Experimental Evidence of Substrate-Induced Artifacts in Atomic Force Microscopy Nanomechanics of Viscoelastic Cells

Atomic force microscopy (AFM) is a pivotal tool for probing the nanomechanical properties of cells, yet the influence of substrate rigidity on viscoelastic samples such as biological material has remained largely experimentally unverified. The present study demonstrates that the stiffness of the underlying substrate significantly alters the forces measured by AFM on single cells, leading to an artificial overestimation of cellular modulus when using traditional, not corrected, semi-infinite contact mechanics models. By employing a finite-thickness rheological model with bottom-effect corrections, we show that the apparent increase in modulus with applied force observed in both cytoplasmic and nuclear regions of HeLa cells is an artifact of substrate interactions. Our findings reveal that the contact radius-to-cell height ratio governs this effect. These results demonstrate the necessity of incorporating substrate corrections in AFM-based mechanical analyses to ensure accurate measurements of cellular properties.