Prior to atomic force microscopy or AFM measurements, mount the cantilever onto a glass block. Insert a Petri dish filled with PBS into the holder of the motor stage and insert the glass block into the AFM head. Then position the AFM head over the Petri dish so that the cantilever is immersed into the buffer and align the laser manually.
In the JPK software, press the approach and acquire button to advance the cantilever onto a hard surface. Once the single force-distance curve is acquired, open the calibration manager and under method, select contact-based. Then adjust the room temperature to 20 degrees Celsius.
Now zoom in on the curve and select the linear part for the sensitivity fit. Click on the sensitivity check box and retract the cantilever 200 micrometers from the surface. Then select the infinity symbol checkbox and click Run Thermal Noise.
Next, zoom in on the resulting frequency graph and define a range using the right-click button of the mouse. Select the Spring Constant check box and close the calibration manager. Insert the Petri dish containing the regenerated axolotl limb embedded in the tissue block and culture medium into the dish holder of the AFM.
Acquire an overview image of the tissue block in bright-field mode. Zoom into the region of interest and adjust the exposure time and focus. Set up the grid parameters for indentation measurements and record an array of force-distance curves.
For data processing in the PyJibe software, open the file containing the force displacement curve. Select the preprocess tab and under the contact point estimation, select piecewise fit with line and polynomial. Then select the fit tab and under model, select spherical indenter, Under method, select nelder.
For viscoelastic analysis in the PyJibe software, open the file containing the force distance curve. Select the preprocess tab and under the contact point estimation, select piecewise fit with line and polynomial. Then select the fit tab and under model, select the Hertz model corrected for viscoelasticity using KVM model.
Under method, select leastsq. Set the accurate indenter radius to 10 micrometers and approach speed to 7.5 micrometers per second. Press Apply Model and Fit All to obtain apparent and unrelaxed Young's moduli, apparent viscosity, and Maxwell element relaxation time.
Apparent Young's modulus measurements of the radius and ulna in intact limbs showed no significant difference. During the histolysis phase, the apparent Young's moduli of the radius and ulna dramatically decreased to 0.03 and 0.13 kilopascals respectively. The apparent Young's moduli in the center of intact cartilage were higher than in the periphery.
At the histolysis stage, apparent Young's modulus measurements showed no significant difference between the cartilage center and periphery. During cartilage condensation, apparent Young's moduli significantly increased to 0.77 kilopascals representing intermediate stiffness values. Unrelaxed moduli showed substantial differences across intact tissue during histolysis and in condensing cartilage.
The apparent Young's moduli were highly similar to unrelaxed moduli, indicating a predominantly elastic response. Apparent viscosity was significantly lower during histolysis compared to intact tissue and condensing cartilage.
