10. When all the force curves from different ROIs are exported to .
SPM files, create a map by overlapping the images with the
regions marked, which were used for the force curve extraction
(Fig. 5a). Then make the list with different ROIs in the images
and corresponding names of the .SPM files (Fig. 5b).
11. Open the single representative .SPM file, a force curve will be
displayed (Fig. 4c). The morphology of the force curve should
correspond to the model curves (Fig. 2b).
12. Click on the Boxcar filter and press Execute. The curve will be
smoothed (Fig. 2d).
13. Click on Baseline Correction, move two perpendicular lines
over the flat region of the force curve, and click on Execute
(Fig. 2e).
14. Click on Indentation and select the following parameters
(Fig. 2f):
Active curve:
Extended
Fit method
Linearized model
Include adhesive force
Yes
Max force fit boundary
90%
Min force fit boundary
10%
Fit model
Sneddon
15. Click Run Auto Program, select all. SPM files, which were
generated from different regions within the same image,
select a directory. Select Save Report as Excel, and press Run
(depending on the number of. SPM files, it might take a
while).
16. Open the Excel file containing the list with image file names,
the coefficient of determination R
2
, Young’s modulus (MPa),
and other parameters.
17. At first, compare the values obtained from extracting the force
curves in the resin control. The resin values sampled along the
X or Y axis should be similar (see Note 18).
18. Group the values according to the positions in which they are
sampled. Average the Young’s modulus (MPa) values for
different regions and calculate SD and SE and perform statistical tests (Fig. 5b). This enables to compare the mechanical
properties among different regions (Fig. 5c). Remember that
ä
Fig. 5 (continued) regions along the cell wall. (b) Table showing the list of stiffness averages, SE, and the
sample number in different ROIs indicated in (a). (c) Graphical representation of the stiffness in different ROIs
indicated in (a, b). Adapted from [33] with kind permission of Elsevier
Atomic Force Microscopy to Study Cell Wall Mechanics in Plants
365
SPM files, create a map by overlapping the images with the
regions marked, which were used for the force curve extraction
(Fig. 5a). Then make the list with different ROIs in the images
and corresponding names of the .SPM files (Fig. 5b).
11. Open the single representative .SPM file, a force curve will be
displayed (Fig. 4c). The morphology of the force curve should
correspond to the model curves (Fig. 2b).
12. Click on the Boxcar filter and press Execute. The curve will be
smoothed (Fig. 2d).
13. Click on Baseline Correction, move two perpendicular lines
over the flat region of the force curve, and click on Execute
(Fig. 2e).
14. Click on Indentation and select the following parameters
(Fig. 2f):
Active curve:
Extended
Fit method
Linearized model
Include adhesive force
Yes
Max force fit boundary
90%
Min force fit boundary
10%
Fit model
Sneddon
15. Click Run Auto Program, select all. SPM files, which were
generated from different regions within the same image,
select a directory. Select Save Report as Excel, and press Run
(depending on the number of. SPM files, it might take a
while).
16. Open the Excel file containing the list with image file names,
the coefficient of determination R
2
, Young’s modulus (MPa),
and other parameters.
17. At first, compare the values obtained from extracting the force
curves in the resin control. The resin values sampled along the
X or Y axis should be similar (see Note 18).
18. Group the values according to the positions in which they are
sampled. Average the Young’s modulus (MPa) values for
different regions and calculate SD and SE and perform statistical tests (Fig. 5b). This enables to compare the mechanical
properties among different regions (Fig. 5c). Remember that
ä
Fig. 5 (continued) regions along the cell wall. (b) Table showing the list of stiffness averages, SE, and the
sample number in different ROIs indicated in (a). (c) Graphical representation of the stiffness in different ROIs
indicated in (a, b). Adapted from [33] with kind permission of Elsevier
Atomic Force Microscopy to Study Cell Wall Mechanics in Plants
365