The analysis of mechanical properties as a function of indentation depth is also the basis for stiffness tomography, introduced as a new imaging AFM functionality in 2009 by Roduit et al. . Typically, as in the AFM force spectroscopy mode, force curves are recorded in relation to the position on the investigated surface (Fig. 4.19a).
The indentation is carried out up to a given value of the load force. Depending on the cell structure probed, if, e.g., stiffer inclusions (like actin filaments) are probed beneath cell membrane the resulting force-indentation relation can manifest as a steeper curve (corresponding to larger Young’s modulus value). Under the assumption that the presence of harder inclusions changes the shape of the force curve significantly, the obtained forceindentation relations can be segmented into slices [51-53]. To each slice the Hertz model is applied and the corresponding Young’s modulus value is calculated. The obtained data are plotted as a function of indentation depth (Fig. 4.19b). The stiffness tomography approach has been initially applied to study the 3D variability in distribution of Young’s modulus in living neurons . The results showed a presence of hard structures, attributed to cortical actin cytoskeleton, whereas softer regions corresponded to surrounding cell membrane. The further applications demonstrated feasibility of the stiffness tomography approach to study the mechanical properties of bacterial membranes, evidencing the presence of stiffer regions lying beneath membrane. The possible explanation involved the hypothesis of various structures accumulations in certain region in the cytoplasm .
Figure 4.19 (a) The idea of stiffness tomography experimental conditions—force curves are indenting the sample surface within the scan area in a pre-defined grid. (b) Force- indentation curves segmentation in stiffness tomography.