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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
- Impact Indentation
- Calibrate the instrumented nanoindenter and adjust default settings to enable dynamic impact experiments on hydrated brain tissues according to the manufacturer's instructions.
- Mount a spherical probe by sliding it onto the pendulum using tweezers.
- Glue a fused quartz sample onto the sample post, which is screwed into the translational stage.
- Go to the Calibration menu and select "Liquid Cell." Follow the software's instructions to make contact with the fused quartz sample.
- Select "Normal" for the Indenter Type and use the default value of 0.05 mN for the Indenter Load. Click "Continue" to perform the calibration for the normal indenter configuration.
- Move the sample stage back by at least 5 mm. Mount the lever arm, which allows the probe to be lowered into the liquid cell, and repeat the liquid cell calibration in the new configuration by selecting "Liquid Cell" for the Indenter Type. Click "Continue" to obtain the Liquid Cell Calibration Factor.
- Activate the Liquid Cell software option by going to the Experiment menu and selecting "Special Options." Use the latest calibration value.
- Increase the capacitor plate spacing, as this will lead to a greater maximum measurable depth, which is necessary when testing highly compliant materials.
- Under the System menu, select "Non Protected Settings" and "Machine Parameters" to change the pendulum test load rate, zero load rate, and standby ramp offset to 0.5 mN/sec, 0.1 mN/sec, and 3 V, respectively.
- With a wrench, turn the three nuts that control the capacitor plate spacing clockwise in small increments.
- After each complete clockwise turn, select "Bridge Box Adjustment" under the Maintenance menu and obtain a good pendulum test, which will require moving the counter-balance weight away from the pendulum.
- Repeat steps 1.1.7.2-1.1.7.3 until the approximate depth calibration reads a value of 70,000 nm/V or higher.
- Position a new limit stop at the bottom of the pendulum that can be switched on and off via a power supply. Retract the original limit stop sitting behind the pendulum to remove a potential obstruction of the pendulum motion and allow for higher impact velocities as well as higher penetration depths into compliant samples.
- Allow the cabinet to reach thermal equilibrium (takes approximately 1 hr).
- While the cabinet equilibrates, go back to the System menu and select "Non-Protected Settings" and "Machine Parameters." Set the depth calibration (dcal) contact velocity to 1 µm/sec, the primary indentation contact velocity to 3 µm/sec, and the ultra-low load contact velocity to 1 µm/sec.
- Under the Calibration menu, perform a standard depth calibration in this new configuration.
- Turn on the power supply for the solenoid and set it to 10 V. Go to the Experiment menu and select "Impact" and "Adjust Impulse Displacement." Follow the software instructions (automatic prompts) to calibrate the swing distance of the pendulum.
- Mount the mouse brain tissue in the liquid cell.
- After harvesting the whole brain, store it immediately in a CO2-independent nutrient medium for adult neural tissue media on ice.
- When the impact indentation setup is fully complete, carefully transfer the brain into a petri dish along with a CO2-independent medium. Slice the brain into 6 mm-thick sections with flat surfaces on either side.
- Adhere the sliced tissue to the aluminum sample post with a thin layer of cyanoacrylate adhesive.
- Slide the liquid cell over the second O-ring on the sample post, and fill the liquid cell with 5 ml of CO2-independent medium to fully immerse the tissue. This sample post is then carefully mounted onto the translational stage inside the instrumented nanoindenter.
- Measure the impact response of the brain tissue.
- If necessary, remove the spherical probe and replace it with the probe of interest without removing the lever arm.
- Under the System menu, select "Non-Protected Settings" and "Machine Parameters." Change the primary impact contact velocity to 5 µm/sec.
- With the sample bath low (-z-direction) and far away from the pendulum (+x direction), move in the -x direction until the tip of the lever arm is properly located above the bath. Move in the +z direction until the tip is fully submerged in the bath and in front of the sample.
- Using the sample stage control window, make contact carefully and then back the stage away from the sample surface by approximately 30 µm.
- Under the Experiment menu, click "Impact" to set up an impact experiment. Choose a specific impulse load that will relate directly to the resulting impact velocity based on the swing distance calibration. Run the scheduled experiment.
- When the pendulum swings back, and the sample surface continues to move to the measurement plane, turn the bottom limit stop switch off.
- Observe as the pendulum swings forward to impact the sample. The displacement of the probe as a function of time will be recorded by the software.
- When the xyz stage window appears, turn the limit stop switch back on.
- Repeat steps 3.4-3.8 to test as many different loads and locations as needed.
- Analyze the acquired displacement vs. time response of the pendulum using customized MATLAB scripts to determine the maximum penetration depth xmax, energy dissipation capacity K, and dissipation quality factor Q.
- Go to the Analysis menu and export the data in a text file.
- Take the time derivative of the displacement profile to obtain velocity as a function of time. Set zero displacement as the contact point xo1.
NOTE: Impact velocity vin is the maximum velocity immediately prior to contact. xmax corresponds to the deformation at which the probe velocity first decreases to zero. xo2, which is equivalent to xr, is the position required to reinitiate contact with the deformed sample in the next cycle. Rebound velocity vout is the velocity at displacement xr. - Define K (unitless) as the energy dissipated by the sample normalized by the sum of the recovered and dissipated sample energies during the first impact cycle. Calculate K based on the intrinsic properties of the pendulum (such as rotational stiffness and damping coefficient), xo1, xmax, xr, vin, and vout.
- Since displacement can be described as a damped harmonic oscillatory motion, fit an exponential decay function to the maxima of the displacement vs. time curve.
- Calculate Q (unitless) as π multiplied by the number of cycles required for the oscillation amplitude to decrease by a factor of e. A higher Q value means a lower energy dissipation rate.