Cell fixation for in-vitro studies is to preserve and study cell or cellular components in a life-like state by conserving essential chemical and physical characteristics of the cells. Such cell state change can be studied by measuring the amount of change in mechanical and surface fluctuation in individual cells before and after the cell fixation process. Atomic Force Microscopy (AFM) and Scanning Ion Conductance Microscopy (SICM) locally probes the mechanical property measurements and measures surface fluctuation on individual cells. For this study, Park Systems scanning probe microscope was used (Park NX12- Bio, Park Systems, Korea) with AFM and SICM capabilities, equipped with an inverted optical microscope (Nikon Corp., Japan) specifically designed for biological applications.
The AFM can gather mechanical properties of a sample as well as cell surface information of soft material samples using SICM. The cell sample used consisted of mouse fibroblast L929 cells (ATCC, USA) cultured in Dulbecco’s modified eagle medium (DMEM; Invitrogen Life Technique, USA) treated with a fixation agent 4% PFA solution for 5 min. The fixed cell samples were rinsed with PBS prior to conducting AFM and SICM experiments. SICM utilizes an ion current that flows between an electrode placed inside a nano-pipette and an external electrode located in a bath solution. SICM can display useful topographical measurements without applying any mechanical force onto the sample surface. To determine the stiffness of live and fixed cell surfaces, AFM force-distance spectroscopy measurements were done on live cells, fixed cells as well as on a solid substrate. Fixed cells exhibited significantly steeper force curve slope compared to that of live cells. Additionally, it showed that the force required for surface indentation is greater for fixed cells than for live cells. The average Young’s modulus values, which indicates the stiffness of fixed cells (77.95 kPa), was greater than that of live cells (8.11kPa). AFM cell stiffness measurements suggested that the acting filamentous structures strongly affected the stiffness. More specifically, it suggests that the PFA fixation process is directly linked to the increase of cell stiffness depending on the number of randomly distributed cross-linking sites available on the cell surface. To obtain cell surface fluctuations, the Ionic Current- Distance, I-D curves were obtained using SICM measurements on live cells, fixed cells and a solid substrate. I-D curves exhibited the steepest slope for solid substrates (petri dish) while untreated one had a broader slope. For PFA-treated cells, I-D curves occurred amidst the two. Subsequently it can be concluded that the live cells demonstrated more activity compared to those of fixed cells. The PFA treatment process resulted in smaller surface fluctuations for fixed cells that results in cross-linking of proteins between the membrane and cytoplasmic proteins. A fundamental mechanical difference was demonstrated by comparing live cells and fixed cells with PFA. The atomic force microscopy and scanning ion conductance microscopy measurements revealed a definite transition in surface fluctuation and elastic modulus of cells when exposed to PFA. Proceeding complete fixation with PFA, cell surface fluctuation was decreased as compared to that of a live cell while the Young’s modulus was increased by five-fold. These findings provide key insights into how cells react to chemical treatment with PFA.
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