Atomic force microscopy imaging of living cells: a preliminary study of the disruptive effect of the cantilever tip on cell morphology

Atomic force microscopy (AFM), with the capability of operating in aqueous solution, has great potential as a tool for imaging living cells in their physiological environments and for studying biologically important dynamic cellular processes in real time, with molecular resolution [1], [2], [3]. In addition, AFM can be used to study quantitatively cell elasticity and molecular interaction forces. Currently, real-time monitoring of dynamic events of living cells, cell–cell interactions and morphological changes of the cells in response to intracellular and extracellular stimuli has been the focus of a number of AFM studies [4], [5], [6], [7], [8], [9]. However, most of the AFM studies of living cells reported so far have been conducted using the contact mode, where the tip physically contacts the cell surface during scanning and exerts certain forces onto the cell surface. The scanned cells inevitably suffer various degrees of structural deformation or damage because of their intrinsic softness, which may provoke significant biological changes inside the cell. Furthermore, when AFM is used to monitor dynamic cellular events, there are difficulties in determining whether the changes in cell morphology observed are authentic, arising from dynamic cellular movement, or are artificial, induced by the perturbing action of the AFM cantilever [5], [10]. To make matters worse, the interactions between the cantilever tip and the cell surface are so complex that there is no simple way to control the tip–cell interactions and to eliminate the disruptive effect of the scanning cantilever. This has become a significant technical hurdle in AFM imaging of living cells [1], [2], [3]. Therefore, for further exploration of AFM applications in the study of important dynamic cellular processes, the disruptive effect of the cantilever on cell morphology must be investigated, and more AFM studies are necessary to establish whether a relationship exists between cell morphology and disruptive effect of the cantilever.

In this study, we investigated the disruptive effect of the scanning cantilever tip on cell morphology. We scanned a large number of samples under identical experimental conditions in order to identify what kinds of changing patterns in cell morphology generally induced by the scanning cantilever in the contact mode and to find out how long cells remain viable under normal AFM imaging conditions. Two cell lines, AV12 (a Syrian hamster cell line) and SK–N–SH (a human neuroblastoma cell line), were used in the experiment because these cells are widely used, in biomedical research, as an expression system for studying cellular functions of neuronal receptors. We expected that the experimental results would provide some guidance for further AFM studying of dynamic cellular events of these two cell lines and for future high-resolution AFM imaging of neuronal receptors expressed in these cells.

SK–N–SH cells were obtained from the American Type Culture Collection (Manassas, USA) and AV12 cells were kindly provided by Eli Lilly Corporation (Indianapolis, USA). Sterilized glass coverslips of 15 mm in diameter were placed in cell culture dishes containing freshly passaged SK–N–SH or AV12 cells. The cells were cultured in conventional culture media in all cases and incubated at 37°C with 5% CO₂. After 2 days, the glass coverslips were mounted for AFM imaging.

AFM imaging of living cells were carried out in 25 mM HEPES-buffered cell culture media (without serum) at room temperature using a Nanoscope IIIa (Digital Instruments Inc., Santa Barbara, CA) equipped with a 120 μm (J type) scanner and an AFM fluid cell. The AFM was operated in the contact mode using a 200 μm long, V-shaped silicon nitride cantilever (Digital Instruments Inc.). The nominal force constant of the cantilever is 0.06 N/m. The image data were collected in the deflection mode with a loading force of typically 1 nN and at a scan rate of typically 0.9 Hz. During scanning, fresh culture media were injected into the AFM fluid cell to maintain the volume of solution, and the setpoint voltage was adjusted to minimize the cantilever loading force.

As it is known, in the contact mode, the cantilever is physically in contact with the cell surface during scanning, and the cantilever exerts both vertical compression and lateral shear forces on the cell surface. The force applied by the cantilever is typically in the range of 1 nN, which is destructive for living cells. Therefore, under normal AFM imaging conditions, cells are inevitably injured to some degree, which will eventually lead to cell death.

Our preliminary experimental results suggest that there is a relationship between sensitivity to the cantilever disruptive effect and cell type and that there are some patterns existing in the change of cell morphology in the two cell lines studied. The AV12 cells were more susceptive to damage caused by the cantilever disruptive force than the SK–N–SH cells. Once the AV 12 cells were injured, significant changes in the cell morphology were immediately observed. Moreover, by examining survival of each AV12 cells in the AFM images with time, we found that the cells were not equally susceptible to be removed by the cantilever when deprived of cell-to-cell or cell-to-substrate adhesion. Instead, the degree of sensitivity varies from cell to cell. We believe that cell adhesion is one of the major physiological factors that control cell susceptibility to the cantilever disruptive effect. The survival of the AV12 cell requires cell adhesion to extracellular matrix (ECM) and cell-to-cell interactions; by contrast, the survival of the SK–N–SH cells requires mainly cell adhesion to the substrate, provided by the stress fibers developed within the cells. When appropriate ECM contacts are absent and cell adhesion to the substrate is degraded, cells suffer injury and undergo cell death processes [12]. The injured cells gradually lose their normal shape, become rounded up and shrink in size, and are removable from the substrate surface.

It should be mentioned that our experiments, like most of AFM studies reported so far [1], [2], [3], have been conducted in a balanced salt buffer and at room temperature that is not equivalent to the environment of cell culture. Together with the cantilever disruptive force, this factor may significantly accelerate the process of cell death and reduce the time window of cell viability. However, these are the conditions commonly used in AFM studies of living cells. Furthermore, we found that the rather strong interactions between the cantilever and the cell surface are often present during scanning, and the damaging effects of the scanning cantilever on the cell surface may start right from the beginning of scan. The observation of the disruptive effect of the scanning cantilever in AFM images depends on the viability of the cell type and the experimental conditions used (e.g., scanning time, loading force, imaging mode, etc.).

Finally, our study has shown that the susceptibility of cells to the AFM cantilever disruption is strongly adhesion-dependent. It is known that the adhesion of a cell to others or to a substrate is mediated by specific receptor–receptor or receptor–ligand bonds or ECM elements. Thus, it may be possible to use biochemical methods to enhance cell adhesion, to investigate further, using AFM, adhesion-mediated cell resistance to the cantilever disruption, and to seek a new sample preparation to improve cell survival.

We studied the disruptive effect of the AFM scanning cantilever on cell morphology. Our experimental results show that the disruptive effect on the cell morphology induced by the AFM cantilever often exists in the contact mode under normal imaging conditions. The cells being scanned suffer injuries to various degrees, depending on the cell type and viability, and the injured cells may undergo remarkable changes in morphology. In general, with prolonged scanning, the cells gradually change their normal shape (e.g., becoming more rounded and shrinking in size), lose their adhesion to the substrate or to the neighboring cells, and detach or are removed from the substrate surface. Our study indicates that the improvement of cell adhesion is a way to promote cell resistance to the cantilever disruptive effect.