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

Abstract

BackgroundCell 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, CPFA, is 4%. However, it is still not quantitatively clear how the conventional protocol of CPFA is optimized.MethodsHere, we investigated the mechanical properties of cell fixation as a function of CPFA by using atomic force microscopy and scanning ion conductance microscopy. The goal of this study is to investigate the effect of CPFA (0–10 wt%) on the morphological and mechanical properties of live and fixed mouse fibroblast cells.ResultsWe found that both Young’s modulus, E, and the fluctuation amplitude of apical cell membrane, am, were almost constant in a lower CPFA (<10−4%). Interestingly, in an intermediate CPFA between 10−1 and 4%, E dramatically increased whereas am abruptly decreased, indicating that entire cells begin to fix at CPFA = ca. 10−1%. Moreover, these quantities were unchanged in a higher CPFA (>4%), indicating that the cell fixation is stabilized at CPFA = ca. 4%, which is consistent with the empirical concentration of cell fixation optimized in biological protocols.ConclusionsTaken 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.