Atomic force microscopy, or AFM cantilever-based nanoindentation, can be used to determine the nanoscale mechanical properties of materials ranging modulus, from kilopascals to gigapascals in both air and fluid. AFM cantilever-based nanoindentation enables co-localized topographical imaging and in situ quantitative mechanical property measurements with nanoscale precision and resolution on a wide range of materials and relevant environments. AFM cantilever-based nanoindentation can be used to differentiate between healthy and disease structures, tissues, or cells that exhibit divergent mechanical properties.
Accurately determine the tip sample contact area and force applied during cantilever-based nanoindentation requires careful calibration of the AFM probe, which is challenging but essential for quantitative nanoscale mechanical property measurements. To begin, select an appropriate atomic force microscopy or AFM probe for nanoindentation of the intended sample based on the medium, expected modulus, sample topography, and relevant feature sizes. Load the probe onto the probe holder and attach the probe holder to the AFM scan head.
Select an appropriate nanoindentation mode in the AFM software that affords user control of individual ramps. Align the laser onto the back of the probe cantilever opposite the location of the probe tip and into the position-sensitive detector, or PSD. Center the laser beam spot on the back of the cantilever by maximizing the sum voltage.
Next, center the reflected laser beam spot on the PSD by adjusting the vertical and horizontal deflection signals to be as close to zero as possible, thereby providing the maximum detectable deflection range for producing an output voltage proportional to the cantilever deflection. Calibrate the deflection sensitivity, or DS, of the probe AFM system. To do this, set up and perform DS calibration indents on Sapphire to achieve approximately the same probe deflection as the planned sample indents since the measured displacement is a function of the tip deflection angle and becomes nonlinear for large deflections.
Repeat the ramp five times. Determine the DS in nanometers per volt or the inverse optical lever sensitivity and volts per nanometer from the slope of the linear portion of the in-contact regime after the initial contact point in the resulting force displacement, or FD, curve. Use the average of the values for maximum accuracy.
If the relative standard deviation exceeds 1%remeasure the DS, as sometimes the first few FD curves are non-ideal due to the initial introduction of adhesive forces. If the probe cantilever's spring constant K is not factory-calibrated, calibrate the spring constant. If the probe does not have a factory-calibrated tip radius measurement, measure the effective tip radius R.If employing the blind tip reconstruction method, image the tip characterization or roughness sample using a slow scan rate and high feedback gains to help optimize tracking of the very sharp features.
Choose an image size and pixel density based on the expected tip radius. Next, use AFM image analysis software to model the probe tip and estimate its end radius and effective tip diameter at the expected sample indentation depth. Upon completing the probe calibration, enter the DS, K, and R values in the software.
Finally, enter an estimate of the sample's Poisson's ratio to convert the measured reduced modulus to the actual sample modulus. If employing a conical or conospherical contact mechanics model based on the tip shape and indentation depth, entering the tip half-angle is necessary. Navigate the sample under the AFM head and engage on the desired region of interest.
Monitor the vertical deflection signal or perform a small initial ramp to verify that the tip and sample are in contact. Adjust the AFM head position slightly upward and ramp again. Repeat until the tip and sample are just out of contact, as evidenced by a nearly flat ramp and minimal vertical deflection of the cantilever.
Once no obvious tip sample interaction is present, lower the AFM head by an amount corresponding to approximately 50%of the ramp size to ensure the probe tip will not crash into the sample while manually moving the AFM head. Ramp again, repeating until a good curve is observed. Adjust the ramp parameters.
Select an appropriate ramp size depending on the sample and desired indentation depth. Then, select an appropriate ramp rate. One hertz is a good starting point for most samples.
Set the number of samples per ramp to achieve the desired resolution of the measurement. Set the X Rotate to reduce the shear forces on the sample and tip by simultaneously moving the probe slightly in the X direction while indenting in the Z direction. Use a value for the X Rotate equal to the offset angle of the probe holder relative to the surface normal.
Next, choose whether to employ a triggered or untriggered ramp. If a triggered ramp is chosen, set the trigger threshold to result in the desired indentation into the sample. Perform an indent in the chosen location.
Select and load the data to be analyzed in the software. Input calibrated values for the spring constant, DS, or inverse optical lever sensitivity, and probe tip radius, along with an estimate for the Poisson's ratio of the sample. Choose a nanoindentation contact mechanics model appropriate for the tip and sample.
Run the fitting algorithm. Check for proper fitting of the FD curves. A low residual error corresponding to an average R-squared near unity indicates a good fit to the chosen model.
If desired, spot check individual curves to visually inspect the curve, model fit, and calculated contact points. Near-ideal FD curves obtained in the air on a resin-embedded loblolly pine sample and in phosphate-buffered saline solution on a mesenchymal stem cell nucleus are shown. With the silicon probe, the tip experienced significant wear relative to its initial pristine state throughout imaging.
With each subsequent image, the tip becomes progressively more rounded. In contrast with the diamond probe, the tip radius did not change within the blind tip reconstruction method's limits, highlighting the diamond's extreme wear resistance. An AFM topography image covering multiple cells with a pseudo 3D depiction and a corresponding modulus map of a loblolly pine sample is shown here.
AFM-derived modulus map generated while obtaining an AFM image shows the mineral inclusion in the center of images is significantly harder than the surrounding organic matrix. The effect of probe tip radius and shape on the appearance of high-aspect ratio features of a Bakken shale sample is shown. Cantilever-based nanoindentation on mesenchymal stem cells and isolated nuclei exhibited no statistical difference in elastic modulus.
The morphology and mechanical properties of cholesterol containing lipid bilayers studied by AFM are demonstrated here. Proper probe calibration and selection of appropriate ramp parameters in contact mechanics models are essential to accurate nanomechanical measurements. In addition to enabling elastic modular measurements, AFM cantilever-based nanoindentation can be used to probe the rupture strength or breakthrough force of phospholipid bilayers under physiological relevant conditions.
AFM cantilever-based nanoindentation has enabled investigation of the effects of structural knockouts, pharmaceutical treatments, and low-intensity vibrations to simulate exercise on the mechanical properties of mesenchymal stem cell nuclei.
