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An AFM-based stiffness clamp for dynamic control of rigidity.

Webster KD, Crow A, Fletcher DA - PLoS ONE (2011)

Bottom Line: Constant force or constant height can be maintained in an AFM experiment through feedback control of cantilever deflection, known respectively as a 'force clamp' or 'position clamp'.We demonstrated the AFM stiffness clamp on two different samples: a contracting fibroblast cell and an expanding polyacrylamide hydrogel.We found that the fibroblast, a cell type that secretes and organizes the extracellular matrix, exhibited a rapid, sub-second change in traction rate (dF/dt) and contraction velocity (dx/dt) in response to step changes in stiffness between 1-100 nN/µm.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Graduate Group, University of California, Berkeley, California, United States of America.

ABSTRACT
Atomic force microscopy (AFM) has become a powerful tool for measuring material properties in biology and imposing mechanical boundary conditions on samples from single molecules to cells and tissues. Constant force or constant height can be maintained in an AFM experiment through feedback control of cantilever deflection, known respectively as a 'force clamp' or 'position clamp'. However, stiffness, the third variable in the Hookean relation F = kx that describes AFM cantilever deflection, has not been dynamically controllable in the same way. Here we present and demonstrate a 'stiffness clamp' that can vary the apparent stiffness of an AFM cantilever. This method, employable on any AFM system by modifying feedback control of the cantilever, allows rapid and reversible tuning of the stiffness exposed to the sample in a way that can decouple the role of stiffness from force and deformation. We demonstrated the AFM stiffness clamp on two different samples: a contracting fibroblast cell and an expanding polyacrylamide hydrogel. We found that the fibroblast, a cell type that secretes and organizes the extracellular matrix, exhibited a rapid, sub-second change in traction rate (dF/dt) and contraction velocity (dx/dt) in response to step changes in stiffness between 1-100 nN/µm. This response was independent of the absolute contractile force and cell height, demonstrating that cells can react directly to changes in stiffness alone. In contrast, the hydrogel used in our experiment maintained a constant expansion velocity (dx/dt) over this range of stiffness, while the traction rate (dF/dt) changed with stiffness, showing that passive materials can also behave differently in different stiffness environments. The AFM stiffness clamp presented here, which is applicable to mechanical measurements on both biological and non-biological samples, may be used to investigate cellular mechanotransduction under a wide range of controlled mechanical boundary conditions.

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Cell contraction rapidly responds to stiffness changes.(a) An AFM was used to expose a single fibroblast cell to dynamically changeable apparent stiffness values with the AFM stiffness clamp. The piezo-controlled substrate was moved in response to deflections of the cantilever, which were precisely measured with an optical lever system. (b) Force and cell height as the cell contracts under different apparent stiffnesses from a typical experiment. A total of 30 cells were tested, all exhibiting the same stiffness-dependent behavior shown above. Each interval is under an apparent stiffness of 3.6, 18, or 90 nN/µm as indicated at the top of the graph. The traction rate and contraction velocity rapidly change with a step change in stiffness. A segmented linear regression fit is plotted highlighting the change in traction rate (inset). Data displayed in (c) and (d) are compiled from this trace. (c) Traction rate increases with apparent stiffness while corresponding contraction velocity decreases. The rates are determined from a linear regression fit where the 95% confidence interval for each slope is within 0.4 nN/min and 20 nm/min for the force and height, respectively. (d) Plot of force versus cell height. The three linear, distinct traces each have slopes that indicate that the desired apparent stiffnesses were achieved. The * marks the trace without any feedback loop. Each interval was translated to begin at the origin for comparison.
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pone-0017807-g004: Cell contraction rapidly responds to stiffness changes.(a) An AFM was used to expose a single fibroblast cell to dynamically changeable apparent stiffness values with the AFM stiffness clamp. The piezo-controlled substrate was moved in response to deflections of the cantilever, which were precisely measured with an optical lever system. (b) Force and cell height as the cell contracts under different apparent stiffnesses from a typical experiment. A total of 30 cells were tested, all exhibiting the same stiffness-dependent behavior shown above. Each interval is under an apparent stiffness of 3.6, 18, or 90 nN/µm as indicated at the top of the graph. The traction rate and contraction velocity rapidly change with a step change in stiffness. A segmented linear regression fit is plotted highlighting the change in traction rate (inset). Data displayed in (c) and (d) are compiled from this trace. (c) Traction rate increases with apparent stiffness while corresponding contraction velocity decreases. The rates are determined from a linear regression fit where the 95% confidence interval for each slope is within 0.4 nN/min and 20 nm/min for the force and height, respectively. (d) Plot of force versus cell height. The three linear, distinct traces each have slopes that indicate that the desired apparent stiffnesses were achieved. The * marks the trace without any feedback loop. Each interval was translated to begin at the origin for comparison.

Mentions: Fibroblast cells are used extensively as a model system to investigate the effect of substrate rigidity [5], [9], [13], [14], [18]. After demonstrating the range and precision of the stiffness clamp algorithm with a hydrogel, we used NIH 3T3 fibroblast cells to investigate how cellular rigidity sensing responds to a reversible step change in stiffness. Figure 4 shows the results of a typical experiment. Cells in suspension were flowed into a chamber and within minutes were brought into contact with both a fibronectin-coated glass surface and a fibronectin-coated tipless AFM cantilever . After a small compressive force established contact, adhesions formed on both surfaces, and the cell contracted (FIG. 4A). Once contraction started we cycled between stiffnesses of , 1, and 5 (3.6, 18, 90 ) every 30 s. We chose a cycle period of 30 s to allow for exchange of cytoskeletal and focal adhesion components (timescale of seconds) but not full reorganization of adhesions or the cytoskeleton (timescale of minutes) [25]. A typical resulting traction force and cell height trace is shown in FIG. 4B.


An AFM-based stiffness clamp for dynamic control of rigidity.

Webster KD, Crow A, Fletcher DA - PLoS ONE (2011)

Cell contraction rapidly responds to stiffness changes.(a) An AFM was used to expose a single fibroblast cell to dynamically changeable apparent stiffness values with the AFM stiffness clamp. The piezo-controlled substrate was moved in response to deflections of the cantilever, which were precisely measured with an optical lever system. (b) Force and cell height as the cell contracts under different apparent stiffnesses from a typical experiment. A total of 30 cells were tested, all exhibiting the same stiffness-dependent behavior shown above. Each interval is under an apparent stiffness of 3.6, 18, or 90 nN/µm as indicated at the top of the graph. The traction rate and contraction velocity rapidly change with a step change in stiffness. A segmented linear regression fit is plotted highlighting the change in traction rate (inset). Data displayed in (c) and (d) are compiled from this trace. (c) Traction rate increases with apparent stiffness while corresponding contraction velocity decreases. The rates are determined from a linear regression fit where the 95% confidence interval for each slope is within 0.4 nN/min and 20 nm/min for the force and height, respectively. (d) Plot of force versus cell height. The three linear, distinct traces each have slopes that indicate that the desired apparent stiffnesses were achieved. The * marks the trace without any feedback loop. Each interval was translated to begin at the origin for comparison.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3050926&req=5

pone-0017807-g004: Cell contraction rapidly responds to stiffness changes.(a) An AFM was used to expose a single fibroblast cell to dynamically changeable apparent stiffness values with the AFM stiffness clamp. The piezo-controlled substrate was moved in response to deflections of the cantilever, which were precisely measured with an optical lever system. (b) Force and cell height as the cell contracts under different apparent stiffnesses from a typical experiment. A total of 30 cells were tested, all exhibiting the same stiffness-dependent behavior shown above. Each interval is under an apparent stiffness of 3.6, 18, or 90 nN/µm as indicated at the top of the graph. The traction rate and contraction velocity rapidly change with a step change in stiffness. A segmented linear regression fit is plotted highlighting the change in traction rate (inset). Data displayed in (c) and (d) are compiled from this trace. (c) Traction rate increases with apparent stiffness while corresponding contraction velocity decreases. The rates are determined from a linear regression fit where the 95% confidence interval for each slope is within 0.4 nN/min and 20 nm/min for the force and height, respectively. (d) Plot of force versus cell height. The three linear, distinct traces each have slopes that indicate that the desired apparent stiffnesses were achieved. The * marks the trace without any feedback loop. Each interval was translated to begin at the origin for comparison.
Mentions: Fibroblast cells are used extensively as a model system to investigate the effect of substrate rigidity [5], [9], [13], [14], [18]. After demonstrating the range and precision of the stiffness clamp algorithm with a hydrogel, we used NIH 3T3 fibroblast cells to investigate how cellular rigidity sensing responds to a reversible step change in stiffness. Figure 4 shows the results of a typical experiment. Cells in suspension were flowed into a chamber and within minutes were brought into contact with both a fibronectin-coated glass surface and a fibronectin-coated tipless AFM cantilever . After a small compressive force established contact, adhesions formed on both surfaces, and the cell contracted (FIG. 4A). Once contraction started we cycled between stiffnesses of , 1, and 5 (3.6, 18, 90 ) every 30 s. We chose a cycle period of 30 s to allow for exchange of cytoskeletal and focal adhesion components (timescale of seconds) but not full reorganization of adhesions or the cytoskeleton (timescale of minutes) [25]. A typical resulting traction force and cell height trace is shown in FIG. 4B.

Bottom Line: Constant force or constant height can be maintained in an AFM experiment through feedback control of cantilever deflection, known respectively as a 'force clamp' or 'position clamp'.We demonstrated the AFM stiffness clamp on two different samples: a contracting fibroblast cell and an expanding polyacrylamide hydrogel.We found that the fibroblast, a cell type that secretes and organizes the extracellular matrix, exhibited a rapid, sub-second change in traction rate (dF/dt) and contraction velocity (dx/dt) in response to step changes in stiffness between 1-100 nN/µm.

View Article: PubMed Central - PubMed

Affiliation: Biophysics Graduate Group, University of California, Berkeley, California, United States of America.

ABSTRACT
Atomic force microscopy (AFM) has become a powerful tool for measuring material properties in biology and imposing mechanical boundary conditions on samples from single molecules to cells and tissues. Constant force or constant height can be maintained in an AFM experiment through feedback control of cantilever deflection, known respectively as a 'force clamp' or 'position clamp'. However, stiffness, the third variable in the Hookean relation F = kx that describes AFM cantilever deflection, has not been dynamically controllable in the same way. Here we present and demonstrate a 'stiffness clamp' that can vary the apparent stiffness of an AFM cantilever. This method, employable on any AFM system by modifying feedback control of the cantilever, allows rapid and reversible tuning of the stiffness exposed to the sample in a way that can decouple the role of stiffness from force and deformation. We demonstrated the AFM stiffness clamp on two different samples: a contracting fibroblast cell and an expanding polyacrylamide hydrogel. We found that the fibroblast, a cell type that secretes and organizes the extracellular matrix, exhibited a rapid, sub-second change in traction rate (dF/dt) and contraction velocity (dx/dt) in response to step changes in stiffness between 1-100 nN/µm. This response was independent of the absolute contractile force and cell height, demonstrating that cells can react directly to changes in stiffness alone. In contrast, the hydrogel used in our experiment maintained a constant expansion velocity (dx/dt) over this range of stiffness, while the traction rate (dF/dt) changed with stiffness, showing that passive materials can also behave differently in different stiffness environments. The AFM stiffness clamp presented here, which is applicable to mechanical measurements on both biological and non-biological samples, may be used to investigate cellular mechanotransduction under a wide range of controlled mechanical boundary conditions.

Show MeSH
Related in: MedlinePlus