title: Mechanical properties of paraformaldehyde-treated individual cells investigated by atomic force microscopy and scanning ion conductance microscopy.
abstract: 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,CPFA, is 4%. However, it is still not quantitatively clear how the conventional protocol ofCPFAis optimized.
  
  
  METHODS
  Here, we investigated the mechanical properties of cell fixation as a function ofCPFAby using atomic force microscopy and scanning ion conductance microscopy. The goal of this study is to investigate the effect ofCPFA(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,am, were almost constant in a lowerCPFA(<10-4%). Interestingly, in an intermediateCPFAbetween 10-1and 4%,Edramatically increased whereasamabruptly decreased, indicating that entire cells begin to fix atCPFA= ca. 10-1%. Moreover, these quantities were unchanged in a higherCPFA(>4%), indicating that the cell fixation is stabilized atCPFA= 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.
authors.affiliation: School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore, 639798 Singapore.
authors.forename: Seong-Oh
authors.initials: SO
authors.lastname: Kim
authors.affiliation: School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore, 639798 Singapore.
authors.forename: Joonhui
authors.initials: J
authors.lastname: Kim
authors.affiliation: Graduate School of Information Science and Technology, Hokkaido University, Sapporo, 060-0814 Japan.
authors.forename: Takaharu
authors.initials: T
authors.lastname: Okajima
authors.affiliation: School of Materials Science and Engineering, Nanyang Technological University, 50 Nanyang Avenue, Singapore, 639798 Singapore.|School of Chemical and Biomedical Engineering, Nanyang Technological University, 62 Nanyang Drive, Singapore, 637459 Singapore.
authors.forename: Nam-Joon
authors.initials: NJ
authors.lastname: Cho
keywords: Atomic force microscopy; Cell fixation; Cellular mechanics; Scanning ion conductance microscopy