title: 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.
authors.affiliation: London Centre for Nanotechnology, University College London, London WC1H 0AH, UK.
authors.forename: Andrew R
authors.initials: AR
authors.lastname: Harris
authors.forename: G T
authors.initials: GT
authors.lastname: Charras
mesh_terms: D000818:Animals; D002460:Cell Line; D004285:Dogs; D004548:Elasticity; D004847:Epithelial Cells / Q000166:cytology*; D018625:Microscopy, Atomic Force / Q000379:methods*; D015203:Reproducibility of Results