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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
  • Wyniki
  • Dyskusje
  • Ujawnienia
  • Podziękowania
  • Materiały
  • Odniesienia
  • Przedruki i uprawnienia

Podsumowanie

We have shown that the etching of nano-architecture into intracortical microelectrode devices may reduce the inflammatory response and has the potential to improve electrophysiological recordings. The methods described herein outline an approach to etch nano-architectures into the surface of non-functional and functional single shank silicon intracortical microelectrodes.

Streszczenie

With advances in electronics and fabrication technology, intracortical microelectrodes have undergone substantial improvements enabling the production of sophisticated microelectrodes with greater resolution and expanded capabilities. The progress in fabrication technology has supported the development of biomimetic electrodes, which aim to seamlessly integrate into the brain parenchyma, reduce the neuroinflammatory response observed after electrode insertion and improve the quality and longevity of electrophysiological recordings. Here we describe a protocol to employ a biomimetic approach recently classified as nano-architecture. The use of focused ion beam lithography (FIB) was utilized in this protocol to etch specific nano-architecture features into the surface of non-functional and functional single shank intracortical microelectrodes. Etching nano-architectures into the electrode surface indicated possible improvements of biocompatibility and functionality of the implanted device. One of the benefits of using FIB is the ability to etch on manufactured devices, as opposed to during the fabrication of the device, facilitating boundless possibilities to modify numerous medical devices post-manufacturing. The protocol presented herein can be optimized for various material types, nano-architecture features, and types of devices. Augmenting the surface of implanted medical devices can improve the device performance and integration into the tissue.

Wprowadzenie

Intracortical Microelectrodes (IME) are invasive electrodes which provide a means of direct interfacing between external devices and the neuronal populations inside the cerebral cortex1,2. This technology is an invaluable tool for recording neural action potentials to improve scientists' ability to explore neuronal function, advance understanding of neurological diseases and develop potential therapies. Intracortical microelectrode, used as a part of Brain Machine Interface (BMI) systems, enables recording of action potentials from an individual or small groups of neurons to detect motor intentions that can be used to produce functional outputs3. In fact, BMI systems have successfully been used for prosthetic and therapeutic purposes, such as acquired sensorimotor rhythm control to operate a computer cursor in patients with amyotrophic lateral sclerosis (ALS)4 and spinal cord injuries5 and restoring the movement in people suffering from chronic tetraplegia6.

Unfortunately, IMEs often fail to record consistently over time due to several failure modes that include mechanical, biological and material factors7,8. The neuroinflammatory response occurring after the electrode implantation is thought to be a considerable challenge contributing to electrode failure9,10,11,12,13,14. The neuroinflammatory response is initiated during the initial insertion of the IME which severs the blood brain barrier, damages the local brain parenchyma and disrupts glial and neuronal networks15,16. This acute response is characterized by the activation of glial cells (microglia/macrophages and astrocytes), which release pro-inflammatory and neurotoxic molecules around the implant site17,18,19,20. The chronic activation of glial cells results in a foreign body reaction characterized by the formation of a glial scar isolating the electrode from healthy brain tissue7,9,12,13,17,21,22. Ultimately, hindering the electrode's ability to record neuronal action potentials, due to the physical barrier between the electrode and the neurons and the degeneration and death of neurons23,24,25.

The early failure of intracortical microelectrodes has brought about considerable research in the development of next generation electrodes, with emphasis on biomimetic strategies26,27,28,29,30. Of particular interest to the protocol described here, is the use of nano-architecture as a class of biomimetic surface alterations for IMEs31. It has been established that surfaces mimicking the architecture of the natural in vivo environment have an improved biocompatible response32,33,34,35,36. Thus, the hypothesis compelling this protocol is that the discontinuity between the rough architecture of the brain tissue and smooth architecture of the intracortical microelectrodes may contribute to the neuroinflammatory and chronic foreign body response to implanted IMEs (for a full review refer to Kim et al.31). We have previously shown that the utilization of nano-architecture features similar to the brain's extracellular matrix architecture reduces astrocyte inflammatory markers from cells cultured on nano-architectured substrates, compared to flat control surfaces in both in vitro and ex vivo models of neuroinflammation37,38. Furthermore, we have shown the application of focused ion beam (FIB) lithography to etch nano-architectures directly onto silicon probes resulted in significantly increased neuronal viability and lower expression of pro-inflammatory genes from animals implanted with the nano-architecture probes compared to the smooth control group26. Therefore, the purpose of the protocol presented here is to describe the use of FIB lithography to etch nano-architectures on manufactured intracortical microelectrode devices. This protocol was designed to etch nano-architecture sized features into silicon surfaces of intracortical microelectrode shanks utilizing both automated and manual processes. These methods are uncomplicated, reproducible, and can certainly be optimized for various device materials and desired feature sizes.

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Protokół

NOTE: Do the following steps while wearing the proper personal protective equipment, such as a lab coat and gloves.

1. Mounting Non-functional Silicon Probe for Focused Ion Beam (FIB) Lithography

NOTE: For the complete procedure describing the fabrication of the SOI wafer with the 1,000 probes, please refer to Ereifej et al.39.

  1. Isolate a strip of 2-3 silicon probes from the silicon on insulator (SOI) wafer containing 1,000 probes. Do not make strips containing more than three silicon probes. This may increase the chances for loose mounting and may cause misalignment resulting in the FIB to etch incorrectly.
    NOTE: Strips/probes not firmly sitting on the aluminum stub can cause two complications: 1) when the stage moves to work on the next section, there will be vibrations and the milling will not be accurate until the probe settles and 2) it can cause a high variation and be out of the focus plane.
    1. While wearing gloves, use fine forceps to put pressure around the probes to break off a small section containing two to three probes.
  2. Carefully clean the silicon probe of all dust and debris prior to FIB etching. Prepare a 6 well polystyrene plate by pipetting 3 mL/well of 95% ethanol into three wells.
    1. Carefully pick up the cut strip of silicon probes using fine tip or vacuum forceps and place it into cell strainer. Place only one strip of silicon probes per strainer to prevent breaking the probes. Place the strainer containing the silicon probes strip into the first well containing 95% ethanol for cleaning. Keep the strainer in the first well for 5 min.
    2. Move the strainer containing the silicon probes from the first well and place it into the second well containing 95% ethanol for another 5 min. Repeat once more in the third well.
    3. Place the strainer containing the cleaned silicon probes onto a polytetrafluoroethylene plate to air dry. Do this step in a sterile hood to avoid contamination from dust.
  3. Place the air-dried strip of silicon probes in a sealed container for transport to the SEM-FIB. Wrap the strainer containing the air-dried samples with a plastic or aluminum foil wrap for transport and/or storage to maintain cleaning.
  4. Use fine tipped or vacuum forceps to carefully pick up the clean strip of silicon probes and place them onto a clean aluminum stub (used for SEM-FIB imaging/etching) to prepare for mounting.
  5. Use a toothpick (or other fine tipped instrument like a thin electrical wire), to place a tiny drop (~10 µL) of silver paint on the edge of the silicon substrate surrounding the probes. Secure the strip down by spreading the silver paint around the sides of the silicon substrate surrounding the probe. Allow the silver paint to dry completely before placing the aluminum stub into the SEM-FIB.
    NOTE: Be careful not to get silver paint on the shank of the electrode because that is the part that will be etched. If the strip of probes is not securely anchored to the aluminum stub, the strip may move during processing or have a different focal plane, thereby resulting in incorrect milling by the FIB. Several strips of silicon probes can be mounted onto the same aluminum stub, making sure there is ample space between the strips to allow for removal from the stub after etching. This will allow more efficient etching of multiple probes using the automated feature described below.

2. Aligning the FIB to the Silicon Probes

  1. Click on vent button in the beam control tab to vent the chamber. Press Shift+F3 to perform home stage. Confirm the selection by selecting the Home Stage button in the popup window.
    NOTE: Running the home stage operation is a preventative step to ensure the stage axis are read correctly by the software and the microscope is in good condition.
  2. After the Home Stage is complete, move the stage to coordinates X = 70 mm, Y = 70 mm, Z = 0 mm, T = 0°, R = 0°. Once the chamber is vented, put on clean nitrile gloves and open the chamber door.
    NOTE: Depending on the previous user's application, it may be necessary to change the stage adapter. The standard stage adapters (e.g., FEI style) can be removed by unscrewing the central bolt counterclockwise and installed by screwing clockwise into the rotation plate of the stage.
  3. Insert the aluminum stub holding the probes into the top of the stage adapter. Secure the aluminum stub by tightening the set screw on the side of the stage adapter. Use the 1.5 mm hex wrench for this task.
  4. Adjust the height of the stage adapter by turning the adapter clockwise to lower it or counterclockwise to raise it. Secure the stage adapter to the rotation plate by turning the locking cone nut clockwise until the nut is secure against the stage rotation plate. Hold the stage adapter with the other hand to prevent the rotation of the adapter and samples while tightening the locking cone nut.
    NOTE: Use the provided height gauge to determine the appropriate height. The top of the aluminum stub should be the same height as the maximum line shown on the height gauge. Over tightening the cone nut can cause damage to the stage and adapter. Only use enough force to secure the samples.
  5. Acquire a navigation camera image. Carefully swing the navigation camera arm open until it stops. The microscope stage will automatically move to a position beneath the camera. Watch the live image shown in Quadrant 3 of the microscope user interface (UI).
    1. Once the brightness level auto adjusts to an appropriate level, acquire the image by pushing the button down on the camera bracket. Be sure to wait for the entire image acquisition to finish, which is indicated by a pause symbol appearing in Quadrant 3 and the illumination of the camera turning off. This takes approximately 10 s. Swing the camera arm back to the closed position. The stage will return to the original position.
  6. Carefully close the microscope chamber door. Watch the CCD camera image in Quadrant 4 while closing the door. Ensure that the samples and stage are a safe distance away from any critical component in the microscope chamber.
  7. Select the down arrow next to the Pump button in the beam control tab. Select Pump with Sample Cleaning button in the UI software to start the chamber vacuum pump and built in plasma cleaner. Ensure the door is sealed by gently pushing on the face of the door while the pump is running. Wait for approximately 8 min for the pumping time and plasma cleaning cycle for the microscope chamber to be completed.
    NOTE: A vacuum seal can be confirmed by gently pulling on the chamber door, which should remain closed if the system is under vacuum.
  8. Once the icon in the bottom right corner of the UI turns green, press the Wake-Up button in the beam control tab which turns on the electron and ion beams. Select quadrant 1 and set the beam signal to electron beam (if not set already), set quadrant 2 to ion beam (if not set already).
    1. Set SEM voltage to 5 kV, set SEM beam current to 0.20 nA, set SEM detector to ETD, set detector mode to Secondary Electron. Set FIB voltage to 30 kV, set FIB beam current to 24 pA, set FIB detector to ICE detector, set detector mode to the secondary electron.
  9. Double click on the silicon probe in the navigation camera image, quadrant 3 to move the stage to the approximate location of the probe. Click on quadrant 1 to select it as the active quadrant and hit the pause button to start SEM scanning. Set the scan dwell time to 300 ns and turn off scan interlacing, line integration, and frame averaging. Set scan rotation to 0 in the beam control tab and right click on the beam shift 2d adjuster and select zero.
  10. Adjust the magnification to the minimum value by turning the magnification knob counterclockwise on the MUI panel. Adjust the image brightness and contrast using the knobs on MUI panel or the Auto Contrast Brightness toolbar icon.
  11. Move the stage by either double-left-clicking the mouse on a feature to center it, or by pressing down the mouse wheel and activating the joystick mouse mode. Move the desired silicon probe to be patterned into the center of the SEM image.
  12. Locate an edge or other features such as a dust particle or scratch. Increase magnification to 2,000x by turning the magnification knob clockwise. Adjust the focus of the SEM by turning the coarse and fine focus knobs on the MUI until the image is in focus. Once the image is in focus, select the Link sample Z to working distance button in the toolbar.
  13. Confirm that operation was completed by looking at the Z-axis coordinate in the navigation tab. The value should be approximately 11 mm. Type in 4.0 mm in the Z axis position and push the Go To button with the mouse or hit the enter key on the keyboard and the stage will move to 4 mm working distance.
  14. Move the stage in X and Y to locate the shoulder of the silicon probe. Position it as close to the center of the SEM as possible. Change the stage tilt to 52˚ by typing in "52" in the T coordinate and hitting enter. Observe whether the shoulder of the probe appears to move up or down in the image. Use the Stage Z slider to bring the shoulder of the probe back to the center of the SEM image. Only adjust the Z position, do not move X, Y, T, or R axis.
  15. Run the built in "xT Align Feature" command located in the stage drop down menu. Use the mouse to click on two points parallel to the edge of the probe. Make sure the horizontal radio button is selected in the popup window and click finish. The stage will rotate to align the probe with the X axis of the stage. Adjust the stage in X,Y using the mouse to put the lower shoulder of the probe in the center of the SEM image again.
    NOTE: The first point should be towards the probe's grip and the second point should be towards the probe's point.
  16. Select the FIB in quadrant 2 and make sure the beam current is still 24 pA. Set the magnification to 5,000x and the dwell time to 100 ns. Type Ctrl-F on the keyboard to set the FIB focus to 13.0 mm. In the beam control tab, right click in the stigmator 2d adjuster and select zero and, also, right click in the Beam Shift 2d adjuster and select zero. Set the scan rotation to 0° and push the auto contrast brightness button in the toolbar.
  17. Look for an image of the probe shoulder in quadrant 2. Use the snapshot tool to acquire an image with the FIB. Confirm the probe shoulder is in the center of the FIB image, if not, double click on the probe shoulder to move it to center. Move the stage to the left by pushing the left arrow key on the keyboard approximately 10-15 times. Take another snapshot and observe whether the probe side is still in the center of the FIB.
    NOTE: IF not, the stage rotation must be adjusted slightly. If the probe is above the image center, the stage must be rotated in the negative direction. If the probe is below center, the stage must be rotated clockwise. Enter a relative compucentric rotation of 0.01 to 0.2 degrees depending on which way is necessary to align the probe.
  18. Repeat steps 2.16 to 2.17 as many times as necessary until the edge of the probe shoulder is perfectly aligned with the X axis of the stage, (the edge stays in the center of the FIB while moving left).
  19. Using the FIB, move the stage back to the lower shoulder of the probe. Save the stage position in the position list by clicking the Add button. Change the FIB beam current to 2.5 nA and make sure the magnification of the FIB is still 5000x. Run the auto brightness contrast function and set the FIB dwell time to 100 ns.
  20. Hit the pause button to start scanning. Adjust the FIB focus and astigmatism, as quickly and precisely as possible, using the Coarse and Fine focus knobs, and the X and Y stigmator knobs on the MUI panel. Hit the pause button to stop the FIB scanning.

3. Writing an Automated Process for Etching

  1. Start the software by locating it in the Windows start menu (i.e., Start\Programs\FEI Company\Applications\Nanobuilder). Position the software window on the side monitor so the UI is not covered up. Open the file for patterning the silicon probes by clicking file and then open. Direct the windows browser to the location of the software script (Supplementary File 1 - the file name is "Case_Western_2000_micron_Final_11H47M_runtime.jbj").
  2. Within the software, select the microscope dropdown menu and select Set stage origin. Within the software, select the microscope dropdown menu and then select Calibrate Detectors.
  3. On the microscope UI, click in Quad 1 once with the mouse to select Quad 1. Ignore the other instructions shown in the popup window, they are not necessary for this project. Click OK to start the calibration. The process will take about 5 min. Make sure the ETD and ICE detectors calibrate. It is ok if any other detectors have calibration failures.
  4. Within the software, select the microscope dropdown menu and choose Execute to start the patterning sequence. When the pattern is complete, close the software.
    NOTE: The software will take over quad 3 and 4 for the patterning and alignment functions. The script will take approximately 12 h to run. While the script is running, do not change any parameter on the microscope.
  5. Hit "Vent" in the microscope UI beam control tab to shut down the microscope beams and start the vent cycle. While the chamber is venting, move the stage to coordinates X = 70 mm, Y = 70 mm, Z = 0 mm, T = 0°, R = 0°. Once the chamber is vented, put on clean nitrile gloves and pull open the chamber door.
  6. Loosen the set screw on the stub adapter using the 1.5 mm hex wrench. Remove the aluminum stub containing the patterned probe from the chamber. Carefully close the microscope chamber door. Watch the CCD camera image in Quadrant 4 while closing the door. Ensure that the stage adapter is a safe distance away from any critical component in the microscope chamber.
  7. Select the down arrow next to the Pump button in the beam control tab. Select Pump button to start the chamber vacuum pump. Ensure the door is sealed by gently pushing on the face of the door while the pump is running.
    NOTE: A vacuum seal can be confirmed by gently pulling on the chamber door, which should remain closed if the system is under vacuum. The pumping time will be approximately 5 min. Only one side of the probe can be etched during a single run.
  8. If the front and back side of the probe requires etching, then carefully remove the etched strip of silicon probes after checking the final etch and imaging the front side (if images are needed). Dissolve the silver paint with acetone, by cautiously dabbing/brushing the acetone on the silver paint. Carefully turn the strip around to the backside, re-mount, align and etch following the steps described above.

4. Checking the Final Etch and Imaging

  1. Once the milling is complete verify the uniformity of the different sections using SEM imaging at a higher magnification.
    NOTE: Imaging at the tilted angle allows a better assessment of the variation in the milling depth. Special attention should be given to the transition regions between the milling locations.
  2. Image the samples again after milling with an optical microscope.
    NOTE: The periodic milled lines result in a refraction effect giving rise to different colors as a function of the imaging angle. If the color is not continuous along with the probe that is a clear indication of the disruption in the milled lines.

5. Mounting a Functional Silicon Probe for FIB Etching

  1. Gently remove the functional silicon electrode from its packaging. Use forceps to carefully lift the plastic protective tab covering the head stage. Start lifting one corner of the tab up from the sticky glue holding it in place and keep lifting until the entire electrode is removed.
  2. Carefully clamp the electrode with hemostats to prepare for mounting into the stereotaxic frame. While holding the covered tab with the forceps, gently place curved hemostats around the green shaft above the silicon shank, with the curved part of the hemostats facing upwards towards the tab. Lock the hemostats in place to ensure the electrode will not drop out of the hemostats.
  3. Gently remove the plastic protective tab covering the head stage. While holding the electrode with the hemostats, carefully clip the electrode into the stereotaxic frame for cleaning.
  4. Fill 3 Petri dishes with 95% ethanol (~10 mL per petri dish). Place the Petri dish under the electrode that is mounted into the stereotaxic frame for cleaning. Slowly lower the electrode by turning the micromanipulator downwards (100 µm/s) so that the shank is submerged into the 95% ethanol.
    NOTE: Be careful not to turn the micromanipulator too fast or too deep, this can cause the electrode to break (i.e., the electrode should not touch the Petri dish).
  5. Leave the electrode shank in the 95% ethanol for 5 min, and then slowly raise the electrode out of the 95% ethanol by turning the micromanipulator upwards (100 µm/s). Repeat this step two more times, for a total of three washes. Allow the electrode to air dry for five minutes.
  6. Use the same technique for mounting the electrode into the stereotaxic frame, to remove the electrode from the stereotaxic frame. Carefully place the hemostats around the shaft of the electrode. Once the hemostats are clasped tight, release the electrode from the stereotaxic frame, return the plastic protective tab covering the head stage, and put the cleaned electrode back into its packaging.

6. Etching Functional Silicon Probe Using FIB

  1. Mount the cleaned functional silicon electrode onto an aluminum stand. Carefully pick up the cleaned functional silicon electrode using forceps and remove the protective tab from the headstage. Place the electrode shank on the aluminum stub so it does not hang over any edge, then using a small piece of Cu or carbon conductive tape, pin the headstage securely to the aluminum stub.
    NOTE: Alternatively, a low-profile clip holder can be used to hold the electrode down. Be careful not to touch the electrode shank.
  2. Following the steps described above (Section 2), position the electrode at the eucentric height and make sure the electrode is at the coincidence point of the SEM and FIB beams. Align the shank with the "X"direction of the stage.
  3. Set the FIB to the optimal current for milling the required the nano-architecture and make sure the focus and stigmation are properly corrected. Prepare an array of lines with desired spacing and length to cover the field of view of the shank (500 µm sections). Adjust the line lengths as etching gets down the shank to the thinner sections.
    NOTE: When etching the functional electrode, it is not possible to add fiducial marks to automate the process. Therefore, moving between the sub-sections (~500 µm) is done manually.
  4. After the milling of the first section is complete, make sure to check the milling quality before moving on to the next section. Repeat step 6.3 to etch the next section of the shank. Align the milled lines from the previous section to the patterns used for the next section to prevent large gaps between runs.

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Wyniki

FIB Etched Nano-architecture on the Surfaces of Single Shank Intracortical Probes
Utilizing the methods described here, intracortical probes were etched with specific nano-architectures following established protocols39. Dimensions and shape of the nano-architecture design described in these methods were implemented from previous in vitro results depicting a decrease in glial cell reactivity when cultured with the nano-architecture design described here37

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Dyskusje

The fabrication protocol outlined here utilizes focused ion beam lithography to effectively and reproducibly etch nano-architectures into the surface of non-functional and functional single shank silicon microelectrodes. Focused ion beam (FIB) lithography allows for the selective ablation of the substrate surface by using a finely-focused ion beam50,51. FIB is a direct-write technique that can produce various features with nanoscale resolution and high aspect rat...

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Ujawnienia

The authors have nothing to disclose.

Podziękowania

This study was supported by the United States (US) Department of Veterans Affairs Rehabilitation Research and Development Service awards: #RX001664-01A1 (CDA-1, Ereifej) and #RX002628-01A1 (CDA-2, Ereifej). The contents do not represent the views of the U.S. Department of Veterans Affairs or the United States Government. The authors would like to thank FEI Co. (Now part of Thermofisher Scientific) for staff assistance and use of instrumentation, which aided in developing the scripts used in this research.

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Materiały

NameCompanyCatalog NumberComments
16-Channel ZIF-Clip HeadstageTucker Davis TechnologiesZC16The headstage and headstage holder may need to be changed, depending on the electrode used. https://www.tdt.com/zif-clip-digital-headstages.html
1-meter cable, ALL spring wrappedThomas Scientific1213F04Any non treated petri dish will suffice. https://www.thomassci.com/Laboratory-Supplies/Cell-Culture-Dishes/_/Non-Treated-Petri-Dishes?q=petri%20dish%20cell%20culture
32-Channel ZIF-Clip Headstage HolderTucker Davis TechnologiesZ-ROD32The headstage and headstage holder may need to be changed, depending on the electrode used. https://www.tdt.com/zif-clip-digital-headstages.html
Acetone, Thinner/Extender/Cleaner, 30mlTed Pella16023https://www.tedpella.com/SEMmisc_html/SEMpaint.htm#anchor16062
Baby-Mixter HemostatFine Science Tools13013-14Any curved hemostat will suffice. https://www.finescience.com/en-US/Products/Forceps-Hemostats/Hemostats/Baby-Mixter-Hemostat
Carbon Conductive Tape, Double CoatedTed Pella16084-7The protocol suggested three options for mounting the functional electrode to the aluminum stub (copper or carbon conductive tape or a low profile clip. We utilized the carbon conductive tape in our study. https://www.tedpella.com/semmisc_html/semadhes.htm
Corning Costar Not Treated Multiple Well Plates - 6 wellSigma AldrichCLS3736-100EAAny non-treated 6 well plate will suffice. https://www.sigmaaldrich.com/catalog/substance/
Dumont #5 Fine ForcepsFine Science Tools11251-30Either this fine forceps or the vacuum pump will suffice. https://www.finescience.com/en-US/Products/Forceps-Hemostats/Dumont-Forceps/Dumont-5-Forceps/11251-30
Ethanol, 190 proof (95%), USP, Decon LabsFisher Scientific22-032-600Any 95% ethanol will suffice. https://www.fishersci.com/shop/products/ethanol-190-proof-95-usp-decon-labs-10/22032600
Falcon Cell StrainerFisher Scientific08-771-1https://www.fishersci.com/shop/products/falcon-cell-strainers-4/087711
FEI, Tescan, Zeiss (also for Philips, Leo, Cambridge, Leica, CamScan), aluminum, grooved edge, Ø32mmTed Pella16148Depending on the SEM machine used, you may need a different size stub. https://www.tedpella.com/SEM_html/SEMpinmount.htm#_16180
Fisherbrand Aluminum Foil, Standard-gauge rollFisher Scientific01-213-101Any aluminum foil will suffice. https://www.fishersci.com/shop/products/fisherbrand-aluminum-foil-7/p-306250
Fisherbrand Low- and Tall-Form PTFE Evaporating DishesFisher Scientific02-617-149Any Teflon plate will suffice, this is used to dry the probes after washing on a surface they will not stick onto. https://www.fishersci.com/shop/products/fisherbrand-low-tall-form-ptfe-evaporating-dishes-12/p-88552
Michigan-style silicon functional electrodeNeuroNexusA1x16-3mm-100-177http://neuronexus.com/electrode-array/a1x16-3mm-100-177/
Model 1772 Universal holderKOPFModel 1772Other stereotaxic frames and accessories will suffice. http://kopfinstruments.com/product/model-1772-universal-holder/
Model 900-U Small Animal Stereotaxic InstrumentKOPFModel 900-UOther stereotaxic frames and accessories will suffice. http://kopfinstruments.com/product/model-900-small-animal-stereotaxic-instrument1/
Model 960 Electrode Manipulator with AP Slide AssemblyKOPFModel 960Other stereotaxic frames and accessories will suffice. http://kopfinstruments.com/product/model-1772-universal-holder/
Parafilm M 10cm x 76.2m (4" x 250')Ted Pella807-5https://www.tedpella.com/grids_html/807-2.htm
PELCO Vacuum Pick-Up System, 220VTed Pella520-1-220Either this vacuum pump or the fine forceps will suffice. http://www.tedpella.com/grids_html/Vacuum-Pick-Up-Systems.htm#anchor-520
PELCO Conductive Silver PaintTed Pella16062https://www.tedpella.com/SEMmisc_html/SEMpaint.htm#anchor16062
SEM FIB FEI Helios 650 NanolabThermo Fisher ScientificHelios G2 650This is the specific focused ion beam and scanning electron microscope used in the protocol. The Nanobuilder software is what it comes with. If a different FIB instrument is used, it may not be completely compatible with the protocol, specifically the steps requiring the Nanobuilder software. https://www.fei.com/products/dualbeam/helios-nanolab/

Odniesienia

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