Cell stiffness measurement by atomic force microscopy

An atomic force microscope (AFM; Bruker Catalyst) combined with an inverted optical microscope (Nikon) was used to measure cell stiffness. The silicon nitride cantilevers with a spring constant k of 0.02 to 0.08 N/m and tip radius of 20 nm were chosen to probe the cells in a 60-mm dish for the experiment at room temperature. The scan size for all measurements was set to 0 to maintain a constant position over a cell, and the tip was brought into contact with the cell at the indicated regions. The force–indentation curves were recorded at 1 Hz to estimate the cell stiffness. For each cell, the force–indentation curves were obtained from at least 5 different cellular locations and the averaged value was used to represent the stiffness for this cell. The force F between the tip and the cell was the product of the cantilever deflection δ and k, i.e., F = k × δ. The Young’s modulus E could be calculated by fitting the force–indentation curves with Hertzian model for a pyramidal tip, i.e., F = 2/π × tan(α) × E/(1 − v²) × d², where α is the half tip angle, the Poisson's ratio v is set to 0.5, and d is the indentation depth. d was kept within 500 nm at 1 Hz to avoid potential substrate effects and cell damage.