Nanowizards 3 bioscience

The AFM employed for this study is JPK NanoWizards 3 BioScience (Berlin, Germany), and it is mounted on an inverted optical microscope (Olympus IX71; Tokoy, Japan), allowing the AFM and optical microscope imaging simultaneously. The criterion for the cantilever selection is that the stiffness of it should be around the range of the compliance of cell sample. It is recommended that for the measurement on a soft and delicate cell, the spring constant of the cantilever should range from $0.01\text{to}0.06N/m$ (Neumann 2008 ). Before indentation, the spring constant of the AFM cantilever was calibrated. A silicon nitride cantilever (Novascan, technologies Inc., Ames, IA, USA) with the spring constant $0.059\text{N/m}$ was used for the cell-tip indentation. The probe is a sphere made by polystyrene, and its radius is $1\mathit{\mu }\text{m}$ . A liquid cell for the AFM cantilever was used to perform the experiments in phosphate buffer solution. A closed-loop system was used to detect the position of the probe in z (vertical) direction during the creep test. The operation of AFM and optical microscope was controlled by JPK’s CellHesion $\circledR$ 200 software. The whole system was mounted on an anti-vibration table (TMC 63-530, USA). The room temperature was $27{}^{\circ }\text{C}$ and variation of room temperature was less than $+/-1.0{}^{\circ }\text{C}$ during experiments.

The module of the AFM employed in this study is JPK NanoWizards 3 BioScience mounted on an inverted optical microscope, allowing the AFM and optical microscope imaging simultaneously. The criterion for cantilever selection is that the compliance of the cantilever should be around the range of the sample compliance. For very soft and delicate cells, the softest cantilevers are available with spring constants ranging from 0.01 to 0.03 N/m (JPK Application Note). Therefore, a silicon nitride cantilever whose spring constants is 0.03 N/m, was chosen for cell-tip indentation in this paper. The probe is a square pyramid tip with a half-opening angle of 25° (half-angle to face), and its radius and height are 10 nm and 2.5–8 μm respectively. During the indentation, the loading and retracting speeds were kept constant at about 2.5 μm/s for all experiments to avoid viscosity effect.