# Morphological Dynamics of Leukemia Cells on TiO\({}_{2}\) Nanoparticle Coatings Studied by AFM
## 3 Results and Discussion
### Statistical Analysis of Morphological Parameters  
To better support the above qualitative observations, we have statistically analyzed a number of parameters to quantify cell morphology, i.e., the mean height of the cells \(z_{\text{m}}\), the contact area between the cell and the substrate \(A\), the cell volume \(V\), and the RMS roughness of the cell surface \(S_{\text{q}}\). As can be seen in Figure 6a, the mean height of the cells decreases significantly upon initial contact with the TiO\({}_{2}\)-m coating, compared to the mean height of the cells on the bare glass substrate. This reduced height is accompanied by an almost threefold increase in the area of contact with the substrate (see Figure 6b), which is evidence for the spreading of the cells on the TiO\({}_{2}\)-m coating. After 20 min of contact, however, the mean height has increased and is now comparable to the glass control (Figure 6a), while the contact area is now right between the glass control and 10 min contact with the TiO\({}_{2}\)-m coating (Figure 6b). Notably, at this contact time of 20 min, the volume of the cells is about 50% larger than for all other contact times as well as for the glass control (see Figure 6c). This change in cell volume may be related to some cytoskeletal rearrangement by polymerization and depolymerization of actin filaments and microtubules [39]. This interpretation is further supported by the fact that after 20 min contact with the TiO\({}_{2}\)-m coatings, the membranes of the cells exhibit a drastically increased surface roughness (see Figure 6d), as it was shown that membrane roughness is controlled mostly by actin dynamics [40]. However, the increase in cell volume and membrane roughness may also be caused to some extent by TiO\({}_{2}\)-m internalization. Figure 6: Results of statistical analyses of the morphological parameters of acute lymphoblastic leukemia cells cultured on a glass surface (control) and TiO\({}_{2}\)-m coatings for different times. (**a**) Average mean height of the cells \(z_{\text{m}}\). (**b**) Average contact area with the substrate \(A\). (**c**) Average cell volume \(V\). (**d**) Average RMS roughness of the cell surface \(S_{\text{q}}\). Values represent averages of up to 30 individual cells with the standard deviations given as error bars. Significances with respect to the control are indicated as * (\(p<0.05\)) and *** (\(p<0.001\)). For longer incubation times exceeding 20 min, the cells on the TiO\({}_{2}\)-m coatings appear to approach a new resting state. This state is characterized by a reduction of the mean height of the cells, while their area of contact with the TiO\({}_{2}\)-m coating surface remains constant at the intermediate value observed already at 20 min incubation (see Figure 6a,b). Most interestingly, in this state, both the cell volume (Figure 6c) and the membrane roughness (Figure 6d) are back at their initial values, indicating that the processes of cytoskeleton remodeling and/or TiO\({}_{2}\)-m internalization are coming to an end. This steady state might be related to the pinning of the cell membranes by larger TiO\({}_{2}\)-m nanoparticle aggregates as observed in the AFM images in Figures 3-5 and quantified by the intermediate value of the contact area in Figure 6b. Such a pinning would hinder further membrane retraction and thereby interrupt or at least delay the completion of the first cycle of amoeboid locomotion. It was previously shown that the number of trapped leukemic cells on the TiO\({}_{2}\)-m coatings increases with contact time until it reaches a maximum value at 40 min, after which it decreases again [15]. Therefore, this time point should be chosen for efficient cell trapping. For PDT, however, not just efficient cell trapping but also efficient cell damage by the generated ROS is of utmost importance.