Anisotropic Mechanical Properties of Living Cells Revealed by Integrated Spinning Disk Confocal and Atomic Force Microscopy

Abstract

Recent developments in fluorescent live-cell imaging and biophysical methods have significantly advanced our understanding of the dynamic biochemical and mechanical processes underlying cellular functions such as cell migration. One of most frequently used techniques for assessment of cell mechanical properties is the indentation experiments conducted with the atomic force microscope (AFM), due primarily to AFM's relative ease of operation, its high precision of force measurement, and high spatial resolution. We used the AFM in conjunction with a spinning disk confocal (SDC) microscope to directly visualize AFM indentation of living cells with high spatial and temporal resolution. With novel live cell imaging probes to fluorescently label F-actin, microtubules, and membrane, we were able to directly observe structural changes during the indentation process of a living cell (NIH 3T3 fibroblasts) with a spherical indenter. The most notable observation was a deformation of single apical stress fibers (ASF) under the action of the probe. The presence of ASF (also known as perinuclear actin cap) correlated with low cell height and high stiffness. Moreover, the presence of ASF caused an anisotropic indentation geometry as observed from surface displacement profiles calculated from the SDC images. Isotropic indentation profile was found in cells without ASF: cancerous cells MDA-MB-231 which are naturally lacking actin cap and NIH 3T3 cells in which ASF were disrupted by latrunculin A. Anisotropic indentation behavior suggests an anisotropic deformability and stiffness of the cell. We performed finite element simulations to extract anisotropic properties from the surface displacement data. Simulations suggest a very strong level of anisotropy in cells which role should be further elucidated.