Comparative study of the conditions required to image live human epithelial and fibroblast cells using atomic force microscopy

Abstract

Successful imaging of living human cells using atomic force microscopy (AFM) is influenced by many variables including cell culture conditions, cell morphology, surface topography, scan parameters, and cantilever choice. In this study, these variables were investigated while imaging two morphologically distinct human cell lines, namely LL24 (fibroblasts) and NCI H727 (epithelial) cells. The cell types used in this study were found to require different parameter settings to produce images showing the greatest detail. In contact mode, optimal loading forces ranged between 2-2.8 x 10(-9) and 0.1-0.7 x 10(-9) (N) for LL24 and NCI H727 cells respectively. In tapping (AC) mode, images of LL24 cells were obtained using cantilevers with a spring constant of at least 0.32 N/m, while NCI H727 cells required a greater spring constant of at least 0.58 N/m. To obtain tapping mode images, cantilevers needed to be tuned to resonate at higher frequencies than their resonance frequencies to obtain images. For NCI H727 cells, contact mode imaging produced the clearest images. For LL24 cells, contact and tapping mode AFM produced images of comparable quality. Overall, this study shows that cells with different morphologies and surface topography require different scanning approaches and optimal conditions must be determined empirically to achieve images of high quality.

Real-Time Monitoring of the Effects of Telmisartan on Angiotensin II-Induced Mechanical Changes in Live Mesangial Cells Using Atomic Force Microscopy

“…AFM imaging of live cells is complicated and requires the maintenance of physiologic conditions [20]. AFM cell imaging in air conditions is attributed to the increasing hardness of the cell by dehydration [21].…”

Atomic force microscopy comes of age

“…A critical first step has been to develop a new technology that integrates AFM with TIRF (total internal reflection fluorescence) and FSD (fast-spinning disc) confocal microscopy to investigate cellular behaviour at the subcellular level, thus providing high spatial and temporal resolution (Trache and Lim, 2009). Other necessary considerations for application development include the selection of cantilever tips with spring constants that are appropriate to the cell type under analysis, as well as scanning parameters such as the resonant frequency employed (Murphy et al, 2006). The flexibility of the AFM and its combination with light microscopy techniques, namely DIC (differential interference contrast microscopy) and CLSM, have allowed the organization of adhesion molecules on the surface and their correlation with structures involved in binding to be elucidated (Poole et al, 2004).…”

Observation of angiotensin II-induced changes in tubular epithelial cells utilizing AFM: Angiotensin II-induced changes in tubular cells

“…Murphy M.F. et al reported that successful imaging of live human cells using AFM was influenced by many variables including cell culture conditions, cell morphology, surface topography, scan parameters and cantilever choice [2]. Lee et al successfully observed the angiotensin II (Ang II) induced changes in fixed and live mesangial cell by AFM [3].…”