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Single-cell elasticity measured by atomic force microscopy

Sep 2006
Lauren I. Rugani

The effect of external forces on living cells holds importance in areas of study such as tissue engineering and cancer research. Forces induce biological transformations on the cell that can alter functionality or mechanical behavior. Two techniques that were combined and utilized to study these transformations, atomic force microscopy and confocal optical microscopy, each have disadvantages when used alone. However, when used together, they are sensitive to biological properties and structural changes and enable the investigation of single-cell mechanics.

A single cell is exposed to compression by atomic force microscopy. Membrane deformation followed by the rupture of individual cellular components is evident as the load increases.

Researchers at the University of California in Davis and in Sacramento have combined atomic force microscopy with optical microscopy techniques to explore single-cell compression and its effect on the mechanical behavior of cells. “Investigation of single-cell mechanics is essential for the characterization and control of the mechanical properties and functions of reconstituted tissues, an important task for practical application of tissue engineering,” said Gang-yu Liu, a chemistry professor at the Davis campus.

To avoid staining the cells, most of the experiments were conducted under bright-field optical microscopy with an inverted optical microscope from Olympus America. An Olympus laser scanning confocal microscope was used to image stained cells when the researchers needed a high-resolution visualization of the three-dimensional cellular structure. “Confocal microscopy provides accurate initial imaging for AFM investigation, including the positioning of AFM probes to the designated cell location and simultaneous monitoring of cell deformation,” Liu explained.

Cell deformation was measured with an atomic force microscope from Asylum Research Corp., equipped with a nanopositioning sensor. Silicon cantilevers with a force of either 1.0 N/m or 40 N/m, depending on single-cell elasticity, were used for single-cell compression, force sensing and high-resolution imaging. A 40-μm glass bead was attached to a cantilever, and optical microscopy positioned the cantilever over the center of the cell to monitor the deformation of the cellular components as force was applied.

The researchers created force versus deformation curves for living, dead and fixed cells. For low applied loads, live-cell deformation was found to be elastic and fully reversible, suggestive of an impermeable cell membrane. As the load increased, most cells burst at roughly 30 percent deformation, followed by the rupture of components within the cell, including the nucleus. These specific events correlated with stress peaks in the force-deformation curves.

Force versus deformation curves demonstrate the relative deformation — calculated as cell height change/initial cell height — of living, dead and fixed cells. The stress peak of the living cell is evidence of the membrane rupturing, while dead and fixed cells exhibit smooth curves. Fixed cells require much higher forces than living and dead cells for relative deformation.

In contrast, dead cells exhibited a smooth force-deformation curve, indicative of a uniform process that became irreversible beyond 20 percent deformation resulting from high membrane permeability. Cells fixed to the surface of the slide also exhibited smooth deformations; however, they required much higher forces for compression and displayed full reversibility even after 60 percent deformation because of the cross-linking of membrane proteins.

The researchers found that AFM-based positioning combined with optical monitoring improved accuracy for cell positioning, mechanical perturbation and force measurements. “Combination of the two methods enables high-resolution applications of AFM to be properly directed and utilized in molecular and cellular biology investigations,” Liu remarked. However, the instrumentation is expensive and complex. The team plans to improve the instrumentation and methodology for easier use in biomedical research.

Langmuir, published online July 18, 2006.

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