Atomic force microscopy (AFM) has been used to measure cellular stiffness at different osmolarities to investigate the effect of osmotic pressure on cells. However, substantial direct evidence is essential to clarify the phenomena derived from the experimental results. This study used both the single-point and force mapping methods to measure the effective Young's modulus of the cell by using temporal and spatial information. The single-point force measurements confirmed the positive correlation between cellular stiffness and osmolarity. The force mapping measurements provided local stiffness on the cellular surface and identified the cytoskeleton distribution underneath the plasma membrane. At hyper-osmolarity, the cytoskeleton was observed to cover most of the area underneath the plasma membrane, and the effective Young's modulus on the area with cytoskeleton support was determined to be higher than that at iso-osmolarity. The overall increase in cellular Young's modulus confirmed the occurrence of cytoskeleton compression at hyper-osmolarity. On the other hand, although the average Young's modulus at hypo-osmolarity was lower than that at iso-osmolarity, we observed that the local Young's modulus measured on the areas with cytoskeleton support remained similar from iso-osmolarity to hypo-osmolarity. The reduction of the average Young's modulus at hypo-osmolarity was attributed to reduced cytoskeleton coverage underneath the plasma membrane.

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