Characterization of the Mechanical Properties of Mouse Brain Tissue Using Atomic Force Microscopy

Take a bioadhesive-coated dish with an immobilized mouse brain slice in a pre-warmed medium.

Place it on a pre-warmed atomic force microscope or AFM stage to maintain physiological conditions.

Apply a drop of the medium onto the spherical bead of the AFM cantilever positioned over the AFM head.

Next, reposition the AFM head and submerge it in the medium.

Align the laser beam onto the cantilever to track cantilever deflection using the photodiode.

Lower the AFM probe until it contacts the brain tissue surface.

For creep-compliance measurement, program the AFM probe to apply a constant force.

This force causes the tissue to deform while the cantilever bends. The laser detects cantilever

bending and measures tissue stiffness.

For force relaxation measurement, indent the tissue to a fixed depth and maintain it.

The laser tracks cantilever bending to measure tissue resistance and relaxation over time, enabling precise evaluation of the tissue’s mechanical properties.

Carefully load an AFM probe with a nominal spring constant of 0.03 newtons per meter and a 20 micrometer diameter borosilicate bead into the probe holder. Place a brain slice mounted in a Petri dish onto an AFM stage-mounted heater that has been pre-warmed to 37 degrees Celsius.

Then, add about 2 milliliters of pre-warmed medium.

Next, carefully add a drop of medium onto the AFM probe to protect it from breaking due to surface tension when it is lowered into the medium surrounding the brain slice. Then, reposition the AFM head onto the stage. And begin lowering the head until it is submerged in the medium.

Using the optical microscope, move the stage so that the region of interest is below the calibrated AFM probe. Then, lower the AFM probe to make contact with the surface of the tissue. In order to conduct the creep compliance experiments, construct an applied force function in the software's function editor.

The function consists of a 0.1 second ramp to a 0.05 nanonewton, which is held for 20 seconds, followed by a 1 second ramp down to 0 nanonewton. The software will record data on the AFM probes indentation into the tissue during the applied force function. After running the creep compliance experiment, conduct force relaxation experiments by creating an applied indentation function in the software. Run this function while the software collects data on the force experienced by the AFM probe as it indents into the tissue.