Single-cell parallel plate mechanics by side-view optical microscopy-assisted atomic force microscopy.

The mechanical forces of individual cells are crucial for physiological and pathological processes, and particularly atomic force microscopy (AFM)-based force spectroscopy has become an important and widely used tool for single-cell mechanical measurements. Here, we present a single-cell parallel plate mechanical measurement method based on side-view optical microscopy-assisted AFM by using a wedged probe, which enables visualization of the uniaxial AFM force spectroscopy process of the cell in real time from the vertical cross-sectional view. With this method, AFM whole-cell compression assays with uniaxial loading forces were performed to quantify the Young's modulus of the entire cell, and the effects of experimental parameters (ramp rate of the AFM probe) and drugs (actin inhibitors) on the measured cell Young's modulus were examined. Additionally, by attaching a living cell to the parallel plate of the wedged probe, AFM-based uniaxial single-cell force spectroscopy (SCFS) assays were performed to measure the adhesion forces of individual cells. The study illustrates a promising approach for single-cell mechanical analysis, which will benefit the application of AFM-based force spectroscopy in the field of mechanobiology to reveal additional insights into the regulatory role of mechanical cues in life activities.