The use of focused ion beam lithography, or FIB, allows researchers to improve the cell surface interaction by designing material surfaces that have increased biocompatibility and integration with the native tissue. With FIB, features can be etched on a variety of materials, such as silicon, metals, and polymers. Non-planar surfaces can also be used and post-processing can be performed on individual devices.
In general, this method can be used on other microscopes. However, different microscope manufacturers have unique versions of the patterning software, which must be optimized for each system. Demonstrating the procedure, will be Miss Shreya Mahajan, a graduate student from my laboratory.
To prepare for mounting, use fine-tipped bore vacuum forceps to carefully pick up the clean strip of silicon probes. Place them onto a clean aluminum stub used for scanning electron microscope FIB imaging and etching. Use a toothpick to place a tiny drop of silver paint on the edge of the silicon substrate surrounding the probe.
Secure the strip 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. Next, click on the Vent button in the Beam Control tab to vent the SEM FIB chamber.
Press Shift F3 to perform home stage. Confirm the selection, by selecting the Home Stage button in the pop-up window. After the Home Stage is complete, move the stage to coordinates X=70 millimeters, Y=70 millimeters, Z=0 millimeters, T=0 degrees, R=0 degrees.
Once the chamber is vented, put on clean nitrile gloves and open the chamber door. 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.
Ensure that the height is adjusted appropriately. Use the 1.5 millimeter hex wrench for this task. 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 Three of the microscope user interface. Once the brightness level auto-adjusts to an appropriate level, acquire the image by pushing the button down on the camera bracket.
This takes approximately 10 seconds. Swing the camera arm back to the closed position. The stage will return to the original position.
Carefully close the microscope chamber door. Watch the CCD camera image in Quadrant Four while closing the door. Ensure that the samples and the stage are a safe distance away from any critical component in the microscope chamber.
Select the Pump with Sample Cleaning button in the user interface software to start the chamber vacuum pump and built-in plasma cleaner. Wait for approximately eight minutes for the pumping time and plasma cleaning cycle of the microscope chamber to be completed. Once the icon in the bottom right corner of the user interface turns green, press the Wake Up button in the beam control tab which turns on the electron and ion beams.
Select Quadrant One and set the beam signal to Electron Beam. Then set Quadrant Two to Ion Beam. Now, set the SEM voltage to five kilovolts, the SEM beam current to 0.20 nanoamps, the SEM Detector to ETD, and the Detector Mode to Secondary Electrons.
Set the FIB Voltage to 30 kilovolts, the FIB beam to 24 picoamps, the FIB Detector to ICE Detector, and Detector Mode to the Secondary Electron. Double click on the silicon probe in the navigation camera image, Quadrant Three to move the stage to the approximate location of the probe. Click on Quadrant One to select it as the active quadrant and hit the pause button to start SEM scanning.
Set the scan dwell time to 300 nanoseconds and turn off Scan Interlacing, Line Integration, and Frame Averaging. Set Scan Rotation to zero in the Beam Control tab. Right click on the Beam Shift 2D Adjuster and select zero.
Adjust the magnification to the minimum value by turning the magnification knob counterclockwise on the microscope user interface panel. Adjust the image brightness and contrast using the knobs on the user interface panel or the auto-contrast brightness toolbar icon. Move the stage by double left clicking the mouse on a feature to center it.
Then move the desired silicon probe to be patterned into the center of the SEM image. Locate an edge or other feature, such as a dust particle or scratch. Increase magnification to 2000x by turning the magnification knob clockwise.
Adjust the focus of the SEM by turning the coarse and fine focus knobs on the microscope user interface, until the image is in focus. Once the image is in focus, select the Link sample Z to working distance button in the toolbar. Confirm that operation was completed by looking at the Z-axis coordinate in the navigation tab.
The value should be approximately 11 millimeters. Type in 4.0 millimeters in the Z-axis position and push the Go To button with the mouse. 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 degrees 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 center of the SEM image. Only adjust the Z position. Do not move the X, Y, T, or R axis.
Run the built-in XT Align Feature, located in the stage dropdown menu. Use the mouse to click on two points parallel to the edge of the probe. Mae sure the horizontal radio button is selected in the pop-up window and click Finish.
The stage will rotate to align the probe with the X-axis of the stage. Adjust the stage in XY by using the mouse to put the lower shoulder of the probe in the center of the SEM image again. Select the FIB in Quadrant Two and make sure the beam current is still 24 picoamps.
Set the magnification to 5000X and the dwell time to 100 nanoseconds. Type Control-F on the keyboard to set the FIB focus to 13 millimeters. In the Beam Control tab, right click in the Stigmator 2D adjuster and select zero.
Also, right click in the Beam Shift 2D adjuster, and select zero. Set the scan rotation to zero degrees and click the auto-contrast brightness button in the toolbar. Look for an image of the probe shoulder in Quadrant Two.
Use the snapshot tool to acquire an image in 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 to 15 times. Take another snapshot and observe whether the probe side is still in the center of the FIB. After repeating these steps until the edge of the probe shoulder is perfectly aligned with the X-axis of the stage, use the FIB to 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 nanoamps 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 nanoseconds.
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 user interface panel. Hit the pause button to stop the FIB scanning.
Within the Nanobuilder software, open the file for Patterning the Silicon Probes. Select the Microscope dropdown menu and select Set Stage Origin. Select the Microscope dropdown menu and then select Calibrate Detectors.
On the microscope user interface, click on Quad One once with the mouse to select Quad One. Click OK to start the calibration. The process will take about five minutes.
Make sure the ETD and ICE detectors calibrate. Within the software, select the Microscope dropdown menu and choose Execute to start the patterning sequence. When the pattern is complete, close the software.
Hit Vent in the microscope user interface Beam Control tab to shut down the microscope beams and start the vent cycle. While the chamber is venting, move the stage to the suitable coordinates. Once the chamber is vented, put on clean nitrile gloves and pull open the chamber door.
Loosen the set screw on the stub adapter using the 1.5 millimeter 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 Four while closing the door. Ensure that the stage adapter is a safe distance away from any critical component in the microscope chamber. Shown here are SEM images of the non-functional single-shank silicon probes with FIB-etched nano-architectures along the back side of the shank.
The final dimensions of the etched nano-architecture were 200 nanometer wide parallel lines spaced 300 nanometers apart and had a depth of 200 nanometers. To determine how etching nano-architectures into the probe's surface affects the neuronal density immediately around the implant, the neuronal nucleii were stained and quantified. Neuronal survival is presented as a percentage of the background region from the same animals distances of 50 micron bins away from the implant site.
There were significantly more neurons around the nano-architecture probes at 100 to 150 micron distances from the implant site compared with the smooth control implants at four weeks post-implantation. Nano-architectures were also FIB etched along the back side of functional single shank silicon microelectrodes and electrophysiological measurements were quantified to investigate how etching nano-architectures affects the electrode performance. Electrophysiological results showed increased percentages of channels recording single units from the nano-architecture microelectrodes compared with the smooth control microelectrodes.
No statistical analysis was performed for the nano-architecture microelectrode because only one was implanted for a proof of concept pilot study. FIB etching allows researchers to investigate how the addition of topographical cues on medical devices can improve the cellular response and ultimately, the performance of the device. The breadth of medical devices that these methods can be applied to is limitless, since FIB can be performed on a range of materials, surface geometries, and most importantly, already manufactured devices.
