# The influence of the AFM experimental conditions

The determination of the elastic modulus can be influenced by the way how the AFM force spectroscopy is carried out. Thus, the knowledge how to setup the AFM experiment is vital to obtain reliable data. This includes choice of a size of the grid within which force curves are recorded, number of force curves recorded at one position, where to measure cellular deformability, load speed that is related to loading rate, maximum force load, etc.

The demand of high statistics requires a prolonged poking of every single cell, which can lead either to a damage of cell membrane or to remodeling of actin cytoskeleton (Fig. 4.22).

Figure 4.22 (a) The effect of prolonged poking at the single location on a single cell showing a drop of the elastic modulus value at certain number of a recorded single force curves. (b) The plot showing Young's modulus dependence on the load speed. Reprinted with permission from [55].

Both effects are undesirable and should be avoided since they can lead to changes in the elastic modulus. A convenient way to verify whether there is no influence of the prolonged cell poking in the obtained date is to plot Young’s modulus as a function of the number of consecutive measurements (which is also related to the time elapsed during measurements of consecutive cells). Figure 4.22a presents such data obtained for TD47 breast cells [55]. Purple squares show the modulus calculated from force curves recorded during poking at predefined, constant position. The measured moduli, randomly distributed around the mean value of 1.20 ± 0.28 kPa start to decrease, revealing a breakdown at the curve no. 145 and indicating sudden change in the elastic modulus and thereby strong alterations in actin cytoskeleton. More stable data can be obtained by setting a square area, e.g., 10 pm x 10 pm, within which force curves are acquired (blue dots in Fig. 4.22a). Here, the prolonged poking does not induce/ generate such clearly visible remodeling of actin cytoskeleton as compared to poking at single location.

Young’s modulus determined by AFM is not a constant and absolute value—it is dependent on the loading rate [54, 55, 62]. In the AFM the loading rate is indirectly introduced by the load speed (the speed of cantilever approach, describing how fast a living cell is indented). As one can see from Fig. 4.22b, the effect of the indenting speed (i.e., loading rate) is cell-dependent. Normal breast cells (184 A) are more sensitive to load rate than cancerous MFC7 cells. Similar results have been already reported for another breast cancer pair, i.e., benign MCF10A and cancerous MCF7. Also in this case, MCF7 cancer cells were more deformable (lower modulus) and changed their elastic properties less pronouncedly as compared to benign cells [54].