Assessing micromechanical properties of cells with atomic force microscopy: importance of the contact point

Mechanical properties are obtainable from atomic force microscopy (AFM) indentation force-depth curves, which are calculated from relationships between tip deflection and cantilever position, i.e. deflection curves. Indentation depth is the difference between tip deflections on a rigid and a soft material for the same amount of cantilever advancement, after contact is made. Since the contact point cannot be unequivocally identified from experimental data, there is some uncertainty in estimating material properties. Using simulations, this study examines some important issues related to the influence of contact point identification on estimated material properties. Simulations for linear materials using a typical stiffness for an AFM cantilever demonstrate that certain portions of the post-contact region of deflection curves for soft and very stiff materials can be approximated by quadratic and linear functions, respectively. Based on these findings, we first develop and verify an objective, automatic method to identify the contact point for materials with linear properties. We then assess the effect of misidentifying the contact point, with and without noise. If the contact point is missed by <50 nm, material properties for small indentations are erroneous but the error decreases asymptotically beyond 200 nm of indentation and the correct estimate of material stiffness is obtained. If the contact point is missed by >100 nm, however, the true material properties cannot be estimated accurately. Noise adds to uncertainty in material properties at small indentations but the combined effect of missing the contact point and noise is dominated by the former. Even though the algorithm was developed for linear materials, it is also suitable for certain nonlinear materials making it more generally applicable.