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. 2009 Jul 22;97(2):674-7.
doi: 10.1016/j.bpj.2009.05.010.

Stiffness tomography by atomic force microscopy

Charles Roduit  1 , Serguei Sekatski, Giovanni Dietler, Stefan Catsicas, Frank Lafont, Sandor Kasas
Affiliations

Stiffness tomography by atomic force microscopy

Charles Roduit et al. Biophys J. .

Abstract

The atomic force microscope is a convenient tool to probe living samples at the nanometric scale. Among its numerous capabilities, the instrument can be operated as a nano-indenter to gather information about the mechanical properties of the sample. In this operating mode, the deformation of the cantilever is displayed as a function of the indentation depth of the tip into the sample. Fitting this curve with different theoretical models permits us to estimate the Young's modulus of the sample at the indentation spot. We describe what to our knowledge is a new technique to process these curves to distinguish structures of different stiffness buried into the bulk of the sample. The working principle of this new imaging technique has been verified by finite element models and successfully applied to living cells.

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Figures

Figure 1
Figure 1
FD curves are recorded by indenting (pushing) the tip of the AFM into the sample (a) and by plotting the deformation of the cantilever as a function of the height (b). A hard sample produces a steeper curve (b, dashed red line) than a softer one (b, dashed blue line). In the case where the sample contains harder inclusions (c, red rectangle) located at the L1 level, the FD curve follows the same path as in a (d, green line) initially, but starts to adopt a steeper path just before reaching the L1 level (d, red line). The dashed green line represents the path the curve should follow in the absence of inclusions. By dividing the curve in small segments and by analyzing their individual slope one can detect the presence of the inclusion (d, red and green horizontal bar).
Figure 2
Figure 2
Simulation of the indentation process by using the finite elements method. The sample contains inclusions (a) colored in red that have a Young's modulus tree times higher than the bulk of the sample colored in blue. The AFM tip and the spots where indentation was simulated are also represented in blue. During the indentation process the sample deforms as depicted in b. The displacement magnitude is displayed in false colors according to the color bar. (c) The stiffness tomography analysis results. The false colors represent the stiffness in arbitrary units according to the color bar. (d and f) Shows similar simulation using three times stiffer and three times softer platforms with their resulting stiffness tomography in e and g, respectively. The same data scale is used between e and g. One can notice that the stiffness difference appear less contrasted in the case of soft platforms. Arrows in g points to stiffness differences induced by the presence of soft platforms.
Figure 3
Figure 3
(a–d) Stiffness tomography images obtained on living neurons. The stiffness, calculated according to the Hertz model, is coded in false colors. Due to the lack of a more accurate model, only color differences are relevant. One can notice the presence of “red” harder inclusions underneath the membrane. The yellow color of the surface of the cells is arbitrary and do not correspond to any stiffness value. The graphs e and f represent the average stiffness of fibroblast as a function of the depth underneath the cell membrane. (e, black curve) Stiffness before the injection of cytochalasin. (e, red curve) Corresponds to the stiffness after the injection. One can notice that in average the cortical part of the cell located under 180 nm became softer after the cytochalasin injection (p < 0.05, two-tailed t-test). (f) The same experiment carried out by injecting the imaging buffer instead of cytochalasin.
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