Figure 4.

Applicability of the method to nonadherent HFF cells. (A) Schematic representation of a nonadherent cell being slightly deformed by a tipless AFM cantilever. When deforming the spherical cell, changes in laser location in the photodetector are acquired and transformed to deflection in length units. F is the applied normal force, d is the cantilever deflection, Z is the piezo movement, k_c is the cantilever spring constant, R is the initial cell radius, and h is the cortical actin thickness. (B) Velocity-dependent compression force curves performed on the same HFF cell. Successive curves show negligible viscous losses with negligible deviation from each other for deformations <∼400 nm. (C) Typical stress-relaxation curve performed on a nonadherent untreated HFF cell. The tipless cantilever approaches and is pushed against the spherical cell until a deformation of ∼500 nm is reached, then the cantilever is held at a constant height for 10 s. The marked area shows the comparison of rapid compression 4 μm/s (equal to 1 s compression of cell) to the region of fast decay in force immediately after holding the cantilever height constant. The decay is moderately small, ≤20% the maximum force. (D) Acquired force-distance curve on a HFF cell. (Inset) Bright field image of the HFF cells to identify the location and viability as well as to determine the cell radius. The red slope in the plot shows the linear region that is fitted to determine the actin cortex tension and the intracellular pressure of each cell. (E) Cortical actomyosin tension of untreated HFF cells extracted at different Z-piezo distances from 0 to 600 nm. It can be observed that from 100 to 400 nm these are not statistically different (p > 0.05) in extracted cortex tension, confirming that our method is robust for quantitatively estimating the mechanical properties for small deformations. For Z-distance of 500 nm or greater, the estimated tension increases, demonstrating significant differences using a one-way analysis-of-variance test (p < 0.05). (F) Cortical actomyosin tension of untreated HFF cells measured at different spreading stages. The progressive cell spreading and adhesion is observed by phase contrast (inset). At early stages (time 0–20 min) there are no statistically significant differences (p > 0.05) in extracted cortical tension, confirming that weak adhesion contributions are negligible. However, when cells exhibit lamellipodia and filopodia projections, the measured tension significantly increases—demonstrating that adhesion contribution cannot be ignored at advanced spreading stages (time >30 min). To see this figure in color, go online.