16. When the AFM indentation experiment is finished, save the
images and withdraw the cantilever. The software can be
closed but returning will require to repeat the calibration
and to follow the instructions described in Subheading 3.3.
17. All the files can be opened in NanoScope Analysis 150 software
(Fig. 4). Every scan is saved in two files: one consists of the
images in different moduli (see Subheading 3.4, step 9) and
another consists of the image and force-indentation curves
(.PFC file). This allows to extract the information from the
images and investigate the properties within specific ROIs.
3.5 Extracting
Apparent Elastic
Modulus from Force
Curves
1. Start NanoScope Analysis 150 or newer.
2. Open the file containing different images (see Subheading 3.4,
step 9), click on DMT Modulus (Derjaguin-Muller-Toporov
modulus, apparent elastic modulus) image [39]. In this image,
the elasticity is represented by a color gradient in which lighter
pixels represent a stiffer area while darker ones indicate a more
elastic area (Fig. 4b) (see Note 15).
3. Click on Journal Quality Export, adjust the Export Type (TIFF
recommended), Font, Background color, etc., and click on
Export.
4. Open the image file containing the scanned area and force
curves (file with. PFC extension) (see Note 16).
5. Create a folder in a specific directory for all the files to be
generated within the same image.
6. In Force Curve Mode, click on Range and select the ROI on the
image. The image will display the ROI from where the force
curves are selected. Save the image and click on Save curves.
7. For each force curve, one file will be generated with a specific
name (file with. SPM extension).
8. Select all the ROIs by placing squares consecutively along and
across entire cell walls (Fig. 5a). Select the regions as big as
possible but within the wall (see Note 17).
9. To make sure that the resin polymerizes equally and it is not
affecting the mechanical properties of the sample, select the
force curves in the resin alone (using the range tool, place the
lines along and across the scan). The properties among different places of the resin alone should not differ.
ä
Fig. 4 (continued) lighter and more elastic areas darker. (c–f) Representative force indentation curves shown
as the function of the force (nN) over separation (μm). The approach (extended) curve is in blue and the
withdraw (retracted) curve in red. (c) Raw force curves. (d) Smoothed force curves after Boxcar filter was
executed. (e) Force curves after Baseline Correction. (f) Fitting the model to the force curves allows to extract
apparent elastic modulus (E a )
Atomic Force Microscopy to Study Cell Wall Mechanics in Plants
363
images and withdraw the cantilever. The software can be
closed but returning will require to repeat the calibration
and to follow the instructions described in Subheading 3.3.
17. All the files can be opened in NanoScope Analysis 150 software
(Fig. 4). Every scan is saved in two files: one consists of the
images in different moduli (see Subheading 3.4, step 9) and
another consists of the image and force-indentation curves
(.PFC file). This allows to extract the information from the
images and investigate the properties within specific ROIs.
3.5 Extracting
Apparent Elastic
Modulus from Force
Curves
1. Start NanoScope Analysis 150 or newer.
2. Open the file containing different images (see Subheading 3.4,
step 9), click on DMT Modulus (Derjaguin-Muller-Toporov
modulus, apparent elastic modulus) image [39]. In this image,
the elasticity is represented by a color gradient in which lighter
pixels represent a stiffer area while darker ones indicate a more
elastic area (Fig. 4b) (see Note 15).
3. Click on Journal Quality Export, adjust the Export Type (TIFF
recommended), Font, Background color, etc., and click on
Export.
4. Open the image file containing the scanned area and force
curves (file with. PFC extension) (see Note 16).
5. Create a folder in a specific directory for all the files to be
generated within the same image.
6. In Force Curve Mode, click on Range and select the ROI on the
image. The image will display the ROI from where the force
curves are selected. Save the image and click on Save curves.
7. For each force curve, one file will be generated with a specific
name (file with. SPM extension).
8. Select all the ROIs by placing squares consecutively along and
across entire cell walls (Fig. 5a). Select the regions as big as
possible but within the wall (see Note 17).
9. To make sure that the resin polymerizes equally and it is not
affecting the mechanical properties of the sample, select the
force curves in the resin alone (using the range tool, place the
lines along and across the scan). The properties among different places of the resin alone should not differ.
ä
Fig. 4 (continued) lighter and more elastic areas darker. (c–f) Representative force indentation curves shown
as the function of the force (nN) over separation (μm). The approach (extended) curve is in blue and the
withdraw (retracted) curve in red. (c) Raw force curves. (d) Smoothed force curves after Boxcar filter was
executed. (e) Force curves after Baseline Correction. (f) Fitting the model to the force curves allows to extract
apparent elastic modulus (E a )
Atomic Force Microscopy to Study Cell Wall Mechanics in Plants
363