Experimental validation of atomic force microscopy-based cell elasticity measurements

Abstract

Atomic force microscopy (AFM) is widely used for measuring the elasticity of living cells yielding values ranging from 100 Pa to 100 kPa, much larger than those obtained using bead-tracking microrheology or micropipette aspiration (100-500 Pa). AFM elasticity measurements appear dependent on tip geometry with pyramidal tips yielding elasticities 2-3 fold larger than spherical tips, an effect generally attributed to the larger contact area of spherical tips. In AFM elasticity measurements, experimental force-indentation curves are analyzed using contact mechanics models that infer the tip-cell contact area from the tip geometry and indentation depth. The validity of these assumptions has never been verified. Here we utilize combined AFM-confocal microscopy of epithelial cells expressing a GFP-tagged membrane marker to directly characterize the indentation geometry and measure the indentation depth. Comparison with data derived from AFM force-indentation curves showed that the experimentally measured contact area for spherical tips agrees well with predicted values, whereas for pyramidal tips, the contact area can be grossly underestimated at forces larger than ∼0.2 nN leading to a greater than two-fold overestimation of elasticity. These data suggest that a re-examination of absolute cellular elasticities reported in the literature may be necessary and we suggest guidelines for avoiding elasticity measurement artefacts introduced by extraneous cantilever-cell contact.

Mechanical Criterion for the Rupture of a Cell Membrane under Compression

“…In our simulations of cell indentation, the endothelial cell is modeled as a nearly incompressible (14,27) hyperelastic neo-Hookean (13,28,29) material, with a Poisson's ratio of 0.49 and a Young's modulus of 1 kPa. The microindenter's spherical tip is considered infinitely rigid compared to the cell.…”

Nanomechanical and topographical imaging of living cells by atomic force microscopy with colloidal probes

“…40 Moreover, it has been demonstrated that it is possible to detect length and grafting density of the brush layer on living cells, 65,66 or even to distinguish between cancerous and healthy cells' brushes, 67 thanks to the sensitivity of micrometric spherical probes provided by their wider surface area.…”

Atomic Force Microscopy in Characterizing Cell Mechanics for Biomedical Applications: A Review

“…The first factor is the AFM probe, such as tip shape and spring constant of cantilever. Harris and Charras [71] used both standard conical cantilevers and the same cantilevers modified with a spherical tip to measure cell Young's modulus, showing that Young's modulus measured by conical tips was 2-3 fold larger than that measured by spherical tips. Vargas-Pinto et al [72] have also shown that the Young's modulus of human umbilical vein endothelial cells (HUVEC) and Schlemm's canal (SC) endothelial cells measured by spherical tips (0.71 ± 0.16 kPa and 0.94 ± 0.06 kPa) was significantly smaller than that measured by conical tips (3.23±0.54 kPa and 6.67±1.07 kPa).…”