This work was supported by the &#34;Biomedical Integrated Technology Research&#34; project through a grant provided by GIST in 2017.

1. Electrical Insulation of Hypodermic Needle
NOTE: A transparent heat shrink tube (HST) is employed for the electrical insulation of a hypodermic needle that is 720 &#181;m in diameter and 32 mm in length. The HST is made of polyethylene terephthalate (PET), which shows good chemical resistance to most acids and bases, excellent mechanical durability, and biocompatibility. The initial inner diameter and wall thickness of the HST are 840 &#181;m and 25 &#181;m, respectively. The diameter of the HST tends to be reduced by more than 50% at a temperature of 100 &#176;C, with even greater reduction at higher temperatures up to 190 &#176;C. Note that PET HST is a thermosetting material that has the property of becoming permanently hard and rigid when cured. The size of the hypodermic needle and shrink tube can be adjusted depending on the research purpose and applications. The overall fabrication process is graphically summarized in Figure 1.
<ol>
	<li>Cut the HST to a length of 3 cm. Adjust the length of the tube depending on the penetration depth of the hypodermic needle.</li>
	<li>Insert the hypodermic needle into the cut HST.</li>
	<li>Shrink the tube using a heat gun at a temperature of 150 &#176;C, which is set to prevent unwanted additional contraction when dehydration is carried out at 105 &#176;C in the cleaning process (in step 1.6).</li>
	<li>Separate the hypodermic needle from its hub.</li>
	<li>Clean the hypodermic needle insulated by the HST in a deionized (DI) water bath (20 &#176;C) with ultrasonic agitation at 30 kHz and 350 W power.</li>
	<li>Dehydrate the hypodermic needle insulated by the HST on a hotplate at 105 &#176;C for 10 min.</li>
</ol>
2. Au Deposition Using Sputtering
NOTE: In this study, the sputtering process that is available is used to deposit an Au layer for electrodes, although an e-beam evaporation process can be an alternative method. It has been confirmed that the temperature rise induced in the sputtering process rarely causes additional shrinkage of the HST. However, a process that continues for more than several minutes might heat the HST above the initial shrinkage temperature. This can cause additional shrinkage of the HST, resulting in an increase in the fabrication margin from the tip.
<ol>
	<li>Arrange the cleaned hypodermic needles insulated by the HST side by side on a glass slide using double-sided tape for Cr/Au deposition.</li>
	<li>Using sputtering equipment, deposit Cr/Au on the cleaned hypodermic needles insulated by the HST. 
	NOTE: In this case, the thicknesses of Cr and Au were 10 nm and 100 nm, respectively (Cr was used for the adhesion layer between the HST and the Au layer).
	<ol>
		<li>Arrange as many needles as possible in order to reduce production cost and production time. Use the sputtering conditions below to deposit 10 nm Cr and 100 nm Au.</li>
		<li>For Cr sputtering, set Cr target diameter: 4 inch, RF power: 300 W, argon pressure: 5 mTorr, and shutter open time: 20 s (10 nm).</li>
		<li>For Au sputtering, use Au target diameter: 4 inch, DC power: 300 W, argon pressure: 10 mTorr, and shutter open time: 80 s (100 nm).</li>
	</ol>
	</li>
</ol>
3. Spray Coating
NOTE: A low-viscosity (14 cp) photoresist is used in the spray coating process to increase spray efficiency. The photoresist can be easily coated on the Au-sputtered needle only when the needle is heated.
<ol>
	<li>Fix one of the Au-sputtered hypodermic needles on a glass slide using double-sided tape.</li>
	<li>Place the slide glass on a chuck of the spray coater that is being heated at 100 &#176;C. Wait 2 - 3 min until the needle is sufficiently heated.</li>
	<li>Spray the photoresist on the Au-sputtered needle while heating the needle at 100 &#176;C. Perform the spray-coating process using the following conditions. Set nozzle diameter: 400 &#181;m, nozzle moving speed: 70 mm/s, spray pressure: 500 kPa, and distance between chuck and nozzle: 13.5 cm.</li>
	<li>After spray coating is finished, leave the glass slide on the chuck at 100 &#176;C for 3 min to perform a soft baking process.</li>
	<li>Inspect the result using a microscope set to 100X magnification to determine whether the photoresist is uniformly coated on the Au-sputtered needle.</li>
</ol>
4. UV Exposure and Developing
NOTE: In general, prior to UV exposure, a flexible film photomask is attached to a flat transparent plate to remove the air gap between the photomask and the sample to be exposed to UV light. However, in this study, the photomask is used without the flat transparent plate to realize direct metal patterning on the curved surface of the hypodermic needle. The photomask can conformably bent along the curve of the hypodermic needle to achieve the best patterning resolution feasible with the contact aligner. The bending allows the flexible photomask to keep the contact area between the photomask and the curved surface of the hypodermic needle as large as possible. Taking a wet etching process (not a lift-off process) for metal patterning into consideration, the use of a positive photoresist is more advantageous than the use of a negative photoresist. This is because the entire area except the electrode pattern is transparent, thereby providing a wide field of view to readily align the electrode pattern with the center of the needle.
<ol>
	<li>To minimize wedge error, slowly lift a freely movable sample-holding plate until it fully contacts the fixed photomask-holding plate. Then, fix the sample-holding plate using a pneumatic pump.
	<ol>
		<li>Carry out this process to possibly avoid undesirable patterns, which may be formed by the scattering of UV light in the air gap, and caused by the incomplete contact between the sample and photomask. 
		NOTE: In addition, the minimization of wedge error ensures that the photoresist-coated hypodermic needle does not move when it contacts a film photomask in the next alignment step, even though the contact surface of the hypodermic needle has a round shape.</li>
	</ol>
	</li>
	<li>Place the photoresist-coated hypodermic needle on the sample-holding plate of the aligner.</li>
	<li>Align the projected image of the photoresist-coated hypodermic needle with the alignment pattern of the film photomask. 
	NOTE: In this case, the alignment pattern of the film photomask was designed as two parallel lines at a distance of 800 &#181;m, considering the thickness of the HST and coated photoresist.
	<ol>
		<li>Align two boundary lines of the projected image with two parallel alignment lines of the photomask (Figure 1e); thus, the photoresist-coated hypodermic needle can be positioned at the center of two parallel alignment lines, with an alignment error of 10 &#181;m or less.</li>
		<li>Monitor the alignment process in real-time through the display monitor connected to the charge-coupled device (CCD) camera and microscope.</li>
	</ol>
	</li>
	<li>Bring the photoresist-coated hypodermic needle into contact with the fixed flexible photomask by slowly lifting the needle towards the photomask.</li>
	<li>Carry out UV exposure for 30 s (UV intensity: 15 mJ/cm2) and follow this by the developing process for 3 min.</li>
	<li>Rinse the developer out of the sample using DI water.</li>
	<li>Inspect the result through a microscope set to 200X magnification to determine whether the photoresist is clearly patterned on the Au-sputtered hypodermic needle. If the exposed photoresist is not perfectly removed after the developing process, repeat the developing process at 30 s intervals.</li>
</ol>
5. Cr/Au Wet Etching
CAUTION: Avoid skin/eye contact with the Cr and Au wet etchants.
<ol>
	<li>Use a tweezer to detach the sample (photoresist-patterned hypodermic needle) fixed on the glass slide.</li>
	<li>Immerse the sample into the Au wet etchant for 1 min.</li>
	<li>Rinse the Au etchant out of the sample using DI water.</li>
	<li>Inspect the result through a microscope set to 200X&#160;magnification. If the gold to be removed still remains, repeat the wet etching process at 10 s intervals. Excessively long wet etching time makes the interdigitated electrode (IDE) thinner.</li>
	<li>Immerse the sample into the Cr etchant for 30 s.</li>
	<li>Rinse the Cr etchant out of the sample using DI water.</li>
</ol>
6. Removal of Residual Photoresist and Passivation
<ol>
	<li>Immerse the sample (metal-patterned hypodermic needle) into an acetone solution for 1 min.</li>
	<li>Rinse the sample with DI water and dehydrate it on a hot plate at 105 &#176;C for 10 min.</li>
	<li>For electrical passivation of the connection lines, cut the shrink tube so that it is 2 - 3&#160;mm longer than the electrode (20 mm, the maximum depth to penetrate), as shown in Figure 2, because the length of the HST will be reduced after the HST shrinks.</li>
	<li>After positioning the HST as far as possible from the end of the IDE, raise the temperature of the HST using a heat gun at 150 &#176;C to tightly passivate the needle.</li>
</ol>

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The authors have nothing to disclose.

We demonstrated that photolithography using spray coating and a film photomask is a feasible method to fabricate fine IDEs on the curved surface of a hypodermic needle with a small diameter of less than 1 mm. Both the width and the gap of the IDEs are as low as 20 &#181;m, and the fabrication margin from the tip is as small as 680 &#181;m. Within the protocol, the alignment process, including wedge error removal, is a critical step. The production yield was over 90% when the EoN was manufactured individually through a rigorous alignment process. This indicates that the proposed fabrication method has the potential to be developed for bulk production at an affordable price.
The discrimination capability of the EoN has been previously verified for PBS, porcine fat/muscle tissues, and even human renal tissues6,23,24. One clinical application is for surgery that involves the partial resection of tumors to preserve as much normal tissue as possible by detecting the surgical margin between the normal and lesion tissues25. Furthermore, the EoN is expected to be utilized in other clinical applications such as thyroid/prostate biopsies and anesthesia drug delivery in a spinal space.
Although the width and gap of the IDEs were fabricated to be 20 &#181;m in the present study, they can be reduced to 10 &#181;m once the resolution of printable film photomasks increases. Another way to reduce the dimensions of the gap and width of the IDEs is to transfer smaller patterns of a chrome mask to a flexible film using the photolithography process. Meanwhile, the wall thickness of the HST can be reduced from 25 &#181;m to a smaller size that is commercially available. A smaller HST with a wall thickness of 6 &#181;m was experimentally verified to be used for electrical insulation and the passivation layer using the same fabrication process. This will facilitate insertion experiments into animal tissues and even lessen the pain of patients in clinical use.
The fabrication method using the photolithography process can be developed for bulk production with a high yield at an affordable price by arranging many hypodermic needles together and by designing a photomask array. Another feasible method for bulk production is to use an array of shadow mask molds made by a 3D printer with high resolution as shown in Figure 6. The shadow mask can physically block the area where metal deposition is unwanted during a physical deposition process, such as sputtering and/or evaporation. Cr/Au deposited on the shadow mask can be readily removed using Cr/Au wet etchant for reuse of the shadow mask. The expected limitations to be addressed are as follows: 1) a 3D printer with high resolution is required, 2) the materials used in the 3D printer should be chemically resistant to the Cr/Au wet etchant for reuse of the shadow mask, and 3) the materials used in the 3D printer should not deform at temperatures above 150 &#176;C that might be induced during the sputtering process. The next plan of the present study is to develop the bulk production method at an affordable price and to verify the applicability of the EoN in spinal anesthesia and thyroid/prostate biopsies.

Hypodermic needles are widely utilized in hospitals for biopsies and drug delivery because they are inexpensive and easy to use. They also have excellent mechanical properties despite their thin diameter and a sharp-edged structure suitable for invasion. During a biopsy, the target tissues are sampled in the hollow of the hypodermic needle with ultrasonography guidance1. Although ultrasonography is free of radiation, safe for fetuses and pregnant women, and provides real-time imaging, it is difficult to see organs that are deep within the body, especially in the case of obese patients because ultrasonic waves cannot penetrate air or fat tissues2. In addition, a surgeon cannot acquire depth information from the two-dimensional ultrasonography that is conventionally utilized in the majority of hospitals, resulting in the need for multiple biopsies if physicians lack skill or experience. In drug delivery for spinal anesthesia, physicians determine that the needle has reached the spinal space if the cerebrospinal fluid (CSF) flows backward into the syringe while carefully inserting the needle into the patient&#39;s back. After confirming the reflux of CSF, the anesthesia drug is injected into the spinal space3. However, physicians risk penetrating or cutting off nerve fibers in the spinal space, causing severe pain to patients and even paraplegia4,5. Thus, this procedure also requires a skillful physician. One solution to overcome and mitigate the aforementioned difficulties is to add a navigation function to the hypodermic needle so that objective information on the needle&#39;s position can be provided. This would help a physician readily perform a biopsy, drug delivery, and even a surgery without relying on their empirical judgment only.
In order to electrically localize the target tissues in the body, a hypodermic needle incorporating an electrical impedance spectroscopy (EIS) sensor has been introduced as EIS-on-a-needle (EoN)6. The EIS sensor is currently utilized in the field of biomedical engineering for applications such as DNA detection7,8,9, bacteria/virus detection10,11,12, and analysis on cells/tissues13,14,15,16,17,18,19,20,21,22. The EoN can discriminate between dissimilar materials in a frequency domain based on their electrical conductivity and permittivity. The discrimination capability of the EoN was verified for various concentration levels of phosphate buffered saline (PBS)23, porcine fat/muscle tissues6,23, and even human renal normal/cancer tissues24,25. This capability of the EoN is expected to considerably increase the biopsy accuracy by locating the target tissues based on the differences in electrical impedance between the target lesion tissues and the neighboring normal tissues. In a similar manner, investigating differences in the electrical impedance between the drug injection space (spinal or epidural space) and surrounding tissues can help physicians deliver an anesthesia drug at the exact target location. Furthermore, the EoN can be utilized to electrically stimulate the brain/muscle as well as to determine an optimal surgical margin during surgeries that involve the partial resection of a tumor, such as partial nephrectomy, to preserve as much normal tissue as possible.
One of the biggest challenges in the realization of the EoN is the fabrication of electrodes on the curved surface of a hypodermic needle having a small radius of curvature. Direct metal patterning using a conventional photolithography process has been regarded as unsuitable for the fabrication of micro-sized electrodes on a curved substrate with a diameter of several millimeters or less. So far, various methods, including conformal printing26, flexible dry film photoresist27, the microfluidic method28, nanoimprint lithography29, and substrate-rotating lithography30, have been introduced to fabricate metal/polymer patterns on a curved surface. However, there are still limitations due to the EoN requirements, such as the required substrate with a diameter of less than 1 mm, total electrode length of 20 mm or more, width and gap of electrodes ranging in tens of micrometers, and high volume production.
In the present study, direct metal patterning by employing photoresist spray coating and a flexible film photomask is proposed to realize micro-sized electrodes on the curved surface of a hypodermic needle. The diameter of the needle is as small as 720 &#181;m (22-gauge), which is widely used for biopsies and drug delivery in hospitals. The production yield of the proposed fabrication method is also evaluated to determine the feasibility of bulk production at an affordable price.

<table>
	<tbody>
		<tr>
			<td>Heat shrink tube</td>
			<td>VENTION MEDICAL, Inc.</td>
			<td>103-0655</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic needle (22G)</td>
			<td>HWAJIN MEDICAL co. ltd</td>
			<td>-</td>
			<td>http://www.hwajinmedical.com</td>
		</tr>
		<tr>
			<td>Heat gun</td>
			<td>Weller</td>
			<td>WHA600</td>
			<td>http://www.weller-tools.com/en/Home.html</td>
		</tr>
		<tr>
			<td>Ultrasonic cleaner</td>
			<td>HWASHIN INSTRUMENT CO, LTD.</td>
			<td>POWERSONIC 620-</td>
			<td>http://www.hwashin.net</td>
		</tr>
		<tr>
			<td>Hotplate</td>
			<td>AS ONE Corporation</td>
			<td>006560</td>
			<td></td>
		</tr>
		<tr>
			<td>Sputtering</td>
			<td>A-Tech System. Ltd.</td>
			<td>ATS/SPT/0208F</td>
			<td>http://www.atechsystem.co.kr</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Paul Marienfeld GmbH &#38; Co. KG</td>
			<td>1000412</td>
			<td></td>
		</tr>
		<tr>
			<td>Spray coater</td>
			<td>LITHOTEK</td>
			<td>LSC-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoresist</td>
			<td>AZ electronic materials</td>
			<td>GXR 601</td>
			<td>http://www.merck-performance-materials.com/en/index.html</td>
		</tr>
		<tr>
			<td>Developer (solution)</td>
			<td>AZ electronic materials</td>
			<td>MIF 300</td>
			<td>http://www.merck-performance-materials.com/en/index.html</td>
		</tr>
		<tr>
			<td>Aligner</td>
			<td>MIDAS SYSTEM CO.,Ltd.</td>
			<td>MDA-400M</td>
			<td>http://www.midas-system.com</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>NIKON Corporation</td>
			<td>L200</td>
			<td>http://www.nikonmetrology.com</td>
		</tr>
		<tr>
			<td>Au wet etchant</td>
			<td>TRANSENE COMPANY, Inc.</td>
			<td>Au etchant type TFA</td>
			<td>http://transene.com</td>
		</tr>
		<tr>
			<td>Cr wet etchant</td>
			<td>KMG Electronic. Chemicals, Inc.</td>
			<td>CR-7</td>
			<td>http://kmgchemicals.com</td>
		</tr>
		<tr>
			<td>Au target</td>
			<td>Thin films and Fine Materials</td>
			<td>-</td>
			<td>http://www.thifine.co.kr</td>
		</tr>
		<tr>
			<td>Cr target</td>
			<td>Thin films and Fine Materials</td>
			<td>-</td>
			<td>http://www.thifine.co.kr</td>
		</tr>
		<tr>
			<td>Argon gas (99.999%)</td>
			<td>SINIL Gas Co.Ltd</td>
			<td>-</td>
			<td>http://www.sigas.kr</td>
		</tr>
		<tr>
			<td>Acetone solution</td>
			<td>OCI Company Ltd</td>
			<td>-</td>
			<td>http://www.ocicorp.co.kr/company/index.asp</td>
		</tr>
		<tr>
			<td>Impedance analyzer</td>
			<td>Gamry Instruments Inc</td>
			<td>Reference 600</td>
			<td>https://www.gamry.com</td>
		</tr>
		<tr>
			<td>Height Controller</td>
			<td>Mitutoyo Corporation</td>
			<td>192-613</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Life Technologies Corporation</td>
			<td>10010023</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 interdigitated electrodes (IDEs), as shown in Figure 2, result in a larger effective sensing area on a limited surface compared to other shapes of electrodes. The overall length of the IDEs is designed to be 860 &#181;m to detect and analyze the impedance changes at less than 1 mm intervals in the biotissues, which will provide a high locating accuracy in biopsy and drug delivery procedures. The total width of the IDEs is 400 &#181;m, which is a geometrically feasible dimension on the curved surface of the hypodermic needle when using the proposed photolithography process. Both the gap and width of the IDEs are as small as 20 &#181;m, which is close to the minimum dimensions of a commercially available film photomask. The maximum penetration depth of the EoN into biotissues is designed to be 20 mm, considering thyroid/prostate biopsies and spinal anesthesia. The total length of the EoN can be adjusted depending on the application.
As shown in Figure 3, the IDEs are successfully fabricated at the tip of the hypodermic needle with a diameter of 720 &#181;m. Overdose during UV lithography was required to compensate for the UV dose imbalance arising from imperfect contact between the outer portion of the photomask and the curved surface of the hypodermic needle. This will increase the gap and decrease the width of the IDEs in the case of a positive photoresist. To resolve the adverse effect of the dimensional changes, the width and the gap were intentionally designed to be 25 &#181;m and 15 &#181;m on the photomask, respectively. Thereby, both the width and the gap of the IDEs can be successfully fabricated to be 20 &#181;m by optimizing the UV exposure time. The fabrication margin from the tip of the hypodermic needle is as small as 680 &#181;m, which will avoid unnecessarily excessive invasion into biotissues during the electrical impedance measurement. A PET HST was employed as the electrical insulation layer for both the IDEs and the connection lines, and also as the electrical passivation layer for the connection lines. The HST features low electrical conductivity/permittivity, durable mechanical properties compared to a poly(p-xylylene) polymer coating, chemical resistance to most acids and bases, and biocompatibility.
From the standpoint of mechanical durability, device failure (for instance, the insulation layer, passivation layer, and/or electrodes peeling off) was not observed even after penetration into biotissues more than 100 times, whereas a poly(p-xylylene) polymer with a wall thickness of 1.5 &#181;m did not endure penetration into porcine tissues more than 20 times. This indicates that the PET HST showed strong adhesion with the sputtered electrodes as well as high durability for clinical trials. Furthermore, the HST shows good chemical resistance to most acids and bases, which enables the EoN to detect the electrical properties of various kinds of chemicals or biomaterials and keeps the HST durable during electrochemical deposition of Au electrodes using acid solution (H2SO4). In the electrochemical deposition process, the Au electrode layer tends to grow in fractal structures, which allows the effective area of the sensing electrodes to significantly increase on the limited surface area of the needle to achieve a higher sensitivity.
To evaluate the discrimination capability of the EoN and its depth profiling capability into biotissue, various concentration levels of PBS and four-layered porcine tissue were employed, respectively23. The impedance analyzer was connected to both the EoN and a laptop, as shown in Figure 4. To carry out depth profiling into the four-layered porcine tissue, the EoN was fixed to the height controller, with a resolution of 10 &#181;m. The various concentration levels of PBS were prepared as 1x, 0.5x, 0.25x, 0.125x, and 0.0625x, by serially diluting the 1x PBS with DI water. The lengths of the IDEs and connection lines used in the experiment was 300 &#181;m and 28 mm, respectively. As shown in Figure 5a, the EoN could successfully discriminate various concentration levels of PBS. Because the 1x PBS was diluted serially with DI water, the electrical conductivity of diluted PBS decreased because of the very small conductivity of DI water. Thus, the magnitude of impedance increased as the concentration level of PBS decreased. Based on the discrimination capability of EoN, the depth profiling of the four-layered porcine tissue was carried out at the frequency of 1 MHz, which was decided as the optimal frequency in our previous research. The EoN was inserted into the four-layered porcine tissue at increments of 1 mm. As shown in Figure 5b, the magnitude of impedance measured from fat tissue was clearly discriminated from that of muscle tissue, according to the penetration depth of the EoN.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56622/56622fig1.jpg" /> 
Figure 1: Schematic of overall EIS-on-a-needle (EoN) fabrication process. (A) Preparation of hypodermic needle, (B) Electrical insulation of hypodermic needle using heat shrink tube (HST, wall thickness: 25 &#181;m), (C) Cr/Au deposition using sputtering or evaporator, (D) Spray coating of photoresist (positive type), (E) Alignment process of film photomask and photoresist-coated hypodermic needle followed by UV exposure.The film photomask includes patterns of interdigitated electrodes (IDEs) and alignment line, (F) development process, (G) Cr/Au wet etching, (H) Removal of residual photoresist using acetone, and (I) Passivation on connection lines using HST. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56622/56622fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56622/56622fig2.jpg" /> 
Figure 2: Detailed structural schematic of the EoN. The shape of the sensing electrodes was designed to be fine interdigitated electrodes to secure a larger effective sensing area on the limited surface of the hypodermic needle. A PET heat shrink tube (HST) was used as the electrical insulation layer for both the interdigitated electrodes (IDEs) and the connection lines, and was also used as the electrical passivation layer for the connection lines. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56622/56622fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56622/56622fig3.jpg" /> 
Figure 3: Microscopic images of the successfully fabricated EoN. Both the width and gap of the IDEs are as low as 20 &#181;m. The overall length and width of the interdigitated electrodes (IDEs) are 860 &#181;m and 400 &#181;m, respectively. The fabrication margin from the tip is as small as 680 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56622/56622fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56622/56622fig4.jpg" /> 
Figure 4: Images of experimental setup. To evaluate the discrimination capability of the EoN and its depth profiling capability into the biotissue, various concentration levels of PBS and four-layered porcine tissue were employed, respectively. To carry out depth profiling into the four-layered porcine tissue, the EoN was fixed on the height controller with a resolution of 10 &#181;m. The various concentration levels of PBS were prepared as 1x, 0.5x, 0.25x, 0.125x, and 0.0625x, by serially diluting the 1x PBS with deionized (DI) water. (a) overall setup, (b) EoN immersed in the PBS, and (c) four-layered porcine tissue. This figure has been modified from the previously published study23. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56622/56622fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56622/56622fig5.jpg" /> 
Figure 5: Experimental results using PBS and four-layered porcine tissue. Evaluation of the discrimination capability of EoN using (a) various concentration levels of PBS and (b) four-layered porcine tissue. Because the 1x PBS was diluted serially with DI water, the electrical conductivity of diluted PBS decreased with increased dilution because of the low conductivity of DI water. Thus, the magnitude of impedance increased as the concentration level of PBS decreased. The depth profiling of the porcine tissue was carried out at the frequency of 1 MHz, which was determined to be the optimal frequency in our previous study23. The magnitude of impedance measured from fat tissues was clearly discriminated from that of muscle tissues according to the penetration depth of the EoN. F1, F2, M1, and M2 represent fat1, fat2, muscle1, and muscle2 shown in Figure 4 (c), respectively. This figure has been modified from the previously published study23. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56622/56622fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56622/56622fig6.jpg" /> 
Figure 6: Schematic of a shadow mask to deposit metal electrodes for bulk production. The shadow mask can be made using a 3D printer with fine resolution. The shadow mask can physically block the area where metal deposition is unwanted during a physical deposition process, such as sputtering and/or evaporation. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56622/56622fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Fabrication of Fine Electrodes on the Tip of Hypodermic Needle Using Photoresist Spray Coating and Flexible Photomask for Biomedical Applications at JoVE.com

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

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 ...

fabrication-fine-electrodes-on-tip-hypodermic-needle-using

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.5K Views.  Gwangju Institute of Science and Technology (GIST). The fabrication method for fine interdigitated electrodes (gap and width: 20 µm) at the tip of a hypodermic needle (diameter: 720 µm) is demonstrated using a spray coating and flexible film photomask in the photolithography process.

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

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. 
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are immense healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression in hippocampal slice cultures, as a means to develop novel therapeutic targets.

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options.  We seek to define how neural immune ...

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments in vision research require displays with more than 12 bits of luminance resolution. Several solutions are available. Bit++ 1 and DataPixx 2 use the Digital Visual Interface (DVI) output from graphics cards and high resolution (14 or 16-bit) digital-to-analog converters to drive analog display devices. The VideoSwitcher 3 described here combines analog video signals from the red and blue channels of graphics cards with different weights using a passive resister network 4 and an active circuit to deliver identical video signals to the three channels of color monitors. The method provides an inexpensive way to enable high-resolution monochromatic displays using conventional graphics cards and analog monitors. It can also provide trigger signals that can be used to mark stimulus onsets, making it easy to synchronize visual displays with physiological recordings or response time measurements.
Although computer keyboards and mice are frequently used in measuring response times (RT), the accuracy of these measurements is quite low. The RTbox is a specialized hardware and software solution for accurate RT measurements. Connected to the host computer through a USB connection, the driver of the RTbox is compatible with all conventional operating systems. It uses a microprocessor and high-resolution clock to record the identities and timing of button events, which are buffered until the host computer retrieves them. The recorded button events are not affected by potential timing uncertainties or biases associated with data transmission and processing in the host computer. The asynchronous storage greatly simplifies the design of user programs. Several methods are available to synchronize the clocks of the RTbox and the host computer. The RTbox can also receive external triggers and be used to measure RT with respect to external events. 
Both VideoSwitcher and RTbox are available for users to purchase. The relevant information and many demonstration programs can be found at <a href="http://lobes.usc.edu/">http://lobes.usc.edu/</a>.

Conventional computer hardware can not generate visual stimuli with sufficiently high grayscale resolution and measure response times with sufficient accuracy. We describe how to use the VideoSwitcher to produce high-resolution monochromatic displays, and the RTbox to measure response times with high accuracy on conventional computer hardware.

Display systems based on conventional computer graphics cards are capable of generating images with 8-bit gray level resolution. However, most experiments ...

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery. Here we provide useful information for surgeons who are learning the process of implanting electrode systems. We demonstrate the implantation procedure of both a penetrating and a surface electrode. The surgical process is described from start to finish, including detailed descriptions of each step throughout the procedure. It should also be noted that this video guide is focused towards procedures conducted in rodent models and other small animal models. Modifications of the described procedures are feasible for other animal models.

We provide useful information for surgeons who are learning the process of implanting chronic neural recording electrodes. Techniques for both penetrating and surface electrode systems are described in a rodent animal model.

The success of long-term electrophysiological recordings often depends on the quality of the implantation surgery.  Here we provide useful information for ...

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. Neurons typically receive multiple excitatory and inhibitory afferent and/or efferent inputs that integrate with each other, and the ultimate measured response properties of the neuron are driven by the neural integrations of these inputs. To study information processing in neural systems, it is necessary to understand the various inputs to a neuron or neural system, and the specific properties of these inputs. A powerful and technically relatively simple method to assess the functional role of certain inputs that a given neuron is receiving is to dynamically and reversibly suppress or eliminate these inputs, and measure the changes in the neuron's output caused by this manipulation. This can be accomplished by pharmacologically altering the neuron's immediate environment with piggy-back multibarrel electrodes. These electrodes consist of a single barrel recording electrode and a multibarrel drug electrode that can carry up to 4 different synaptic agonists or antagonists. The pharmacological agents can be applied iontophoretically at desired times during the experiment, allowing for time-controlled delivery and reversible reconfiguration of synaptic inputs. As such, pharmacological manipulation of the microenvironment represents a powerful and unparalleled method to test specific hypotheses about neural circuit function.
Here we describe how piggy-back electrodes are manufactured, and how they are used during in vivo experiments. The piggy-back system allows an investigator to combine a single barrel recording electrode of any arbitrary property (resistance, tip size, shape etc) with a multibarrel drug electrode. This is a major advantage over standard multi-electrodes, where all barrels have more or less similar shapes and properties. Multibarrel electrodes were first introduced over 40 years ago 1-3, and have undergone a number of design improvements 2,3 until the piggy-back type was introduced in the 1980s 4,5. Here we present a set of important improvements in the laboratory production of piggy-back electrodes that allow for deep brain penetration in intact in vivo animal preparations due to a relatively thin electrode shaft that causes minimal damage. Furthermore these electrodes are characterized by low noise recordings, and have low resistance drug barrels for very effective iontophoresis of the desired pharmacological agents.

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

Iontophoresis of neural agonists and antagonists during extracellular in vivo recordings is a powerful way to manipulate a neuron&#x2019;s microenvironment. These manipulations can most easily be done via piggy-back multibarrel electrodes. Here we describe how to manufacture them and use them during auditory recordings.

In vivo recordings from single neurons allow an investigator to examine the firing properties of neurons, for example in response to sensory stimuli. ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects foraging for food or searching for mates3: the olfactory search is constrained by the physics of turbulent transport4. The concentration landscape of wind borne odors is discontinuous and consists of sporadically located patches. A pre-requisite to olfactory search is that intermittent odor patches are detected. Because of its high speed and sensitivity5-6, the olfactory organ of insects provides a unique opportunity for detection. Insect antennae have been used in the past to detect not only sex pheromones7 but also chemicals that are relevant to humans, e.g., volatile compounds emanating from cancer cells8 or toxic and illicit substances9-11. We describe here a protocol for using insect antennae on autonomous robots and present a proof of concept for tracking odor plumes to their source. The global response of olfactory neurons is recorded in situ in the form of electroantennograms (EAGs). Our experimental design, based on a whole insect preparation, allows stable recordings within a working day. In comparison, EAGs on excised antennae have a lifetime of 2 hr. A custom hardware/software interface was developed between the EAG electrodes and a robot. The measurement system resolves individual odor patches up to 10 Hz, which exceeds the time scale of artificial chemical sensors12. The efficiency of EAG sensors for olfactory searches is further demonstrated in driving the robot toward a source of pheromone. By using identical olfactory stimuli and sensors as in real animals, our robotic platform provides a direct means for testing biological hypotheses about olfactory coding and search strategies13. It may also prove beneficial for detecting other odorants of interests by combining EAGs from different insect species in a bioelectronic nose configuration14 or using nanostructured gas sensors that mimic insect antennae15.

We describe a protocol for using insect antennae in the form of electroantennograms (EAGs) on autonomous robots. Our experimental design allows stable recordings within a day and resolves individual odor patches up to 10 Hz. The efficiency of EAG sensors for olfactory searches is demonstrated in driving a robot toward an odor source.

Robots designed to track chemical leaks in hazardous industrial facilities1 or explosive traces in landmine fields2 face the same problem as insects ...

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

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 ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

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

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Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Enabling High Grayscale Resolution Displays and Accurate Response Time Measurements on Conventional Computers

Surgical Implantation of Chronic Neural Electrodes for Recording Single Unit Activity and Electrocorticographic Signals

Manufacturing and Using Piggy-back Multibarrel Electrodes for In vivo Pharmacological Manipulations of Neural Responses

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Using Insect Electroantennogram Sensors on Autonomous Robots for Olfactory Searches

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

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

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development