This method can help answer key questions in the field of neuroscience, such as how aging processes manifest in the brain, how the brain develops, and how brain diseases originate and progress. The main advantage of this technique is that mesh electronics do not elicit a chronic immune response in gliosis once implanted into the brain and allows for a seamless integration with the surrounding neural tissue. Demonstrating the procedure will be Tao Zhou, a graduate student in the Lieber Group, and Jung Min Lee, a post-doc in the Lieber Group.
To begin the procedure, load a clean silicon wafer into a thermal evaporator and evaporate 100 nanometers of nickel. Next, spin coat SU-8 2000.5 negative photoresist onto the wafer at 400 revolutions per minute for an approximate SU-8 thickness of 400 to 500 nanometers. Load the wafer into a mask aligner to expose the SU-8 with photolithography mask one corresponding to the bottom mesh SU-8 layer.
Expose at I-line dose of 100 millijoules per square centimeter. Them, immerse the wafer ina tray of SU-8 developer. Gently agitate the solution for two minutes, until the mesh pattern in the SU-8 has been fully developed.
Then, rinse the wafer in a tray of isopropyl alcohol for one minute and blow dry. Hard bake the wafer on a hot plate at 180 degrees celsius for one hour. Afterward, spin coat LOR 3A onto the wafer at 4, 000 revolutions per minute for an approximate thickness of 300 nanometers.
Spin coat S1805 positive photoresist at 4, 000 revolutions per minute for an approximate thickness of 500 nanometers. Subsequently, load the wafer into a mask aligner to expose the S1805 with photolithography mask two corresponding to the metal interconnects and input, output pads. Exposed at H-line dose at 40 millijoules per square centimeter, then immerse the wafer in a tray of CD-26 photoresist developer.
Gently agitate the solution for one minute until the metal interconnects pattern has been fully developed. Rinse the wafer in a tray of deionized water for one minute and blow it dry. Next, thermally evaporate three nanometers of chromium followed by 80 nanometers of gold.
Immerse the wafer into a flat beaker of remover PG for approximately three hours, until the metal has fully undercut, leaving the metal only in the desire interconnect and input, output pad regions of mesh electronics. Repeat spin coating, lithography, evaporation, and lift off to leave three nanometers of chromium and 50 nanometers of platinum in the electrode regions. Subsequently, repeat spin coating and photolithography of SU-8 to define the top SU-8 mesh layer.
Treat the wafer with oxygen plasma at 50 watts for one minute. Afterward, immerse the wafer into nickel etch in solution for approximately three hours, until the mesh electronics have completely been released from the silicon wafer. Use a pasteur pipette to transfer the released mesh electronics probes from nickel etching to a 100 milliliter beaker of deionized water.
Then, transfer the mesh electronics to a fresh beaker of deionized water at least three times to ensure rinsing. To load the mesh electronics into a needle, insert a glass capillary needle into the 100 milliliter beaker of PBS containing the mesh electronics. Position the end of the needle near the input, output pads of a mesh electronics probe and manually retract the syringe to draw a mesh electronics probe into the needle.
Push and pull the syringe plunger while it is still immersed in saline to adjust the position of mesh electronics within the needle. To inject mesh electronics into a mouse brain, use a dental drill and stereotaxic frame to open a craniotomy at the desired coordinates on the skull. Open a second craniotomy away from the injection site for the insertion of a stainless steel graphing screw or wire.
Then, fix a clamping substrate to the skull with dental cement. An approximately one millimeter wide cut in the substrate improves the reliability of the folding step later in the procedure. Afterward, mount the pipette holder with the needle containing mesh electronics into the stereotaxic frame using a right angle end clamp.
Attach the side outlet of the pipette holder to a five milliliter syringe fastened in a syringe pump with a 0.5 to one meter long capillary tubing. Then, use the stereotaxic frame to position the tip of the needle at the desired starting location within the brain. Position the camera to display the top of the mesh electronics probe within the glass needle.
Initiate the flow by setting the syringe pump to a low speed and pressing start. Slowly increase the flow rate if the mesh electronics probe does not move within the needle. As the mesh electronics probe starts to move within the needle, use the stereotaxic frame to retract the needle at the same rate with which the mesh electronics probe is being injected using the marked original position of mesh electronics as a guide.
Continue flowing saline and retracting the needle until the needle has exited the skull. Then, stop the flow from the syringe pump. In this procedure, use the stereotaxic frame to carefully guide the needle to the flat, flexible cable clamping substrate in across the gap, flowing the solution with the syringe pump to generate a slack in the mesh electronics interconnects.
Once the needle is above the clamping substrate and across the gap, resume the flow at a fast rate to inject the mesh electronics input, output pads onto the clamping substrate. Using tweezers and a pipette of deionized water, bend the input, output pads to a 90 degree angle as close to the first input, out pad as possible. Once the input, output pads are aligned and folded and at the 90 degree angle to the mesh stem, dry them in place with gently flowing compressed air.
Cut the clamping substrate at a strait edge approximately 0.5 to one millimeters from the edge of the input, output pads. Also, cut off extraneous parts of the clamping substrate that will hinder the insertion into the PCB mounted 32 channel zif connector. Then, insert the input, output pads into the zif connector onto the PCB and close the latch.
Use measurement electronics to measure the impedance between the channels and the ground screw to confirm successful interfacing. If the impedance values are too high, unlatch the zif connector, adjust the insertion, and retest until successful connection is confirmed. Subsequently, cover the zif connector and exposed mesh electronics interconnects with dental cement for protection.
Flip the PCB at the gap in the substrate and fix the PCB with cement onto the mouse skull. For freely moving recordings, release the mouse from the restrainer after inserting the preamplifier PCB and grounding the reference screw. Record for the desired length of time using the data acquisition system while the mouse behaves freely.
Shown here are the representative local field heat maps from the 32 channel mesh electronics probes injected into the mouse hippocampus and somatosensory cortex. Data were recorded while the mouse freely explored its cage at two months and four months post injection. Local field potential amplitude is color coded according to the color bar at the right.
High pass filter traces, black, showing spiking activity are overlayed on the spectrogram for each of the 32 channels. Here are the spike isolated after sorting the data plotted. Single unit spiking activity was detected on 26 of the 32 channels, both two months post injection and four months post injection.
While attempting this procedure, it's important to remember to test the I/O interfacing to confirm successful electrical connection to the mesh electronics probe. The measure impedance helps troubleshoot the source of any problems and is critical to identify issues with interfacing or fabrication. Following this procedure, histology can be performed in order to study the cellular environment around each recorded electrode.
Unlike conventional, rigid brain probes, mesh electronics can be left in the tissue during histology, making it possible to precisely correlate electrophysiology data with immunohistochemical analysis.
