Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

Summary

This paper demonstrates a protocol to characterize the mechanical properties of living cells by means of microindentation using an Atomic Force Microscope (AFM).

Abstract

Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed.

Introduction

Mechanical properties, especially stiffness, of individual cells and their surrounding extracellular matrices (ECM) are critical for many biological processes including cell growth, motility, division, differentiation, and tissue homeostasis.¹ It has been demonstrated that cell mechanical stiffness is mainly determined by the cytoskeleton, especially the networks of actin and intermediate filaments and other proteins associated with them.² Results from mechanical tests on in vitro networks of actin and intermediate filaments suggest that the cell mechanics is largely dependent on the cytoskeletal structure and the pre-stress in t....

Protocol

1. Calibrate the Spring Constant of Cantilever

1. Load the cantilever into the AFM according to the manufacturer's instructions. It is necessary to clean the cantilever holder with ethanol before any experiments. This will help limit bacterial contamination to culture during the AFM measurements.
2. Calibrate InvOLS (Inverse Optical Lever Sensitivity). This parameter describes the amount of photodiode response (Volts) per nanometer of cantilever deflection.
3. Load a clean glass slide onto the sample stage, then install the AFM head and adjust the laser beam and alignment according to the manufacturer's instructions. Engage the AFM tip on the gl....

Results

Figure 2a shows three representative force curves taken from 3T3 fibroblasts cultured on plastic surface, polyacrylamide gel of Young's moduli 3,000 Pa and 17,000 Pa, respectively. After carefully identifying the contact points in the curves, the indenting force as function of cell deformation is plotted in Figure 2b. Under a force of magnitude smaller than 0.3 nN, a pyramid shape tip indents 3 micrometers into a cell cultured on a 3 kPa polyacrylamide gel. In contrast, a force more than.......

Discussion

The AFM indentation method has advantages to characterize mechanical properties of living cells. Albeit less sensitive than the magnetic twisting cytometry and optical tweezers, which can measure forces on the piconewton level³², the AFM can detect resistance force from samples ranging from tens of pico-Newton to hundreds of nano-Newton, comparable to range of force that can be applied to cells using a micropipette¹⁹. This range of force fits the needs to create measurable deformations in all types .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Atomic Force Microscope	Asylum Research	MFP3D-BIO