To begin, fix the cantilever holder of an AFM onto the cantilever changing stand. Use watchmaker forceps to mount the selected cantilever on the holder. Tighten the screw on the holder to secure the cantilever.
Place and lock the cantilever holder on the AFM head that is resting on the stand. Use the step motor function in the AFM software to withdraw the AFM head to the highest point. and set that position as 0.0.
Carefully lift the AFM head from its stand, then mount it on the sample stage by placing the legs in their respective slots. To calibrate the cantilever, use the step motor function to bring it close to the sample surface. Then click on Approach in the Contact Mode for Spectroscopy window.
Bring down the cantilever in small increments of 15 micrometers. Press Acquire to capture a force curve. Then select the force curve, open it in the Calibration Manager window and select Contact-Based Mode in the Method section.
Now use the select fit range function and select the cantilever retraction curve for a linear curve fit. Next, check the Sensitivity check box to convert the force unit from volt to Newton. Lift the cantilever by 100 to 200 micrometers in the liquid and select the thermal noise function Again, click on Select Fit Range and fit the thermal noise bell curve with a Lawrence Curve.
After curve fitting, choose the Spring Constant K box. Confirm that the spring constant is close to the manufacturer's value and note it down for future reference. Next place the slide mounted sample of rodent eye retinal vessels or subendothelial matrix on the AFM stage and visualize the retinal vessels.
Select Contact Mode for Spectroscopy in the software's Experiment section. Then set the set point force to 0.5 nano-Newtons and click Approach. Use the stage screws on the AFM stage to carefully position the cantilever probe at a desired location on the sample.
Click Approach to position the cantilever closer to the sample, and then click Acquire to capture the force curves. Finally, save all force curves for analysis. The approach and retraction curves obtained from a retinal capillary isolated from a diabetic mouse were steeper than those obtained from a non-diabetic mouse.
