.</a> J. Vis. Exp.: JoVE. (47), e2586(2011).</li>
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<li>Farago, E., MacIsaac, D., Suk, M., Chan, A. D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+techniques+for+surface+electromyography+signal+quality+analysis%5BTitle%5D)+AND+%22IEEE+Rev.+Biomed.+Eng%22%5BJournal%5D)">A review of techniques for surface electromyography signal quality analysis.</a> IEEE Rev. Biomed. Eng. 16, 472-486 (2023).</li>
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<li>Tankisi, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Standards+of+instrumentation+of+EMG%5BTitle%5D)+AND+%22Clin.+Neurophysiol%22%5BJournal%5D)">Standards of instrumentation of EMG.</a> Clin. Neurophysiol. 131 (1), 243-258 (2020).</li>
<li>Kumar, A., Accorsi, A., Younghwa, R., Mahasweta, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Do%27s+and+don%27ts+in+the+preparation+of+muscle+cryosections+for+histological+analysis%5BTitle%5D)+AND+%22J.+Vis.+Exp.+JoVE%22%5BJournal%5D)">Do's and don'ts in the preparation of muscle cryosections for histological analysis.</a> J. Vis. Exp. JoVE. (99), e52793(2015).</li>
<li>Wang, C., Yue, F., Kuang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Muscle+histology+characterization+using+H%26E+staining+and+muscle+fiber+type+classification+using+immunofluorescence+staining%5BTitle%5D)+AND+%22Bio-Protoc%22%5BJournal%5D)">Muscle histology characterization using H&E staining and muscle fiber type classification using immunofluorescence staining.</a> Bio-Protoc. 7 (10), e2279(2017).</li>
<li>Kreifeldt, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Signal+versus+noise+characteristics+of+filtered+EMG+used+as+a+control+source%5BTitle%5D)+AND+%22IEEE+Trans.+Biomed.+Eng%22%5BJournal%5D)">Signal versus noise characteristics of filtered EMG used as a control source.</a> IEEE Trans. Biomed. Eng. BME-18 (1), 16-22 (1971).</li>
<li>Farina, D., Yoshida, K., Stieglitz, T., Koch, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multichannel+thin-film+electrode+for+intramuscular+electromyographic+recordings%5BTitle%5D)+AND+%22J.+Appl.+Physiol%22%5BJournal%5D)">Multichannel thin-film electrode for intramuscular electromyographic recordings.</a> J. Appl. Physiol. 104 (3), 821-827 (2008).</li>
<li>Muceli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decoding+motor+neuron+activity+from+epimysial+thin-film+electrode+recordings+following+targeted+muscle+reinnervation%5BTitle%5D)+AND+%22J.+Neural+Eng%22%5BJournal%5D)">Decoding motor neuron activity from epimysial thin-film electrode recordings following targeted muscle reinnervation.</a> J. Neural Eng. 16 (1), 016010(2018).</li>
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<li>Zwarts, M. J., Stegeman, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multichannel+surface+EMG%3A+basic+aspects+and+clinical+utility%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">Multichannel surface EMG: basic aspects and clinical utility.</a> Muscle Nerve. 28 (1), 1-17 (2003).</li>
<li>Fabrication and test of robust spherical epimysial electrodes for lower limb stimulation. Aalb. Den. Koch, K. P., Leinenbach, C., Stieglitz, T. IFESS 5th Conf, , (2000).</li>
<li>Uhlir, J. P., Triolo, R. J., Davis, J. A., Bieri, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Performance+of+epimysial+stimulating+electrodes+in+the+lower+extremities+of+individuals+with+spinal+cord+injury%5BTitle%5D)+AND+%22IEEE+Trans.+Neural+Syst.+Rehabil.+Eng%22%5BJournal%5D)">Performance of epimysial stimulating electrodes in the lower extremities of individuals with spinal cord injury.</a> IEEE Trans. Neural Syst. Rehabil. Eng. 12 (2), 279-287 (2004).</li>
<li>Deer, T. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+appropriate+use+of+neurostimulation%3A+new+and+evolving+neurostimulation+therapies+and+applicable+treatment+for+chronic+pain+and+selected+disease+states%5BTitle%5D)+AND+%22Neuromodulation+Technol.+Neural+Interface%22%5BJournal%5D)">The appropriate use of neurostimulation: new and evolving neurostimulation therapies and applicable treatment for chronic pain and selected disease states.</a> Neuromodulation Technol. Neural Interface. 17 (6), 599-615 (2014).</li>
<li>Ortiz-Catalan, M., Brånemark, R., Håkansson, B., Delbeke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((On+the+viability+of+implantable+electrodes+for+the+natural+control+of+artificial+limbs%3A+Review+and+discussion%5BTitle%5D)+AND+%22Biomed.+Eng.+OnLine%22%5BJournal%5D)">On the viability of implantable electrodes for the natural control of artificial limbs: Review and discussion.</a> Biomed. Eng. OnLine. 11, 33(2012).</li>
<li>Sando, I. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Regenerative+peripheral+nerve+interface+for+prostheses+control%3A+electrode+comparison%5BTitle%5D)+AND+%22J.+Reconstr.+Microsurg%22%5BJournal%5D)">Regenerative peripheral nerve interface for prostheses control: electrode comparison.</a> J. Reconstr. Microsurg. 32 (3), 194-199 (2016).</li>
</ol>

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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&#39;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 (&#60;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.

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&#38;E) staining was conducted on the instrumented muscle to assess the biocompatibility of the custom-fabricated electrode.

<table><tbody><tr><td>&lt;strong&gt;Electrode Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>&lt;strong&gt;Quantity &amp;amp; price per electrode&lt;/strong&gt;</td></tr><tr><td>Contact surface</td><td>Prince and Izant PT90/IR10 1.25 mm x 5 mm foil</td><td>Catalog #1040055&amp;nbsp;</td><td>2 per electrode&lt;br/&gt; $7.50 per foil&lt;br/&gt; $15.00 per electrode</td></tr><tr><td>PFA coated stainless-steel electrode lead wire&amp;nbsp;</td><td>A-M Systems Multi-Stranded PFA-Coated Stainless Steel Wire 50.8 &amp;micro;m strand diameter&amp;nbsp;</td><td>Catalog #793500</td><td>Dependent on desired lead length (e.g., 9 inch lead wires x2)&lt;br/&gt; $128 per 25 ft spool&lt;br/&gt; $5.12 per foot&lt;br/&gt; $0.42 per inch (x18)&lt;br/&gt; $7.68 per electrode</td></tr><tr><td>Folding jig&amp;nbsp;</td><td>3D printed&lt;br/&gt; (see .gcode file)</td><td>NA</td><td>NA</td></tr><tr><td>Sealant for electrode body</td><td>Nusil Med-1137 liquid silicone</td><td>Catalog #MED-1137</td><td>1 gram&lt;br/&gt; $344.66 per 2 oz. (59.15 mL)&lt;br/&gt; $5.83 per electrode</td></tr><tr><td>Silicone base</td><td>Implantech Alliedsil Silicone Sheeting-Reinforced, Long Term Implantable (8&amp;rdquo; x 6&amp;rdquo;) .007 thick</td><td>Catalog #701-07&amp;nbsp;</td><td>10mm x 5mm sheet&lt;br/&gt; $225.00 per 8 x 6 inch&lt;br/&gt; $0.36 per electrode (10 mm x 5 mm)</td></tr><tr><td>&amp;nbsp;Thinner for sealant mixture</td><td>Toluene 99.5% ACS Reagent 500mL or Xylene ACS 99.5%</td><td>Catalog #179418-500 ML</td><td>0.75 mL&lt;br/&gt; $25.53 per 500 mL&lt;br/&gt; $0.38 per electrode</td></tr><tr><td>Template for perforating silicone base</td><td>Cutting jig &amp;ndash; 3D printed&lt;br/&gt; (see CAD file)</td><td>NA</td><td>NA</td></tr><tr><td>Custom-fabricated electrode: &lt;strong&gt;$29.25&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Industry standard electrode (EP105 EMG Patch Electrode, 2 contacts, single-sided, 7mm x 4mm, MicroProbe for Life Science): $305.00</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Additional Fabrication Materials&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp; &amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;&amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;&amp;nbsp;</td><td>&lt;strong&gt;Quantity &amp;amp; price per electrode&lt;/strong&gt;</td></tr><tr><td>3D printing software&amp;nbsp;</td><td>Solidworks (Solidworks, 2022)</td><td></td><td></td></tr><tr><td>Micro-Tig welder&amp;nbsp;</td><td>Micro-Tig Welder (CD1000SPM, Single Pulse Research and Light Production Resistance Spot Welder, Sunstone)</td><td>SKU&amp;nbsp;301010</td><td>$3,500</td></tr><tr><td>Ultrasonic bath</td><td>Ultrasonic bath (CPX Series Ultrasonic Bath, Fisherbrand).&amp;nbsp;</td><td>15-337-403</td><td>NA</td></tr><tr><td>&lt;strong&gt;Ex Vivo Testing Materials&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>&lt;strong&gt;Quantity &amp;amp; price per electrode&lt;/strong&gt;</td></tr><tr><td>Data acquisition platform and software</td><td>DigitalLynx 4sX Base Cheetah version 6.0 (Neuralynx Inc.)&amp;nbsp;</td><td>NA</td><td>EMG acquisition hardware and software</td></tr><tr><td>Electrode interface board (EIB)</td><td>EIB, EIB16-QC, Neuralynx Inc.</td><td>31-0603-0007</td><td>NA</td></tr><tr><td>Signal generator</td><td>5 MHz Function Generator, B&amp;amp;K Precision&amp;nbsp;</td><td>&amp;nbsp;4005DDS220V</td><td>$387.46</td></tr><tr><td>Potentiostat</td><td>PGSTAT1 potentiostat (EcoChemie, Utrecht, Netherlands)</td><td>NA</td><td>NA</td></tr><tr><td>Stainless steel screw</td><td>Fine Science Tools</td><td>19010-00</td><td>$98</td></tr><tr><td>&lt;strong&gt;Ex Vivo Testing Materials&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>&amp;nbsp; &amp;nbsp;</td><td>&lt;strong&gt;Quantity &amp;amp; price per electrode&lt;/strong&gt;</td></tr><tr><td>Rodent treadmill&amp;nbsp;</td><td>Exer 3/6 Open Treadmill, Columbus Instruments</td><td>NA</td><td>NA</td></tr><tr><td>Dental cement</td><td>Excel Formula&amp;reg; Pourable Dental Material, St. George Technology Inc.</td><td>#24211</td><td>$125.60</td></tr><tr><td>Light microscope</td><td>Keyence BZ-X800, Keyence Corporation, Osaka, Japan&amp;nbsp;</td><td>NA</td><td>NA</td></tr><tr><td>Motion capture system</td><td>Optitrack Color Camera, Optitrack, NaturalPoint Inc.</td><td>NA</td><td>NA</td></tr><tr><td>Peak detection algorithm</td><td>&amp;ldquo;SciPy.signal.find_peaks - SciPy v1.8.1 Manual&amp;rdquo;, 2022</td><td>NA</td><td>NA</td></tr><tr><td>Python software</td><td>Python Software Foundation. Python Language Reference, version 3.9. Available at http://www.python.org</td><td>NA</td><td>NA</td></tr><tr><td>Rat</td><td>HsdBlu: LE, Envigo</td><td>140</td><td>NA</td></tr><tr><td>Statistical sotware</td><td>GraphPad Prism version 10.0.0 (GraphPad Software, Boston, Massachusetts USA)</td><td>NA</td><td>NA</td></tr></tbody></table>

development of a low-cost epimysial electromyography electrode: a simplified workflow for fabrication and testing

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 &plusmn; 0.005 mV) than uphill (0.031 &plusmn; 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 &plusmn; 0.007 mV, 0.012 &plusmn; 0.007 mV respectively; p &gt; 0.05) and the absence of histological inflammation. Collectively, we provide an updated workflow for the fabrication and testing of low-cost epimysial electrodes.

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 &#60; .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]).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66744/66744fig03.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66744/66744fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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 k&#937;. For comparison, the mean impedance of 10 custom-fabricated epimysial electrodes was 3.61 &#177; 7.95 k&#937; and 1.63 &#177; 1.59 k&#937; at 1 kHz for contact surfaces 1 and 2, respectively.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/66744/66744fig04.jpg" /> 
Figure 4: Impedance. Bode magnitude plot depicting the impedance value in Ohms (&#937;) at ~ 1 kHz for a representative custom-fabricated epimysial electrode.&#160;Channels 1 and 2 pertain to contact surfaces 1 and 2 respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66744/66744fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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 &#177; 0.005 mV) than uphill (0.031 &#177; 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 &#177; 0.007 mV) did not exhibit a significant difference from those at 56 days (0.012 &#177; 0.007 mV, p &#62; 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.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/66744/66744fig05.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66744/66744fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Biocompatibility 
H&#38;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.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/66744/66744fig06.jpg" /> 
Figure 6: Biocompatibility. Histological comparison of control (left) and instrumented (right) vastus lateralis muscles stained with hematoxylin and eosin (40x). Scale bars = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66744/66744fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66744/Supplementary File 1.zip" target="_blank">Please click here to download this File.</a>
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66744/Supplementary File 2.zip" target="_blank">Please click here to download this File.</a>
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66744/Supplementary File 3.zip" target="_blank">Please click here to download this File.</a>
Supplemental Video S1: In vivo testing. EMG signaling of the VL during decline walking (-16&#176;) on a motorized treadmill at 16m/min using the custom-fabricated epimysial EMG electrode. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/66744/Supplemental_Video.zip" target="_blank">Please click here to download this File.</a>

Read this Scientific Article Protocol about Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies at JoVE.com

Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies

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.

Development of a Low-cost Epimysial Electromyography Electrode: A Simplified Workflow for Fabrication and Testing

Electromyography (EMG) is a valuable diagnostic tool for detecting neuromuscular abnormalities. Implantable epimysial electrodes are commonly used to ...

development-low-cost-epimysial-electromyography-electrode-simplified

Nanyang Technological University

Research

JoVE Journal

Neuroscience

394 Views.  University of Michigan, Ann Arbor. 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. 

Video: Author Spotlight: Epimysial Electrode Fabrication and Testing in ACL Injury Studies

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals suffering from chronic medical conditions facile assessment of their immediate physiological state. Furthermore, it could serve as a research tool for analysis of complex, multifactorial medical conditions. In order for such a multianalyte sensor to be realized, it must be minimally invasive, sampling of interstitial fluid must occur without pain or harm to the user, and analysis must be rapid as well as selective.
Initially developed for pain-free drug delivery, microneedles have been used to deliver vaccines and pharmacologic agents (e.g., insulin) through the skin.1-2 Since these devices access the interstitial space, microneedles that are integrated with microelectrodes can be used as transdermal electrochemical sensors. Selective detection of glucose, glutamate, lactate, hydrogen peroxide, and ascorbic acid has been demonstrated using integrated microneedle-electrode devices with carbon fibers, modified carbon pastes, and platinum-coated polymer microneedles serving as transducing elements.3-7,8
This microneedle sensor technology has enabled a novel and sophisticated analytical approach for in situ and simultaneous detection of multiple analytes. Multiplexing offers the possibility of monitoring complex microenvironments, which are otherwise difficult to characterize in a rapid and minimally invasive manner. For example, this technology could be utilized for simultaneous monitoring of extracellular levels of, glucose, lactate and pH,9 which are important metabolic indicators of disease states7,10-14 (e.g., cancer proliferation) and exercise-induced acidosis.15

This article details the construction of a multiplexed microneedle-based sensor. The device is being developed for in situ sampling and electrochemical analysis of multiple analytes in a rapid and selective manner. We envision clinical medicine and biomedical research uses for these microneedle-based sensors.

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

Bioengineering

Scalable Solution-processed Fabrication Strategy for High-performance, Flexible, Transparent Electrodes with Embedded Metal Mesh

Here, the authors report the embedded metal-mesh transparent electrode (EMTE), a new transparent electrode (TE) with a metal mesh completely embedded in a polymer film. This paper also presents a low-cost, vacuum-free fabrication method for this novel TE; the approach combines lithography, electroplating, and imprint transfer (LEIT) processing. The embedded nature of the EMTEs offers many advantages, such as high surface smoothness, which is essential for organic electronic device production; superior mechanical stability during bending; favorable resistance to chemicals and moisture; and strong adhesion with plastic film. LEIT fabrication features an electroplating process for vacuum-free metal deposition and is favorable for industrial mass production. Furthermore, LEIT allows for the fabrication of metal mesh with a high aspect ratio (i.e., thickness to linewidth), significantly enhancing its electrical conductance without adversely losing optical transmittance. We demonstrate several prototypes of flexible EMTEs, with sheet resistances lower than 1 &#937;/sq and transmittances greater than 90%, resulting in very high figures of merit (FoM) &#8211; up to 1.5 x 104 &#8211; which are amongst the best values in the published literature.

This protocol describes a solution-based fabrication strategy for high-performance, flexible, transparent electrodes with fully-embedded, thick metal mesh. Flexible transparent electrodes fabricated by this process demonstrate among the highest reported performances, including ultra-low sheet resistance, high optical transmittance, mechanical stability under bending, strong substrate adhesion, surface smoothness, and environmental stability.

Here, the authors report the embedded metal-mesh transparent electrode (EMTE), a new transparent electrode (TE) with a metal mesh completely embedded in a ...

Engineering

Wireless Intramuscular EMG System for Locomotion Studies in Stroke Model Rats

A Real-Time Wearable Electromyography Measurement System for Small Animals

The intramuscular electromyography (EMG) measurement method for experimental animals has been implemented in various ways. Among these methods, tethering cables to external measurement devices can restrict the movement of experimental animals, while implantable devices may cause unwanted side effects due to the constant presence of a device with considerable size and weight. To address these issues, we propose a low-cost, wireless, detachable EMG measurement system and experimental procedure. This article focuses on the surgical installation of intramuscular wire electrodes with small connectors and the development of the wireless system. Notably, in this system, only the wire electrodes are inserted into the animal's body. Using this system, EMG measurements can be easily performed by attaching the circuit system to a connector installed on the animal's back, with real-time monitoring achievable on a laptop. The proposed method is explained in a detailed, step-by-step manner, followed by a demonstration involving the insertion of intramuscular electrodes into the hindlimbs of a rat. A treadmill experiment is conducted for a locomotion study, and the resulting electrophysiological signals are subsequently obtained and analyzed.

A protocol is presented for the installation and preparation of intramuscular electrodes, along with an animal-mountable, miniaturized measurement device for electromyography (EMG) analysis in locomotion studies. This system enables the wireless transmission of real-time EMG data for investigating locomotion recovery in stroke model rats.

The intramuscular electromyography (EMG) measurement method for experimental animals has been implemented in various ways. Among these methods, tethering ...

Fabrication of High Contact-Density, Flat-Interface Nerve Electrodes for Recording and Stimulation Applications

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic applications. This protocol describes a fabrication technique of a modified cylindrical nerve cuff electrode to meet these criteria. Minimum computer-aided design and manufacturing (CAD and CAM) skills are necessary to consistently produce cuffs with high precision (contact placement 0.51 &#177; 0.04 mm) and various cuff sizes. The precision in spatially distributing the contacts and the ability to retain a predefined geometry accomplished with this design are two criteria essential to optimize the cuff&#39;s interface for selective recording and stimulation. The presented design also maximizes the flexibility in the longitudinal direction while maintaining sufficient rigidity in the transverse direction to reshape the nerve by using materials with different elasticities. The expansion of the cuff&#39;s cross sectional area as a result of increasing the pressure inside the cuff was observed to be 25% at 67 mm Hg. This test demonstrates the flexibility of the cuff and its response to nerve swelling post-implant. The stability of the contacts&#39; interface and recording quality were also examined with contacts&#39; impedance and signal-to-noise ratio metrics from a chronically implanted cuff (7.5 months), and observed to be 2.55 &#177; 0.25 k&#8486; and 5.10 &#177; 0.81 dB respectively.

This article provides a detailed description on the fabrication process of a high contact-density flat interface nerve electrode (FINE). This electrode is optimized for recording and stimulating neural activity selectively within peripheral nerves.

Many attempts have been made to manufacture multi-contact nerve cuff electrodes that are safe, robust and reliable for long term neuroprosthetic ...

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network connectivity, are applicable to an expanding variety of questions in neuroscience. Performing these kinds of analyses in a rodent model requires higher electrode density than traditional screw electrodes can accomplish. While higher-density electroencephalographic montages for rodents exist, they are of limited availability to most researchers, are not robust enough for repeated experiments over an extended period of time, or are limited to use in anesthetized rodents.1-3 A proposed low-cost method for constructing a durable, high-count, transcranial electrode array, consisting of bilaterally implantable headpieces is investigated as a means to perform advanced electroencephalogram analyses in mice or rats.

Procedures for headpiece fabrication and surgical implantation necessary to produce high signal to noise, low-impedance electroencephalographic and electromyographic signals are presented. While the methodology is useful in both rats and mice, this manuscript focuses on the more challenging implementation for the smaller mouse skull. Freely moving mice are only tethered to cables via a common adapter during recording. One version of this electrode system that includes 26 electroencephalographic channels and 4 electromyographic channels is described below.

Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network ...

Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the body by measuring and analyzing differences in the electrical impedance between dissimilar biotissues. This paper describes the fabrication method of fine interdigitated electrodes (IDEs) at the tip of a hypodermic needle using a photoresist spray coating and flexible film photomask in the photolithography process. A polyethylene terephthalate (PET) heat shrink tube (HST) with a wall thickness of 25 &#181;m is employed as the insulation and passivation layer. The PET HST shows a higher mechanical durability compared with poly(p-xylylene) polymers, which have been widely used as a dielectric coating material. Furthermore, the HST shows good chemical resistance to most acids and bases, which is advantageous for limiting chemical damage to the EoN. The use of the EoN is especially preferred for the characterization of chemicals/biomaterials or fabrication using acidic/basic chemicals. The fabricated gap and width of the IDEs are as small as 20 &#181;m, and the overall width and length of the IDEs are 400 &#181;m and 860 &#181;m, respectively. The fabrication margin from the tip (distance between the tip of hypodermic needle and starting point of the IDEs) of the hypodermic needle is as small as 680 &#181;m, which indicates that unnecessarily excessive invasion into biotissues can be avoided during the electrical impedance measurement. The EoN has a high potential for clinical use, such as for thyroid biopsies and anesthesia drug delivery in a spinal space. Further, even in surgery that involves the partial resection of tumors, the EoN can be employed to preserve as much normal tissue as possible by detecting the surgical margin (normal tissue that is removed with the surgical excision of a tumor) between the normal and lesion tissues.

The fabrication method for fine interdigitated electrodes (gap and width: 20 &#181;m) at the tip of a hypodermic needle (diameter: 720 &#181;m) is demonstrated using a spray coating and flexible film photomask in the photolithography process.

We have introduced a fabrication method for electrical impedance spectroscopy (EIS)-on-a-needle (EoN: EIS-on-a-needle) to locate target tissues in the ...

Open-source Toolkit: Benchtop Carbon Fiber Microelectrode Array for Nerve Recording

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This paper presents a cleanroom "light" fabrication process of carbon fiber neural electrode arrays that can be learned quickly by an inexperienced cleanroom user. This carbon fiber electrode array fabrication process requires just one cleanroom tool, a Parylene C deposition machine, that can be learned quickly or outsourced to a commercial processing facility at marginal cost. This fabrication process also includes hand-populating printed circuit boards, insulation, and tip optimization. 
The three different tip optimizations explored here (Nd:YAG laser, blowtorch, and UV laser) result in a range of tip geometries and 1 kHz impedances, with blowtorched fibers resulting in the lowest impedance. While previous experiments have proven laser and blowtorch electrode efficacy, this paper also shows that UV laser-cut fibers can record neural signals in vivo. Existing carbon fiber arrays either do not have individuated electrodes in favor of bundles or require cleanroom fabricated guides for population and insulation. The proposed arrays use only tools that can be used at a benchtop for fiber population. This carbon fiber electrode array fabrication process allows for quick customization of bulk array fabrication at a reduced price compared to commercially available probes.

Here, we describe fabrication methodology for customizable carbon fiber electrode arrays for recording in vivo in nerve and brain.

Conventional peripheral nerve probes are primarily fabricated in a cleanroom, requiring the use of multiple expensive and highly specialized tools. This ...

Conformable Wearable Electrodes: From Fabrication to Electrophysiological Assessment

Wearable electronic devices are becoming key players in monitoring the body signals predominantly altered during physical activity tracking. Considering the growing interest in telemedicine and personalized care driven by the rise of the Internet of Things era, wearable sensors have expanded their field of application into healthcare. To ensure the collection of clinically relevant data, these devices need to establish conformable interfaces with the human body to provide high-signal-quality recordings and long-term operation. To this end, this paper presents a method to easily fabricate conformable thin tattoo- and soft textile-based sensors for their application as wearable organic electronic devices in a broad spectrum of surface electrophysiological recordings. 
The sensors are developed through a cost-effective and scalable process of cutaneous electrode patterning using poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS), the most popular conductive polymer in bioelectronics, on off-the-shelf, wearable substrates. This paper presents key steps in electrode characterization through impedance spectroscopy to investigate their performance in signal transduction when coupled with the skin. Comparative studies are required to position the performance of novel sensors with respect to the clinical gold standard. To validate the fabricated sensors' performance, this protocol shows how to perform various biosignal recordings from different configurations through a user-friendly and portable electronic setup in a laboratory environment. This methods paper will allow multiple experimental initiatives to advance the current state of the art in wearable sensors for human body health monitoring.

Two recent technologies-tattoo and textiles-have demonstrated promising results in cutaneous sensing. Here, we present the fabrication and evaluation methods of tattoo and textile electrodes for cutaneous electrophysiological sensing. These electronic interfaces made of conductive polymers outperform the existing standards in terms of comfort and sensitivity.

Wearable electronic devices are becoming key players in monitoring the body signals predominantly altered during physical activity tracking. Considering ...

Fabrication of Flat-Interface Nerve Electrodes for Recording Neural Activity

To add a shielding layer, remove the large fixture bar, and remove the transparency piece with tweezers. Then, place the shielding sheet in the center of each face of the electrode and gently press it into the electrode.
Then, deposit some uncured silicone into the through-holes, and partially cure it for 30 minutes at 130 degrees Celsius. Once cooled to room temperature, place adhesive tape over the outer ends of the electrode and over the closing flanges, to keep silicone out of these segments.
Then, repeat the process of adding a layer of silicone to cover the entire electrode body. Follow this by placing silicone and transparency sheets over the electrode, and then, secure them with a large fixture bar and cure the silicone.
After curing, to complete the process, remove the excess silicone and the tape. Then, cut windows through the silicone to expose the spacer segments, and use tweezers to remove the spacer segments.
Next, under a microscope, remove the tape and silicone over the tubes, until it is level with the electrode body. Then, carefully cut around the perimeter of the electrode down to the base plate.
Now, taking caution to not damage the electrode, follow the guide diagram to shape the leads&#39; exit sites. Cut out a triangle between each tube pair that goes completely through the electrode layers, and onto the outer side.
Now, cut windows into the silicone that covers the shielding layer. Then, glide the polypropylene suture between the electrode base and the adjacent transparent layer, and use it to delaminate the nearly finished cuff electrode. Finally, cut windows out of the silicone to expose the center contacts and reference contacts.
The fabricated fine electrode was used to record the activity of the sciatic nerve in a dog over 7.5 months. A customized preamplifier and a super beta input instrumentation amplifier were implemented. The electrode was implanted around the nerve by suturing the two free edges together. The cuff flattens the nerve while retaining flexibility in the longitudinal direction.