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.