Mechanical properties of paraformaldehyde-treated individual cells investigated by atomic force microscopy and scanning ion conductance microscopy

Background: Cell fixation is an essential step to preserve cell samples for a wide range of biological assays involving histochemical and cytochemical analysis. Paraformaldehyde (PFA) has been widely used as a cross-linking fixation agent. It has been empirically recognized in a gold standard protocol that the PFA concentration for cell fixation, C_PFA, is 4%. However, it is still not quantitatively clear how the conventional protocol of C_PFA is optimized. Methods: Here, we investigated the mechanical properties of cell fixation as a function of C_PFA by using atomic force microscopy and scanning ion conductance microscopy. The goal of this study is to investigate the effect of C_PFA (0–10 wt%) on the morphological and mechanical properties of live and fixed mouse fibroblast cells. Results: We found that both Young’s modulus, E, and the fluctuation amplitude of apical cell membrane, a_m, were almost constant in a lower C_PFA (<10⁻⁴%). Interestingly, in an intermediate C_PFA between 10⁻¹ and 4%, E dramatically increased whereas a_m abruptly decreased, indicating that entire cells begin to fix at C_PFA = ca. 10⁻¹%. Moreover, these quantities were unchanged in a higher C_PFA (>4%), indicating that the cell fixation is stabilized at C_PFA = ca. 4%, which is consistent with the empirical concentration of cell fixation optimized in biological protocols. Conclusions: Taken together, these findings offer a deeper understanding of how varying PFA concentrations influence the mechanical properties of cells and suggest new avenues for establishing refined cell fixation protocols.