Evidence of the bottom stiffness effect on atomic force microscopy-based cell mechanobiology.

AFM is the dominant method to characterize the nanomechanical properties of cells. These properties are obtained by model fitting. Semi-infinite contact mechanics models predict that the force depends on the cell's mechanical properties, indentation and the tip's geometry. Finite-thickness rheological models predict that the force should depend also on the rigidity of the substrate. The latter property has never been observed experimentally. It would make cells appear stiffer than they are. Here, we designed a force-distance curve experiment to reveal the influence of the rigidity of the substrate on the forces and the apparent moduli measured by AFM. Model fitting by using a semi-infinite power-law rheological model showed an increase of the apparent modulus with increasing force. This behavior was an artifact which disappeared when the force was fit with a bottom-effect correction model. Our findings demonstrated that the force applied on a cell depended intrinsically on the stiffness of the substrate while the mechanical properties (true values) did not.