Influence of cantilever tip geometry and contact model on AFM elasticity measurement of cells.

We have measured the elastic properties of live cells by Atomic Force Microscope (AFM) using different tip geometries commonly used in AFM studies. Soft 4-sided pyramidal probes (spring constant = 12 and 30 mN/m, radius 20 nm), 3-sided pyramidal probes (spring constant = 100 mN/m, radius 65-75 nm), flat (circular) probes (spring constant = 63 mN/m, radius 290 nm) and spherical probes (spring constant = 43 mN/m, radius 5 μm) have been used. Cells (3T3 fibroblasts) having elastic moduli around 0.5 kPa were investigated. We found that cell measured stiffness shows a systematic dependence on tip geometry: the sharper the tip, the higher the average modulus values. We hypothesize that the blunter the tip, the larger the contact area over which the mechanical response is measured or averaged. If there are small-scale stiffer areas (like actin bundles) they will be easier to pick up by a sharp probe. This effect can be seen in the wider distribution of the histograms of the measured elastic moduli on cells. Furthermore, non-linear responses of cells may be present due to the high average pressures applied by sharp probes, which would lead to an overestimation of the Young's modulus. Pressure versus contact radius simulations for the different tip geometries for a 0.5 kPa sample suggested similar average pressure for Bio-MLCTs, PFQNM and cut tips, except spherical tips that showed much lower average pressure at the same 400 nm indentation. However, real data of the cells suggested different results. Using the same indentation depth (400 nm), PFQNM and Bio-MLCTs showed similar average pressure and it decreased for cut and spherical tips. The calculated contact area at 400 nm cell indentation, using the obtained apparent Young's modulus for each tip geometry, showed the following distribution: Bio-MLCTs < PFQNM < cut << spherical. In summary, tip geometry as well as average pressure and tip-sample contact area are important parameters to take into account when measuring mechanical properties of soft samples. The larger the tip radius, the larger the contact area that will lead to a more evenly distribution of the applied pressure.