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All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Atomic Force Microscope (AFM)-enabled Indentation
- Prepare 60 mm-diameter Petri (P60) dishes with a mussel-derived bioadhesive according to the manufacturer's instructions.
- Prepare a stock of neutral buffer solution consisting of 0.1 M sodium bicarbonate in sterile water with an optimal pH of 8.0. Filter-sterilize (0.2 microns) the sodium bicarbonate buffer and store it at 4 °C.
- In a laminar flow hood, mix a solution of 6.25% mussel-derived bio-adhesive and 3.125% sodium hydroxide (NaOH) in the sodium bicarbonate buffer.
- Pipette 100μl of the bio-adhesive solution onto a 60 mm-diameter Petri (P60) dish and use a pipette tip to spread the solution into a 3-5 cm diameter circle.
- Leave P60 dishes uncovered in a laminar flow hood and let the solution dry (~30 min). Wash dishes 1x with phosphate-buffered saline (PBS) and 2x with sterile water. Let dishes air dry in a laminar flow hood and store them in a sealed plastic bag at 4 °C for up to 1 month.
- Calibrate the AFM and set up a brain sample in the AFM. NOTE: Follow AFM calibration instructions as per the manufacturer.
- Carefully load an AFM probe with a nominal spring constant of 0.03 N/m and a 20 μm-diameter borosilicate bead into the probe holder.
- Calibrate the spring constant and inverse optical lever sensitivity (InvOLS) of the AFM cantilever using the thermal tune method.
NOTE: Once the spring constant for an AFM probe is calculated, it should remain constant with repeated use. However, the cantilever InvOLS will need to be recalibrated each time the laser is realigned with the cantilever. Additionally, calibration should be performed against a substrate several orders of magnitude stiffer than the cantilever, such as polystyrene. - Turn on the stage-mounted heater and set the temperature to 37 °C.
- Mount the brain slice onto the P60 dishes.
- Gently pour a 350 μm-thick brain slice and the carbon dioxide (CO2)-independent medium from the round-bottom flask into a P60 dish coated with the mussel-derived bioadhesive.
- Position the brain slice in the center of the P60 dish by gently tilting it. If necessary, slowly pipette the medium from a manual pipette to unfold a brain slice that has folded over on itself, or better yet, position the brain slice in the center of the dish.
- Carefully remove excess media using a P1000 pipette (do not use the vacuum).
- Place a cover on the P60 dish and let the brain slice adhere for 20 min.
- Remove the AFM head, place the brain slice mounted in the P60 dish on the AFM stage, and add ~2 ml pre-warmed CO2-independent medium.
- Carefully add a drop of media onto the AFM probe to protect it from breaking due to surface tension when lowered into the media surrounding the brain slice.
- Reposition the AFM head onto the stage and begin lowering the head until it is submerged into the media.
- Using the top-view charge-coupled device (CCD) camera, reposition the laser onto the cantilever.
NOTE: The alignment of the laser on the cantilever will have changed slightly due to the difference in refractive index of air and medium. - Wait 5 min for the cantilever to adjust to being submerged in a warm liquid, then reset the mirror alignment to a free deflection of 0 V.
- Run a thermal spectrum on the AFM probe according to the manufacturer's instructions. Use the fit of the first thermal peak to recalculate the AFM probe's InvOLS in media.
- Using the optical microscope, move the sample stage so the brain region of interest is below the AFM probe. NOTE: The corpus callosum will appear dark as it is more opaque than the surrounding gray matter. The cortex is superior to the corpus callosum.
- Reset the mirror alignment to a free deflection of 0 V.
- On the Sum and Deflection Meter in the AFM software, click "Engage" to engage the AFM head.
- Using the position dial on the AFM head, lower the head until contact is made between the cantilever and the sample.
- Conduct creep compliance experiments.
- Construct an applied force function in the software's function editor. The force function consists of a 0.1-sec ramp to a set point of 5 nN and holding it for 20 sec, followed by a 1-sec ramp down to an applied force of 0 nN.
- On the Indentation Master Panel, under the indentation method, select "Load" for Indenter Mode, "N" for units, and "Function editor" for Indenter Function.
- In the function editor, on the Segment Parms Panel, create an applied force function segment that starts at 0 nN and ends at 5 nN, with a time of 0.1 sec. Click "Insert -->."
- For the next segment, set the start to 5 nN, end to 5 nN, and time to 20 sec. Click "Insert -->."
- For the final segment, set the start to 5 nN, the end to 0 nN, and the time to 1 sec. Then, click "Draw" and close the Function Editor window.
- On the Force Tab of the Master Panel, check "indenter ramp after trigger" and set the applied force function to trigger after reaching a trigger point of 0.1 V.
- Click "Single Force" at the bottom of the Force Tab of the Master Panel, which will trigger the constructed-applied force function for creep compliance.
- After the single force indentation is finished, raise the AFM head so that it is out of contact with the sample. Then, re-engage the head and re-zero free deflection.
- Reposition the sample stage to locate a new area of interest and lower the AFM head to make contact. NOTE: The AFM head must be retracted from the sample surface when the sample stage is moved. Failure to do so can result in damage to the delicate AFM cantilever.
- Repeat steps until the desired amount of data has been collected.
- Conduct force relaxation experiments.
- Construct an applied indentation function in the software's function editor. The indentation function consists of a 0.1-sec ramp to a set point of 3 μm and holding it for 20 sec, followed by a 1-sec ramp down to an indentation depth of 0 µm.
- On the Indentation Master Panel, under the indentation method, select "Indentation" for Indenter Mode, "m" for units, and "Function editor" for Indenter Function.
- In the function editor, on the Segment Parms Panel, create an applied force function segment that starts at 0 µm and ends at 3 µm, with a time of 0.1 sec. Click "Insert -->".
- For the next segment, set the start to 3 µm, end to 3 µm, and time to 20 sec. Click "Insert -->."
- For the final segment, set the start to 3 µm, the end to 0 µm, and the time to 1 sec. Then click "Draw" and close the Function Editor window.
- On the Force Tab of the Master Panel, check "indenter ramp after trigger" and set the applied force function to trigger after reaching a trigger point of 0.1 V.
- Click "Single Force" at the bottom of the Force Tab of the Master Panel to trigger the constructed-applied indentation function for force relaxation.
- After the single-force indentation is finished, raise the AFM head so that it is out of contact with the sample, then re-engage the head and re-zero deflection.
- Reposition the stage to locate a new area of interest and lower the head to make contact.
- Repeat both measurements until the desired amount of data has been collected.