15. Dry the slides on a warm plate to let water evaporate (usually
takes a few minutes) (see Note 7).
16. Prior to AFM scan, look at the samples under the fluorescent or
polarized microscope. Exclude all the sections which are: not
flat (wrinkling); detached locally from the slide; or overlapping
with each other.
17. Store slides in a box to isolate them from the dust, use compressed air to remove any obstacle on the slide.
3.3 Selecting the
Cantilever and Its
Calibration
1. Connect the cantilever to the holder (Fig. 3a) and to the AFM
head (Fig. 3b) (see Note 8).
2. Open the software NanoScope 8.15.
3. In the Select Experiment window, Choose an Experiment Category (Mechanical Properties), Select Experimental Group
(Quantitative Nanomechanical Mapping) [38], Select Experiment (PeakForce QNM in Air (Large Amplitude), and press
Load Experiment.
4. A new window with Workflow Toolbar on the left will appear
(consists of Setup, Navigation, Check parameters, Engage,
Scan, Ramp, Withdraw).
5. Click on Setup and select the Probe Type (ScanAsyst-Air).
6. Place the AFM head with the attached cantilever on the Laser
Alignment Station (Fig. 3c).
7. Using metallic knobs on the Laser Alignment Station, move
the cantilever tip to the center (Fig. 3c,d).
8. Using the beam’s knobs on the right of the AFM head, align
the laser beam that will be on the top of the cantilever tip. Try
to get the maximum signal sum (a well-aligned sum is around
6.0 V) (Fig. 3c,d).
9. Using the vertical (V) and horizontal (H) detector’s wheels on
the left of the AFM head, align the detector by moving the
mirror so that it will reflect the laser beam in the best possible
way to the detector (Fig. 3c). The laser is seen in the Setup
window as a red spot on a black field crossed with X and Y lines.
Manipulate the V and H wheels in such a way that the red spot
will move to the center (V and H deflection should be close to
0 V) (Fig. 3c).
10. Put a clean slide on the microscope stage, stabilize it with
magnetic clamps, and focus on the slide (see Note 9).
11. Move the AFM head on the microscope stage (Fig. 3e).
12. Go to Navigate and move the cantilever close to the slide.
13. Focus on the cantilever using the objective camera.
Atomic Force Microscopy to Study Cell Wall Mechanics in Plants
357
takes a few minutes) (see Note 7).
16. Prior to AFM scan, look at the samples under the fluorescent or
polarized microscope. Exclude all the sections which are: not
flat (wrinkling); detached locally from the slide; or overlapping
with each other.
17. Store slides in a box to isolate them from the dust, use compressed air to remove any obstacle on the slide.
3.3 Selecting the
Cantilever and Its
Calibration
1. Connect the cantilever to the holder (Fig. 3a) and to the AFM
head (Fig. 3b) (see Note 8).
2. Open the software NanoScope 8.15.
3. In the Select Experiment window, Choose an Experiment Category (Mechanical Properties), Select Experimental Group
(Quantitative Nanomechanical Mapping) [38], Select Experiment (PeakForce QNM in Air (Large Amplitude), and press
Load Experiment.
4. A new window with Workflow Toolbar on the left will appear
(consists of Setup, Navigation, Check parameters, Engage,
Scan, Ramp, Withdraw).
5. Click on Setup and select the Probe Type (ScanAsyst-Air).
6. Place the AFM head with the attached cantilever on the Laser
Alignment Station (Fig. 3c).
7. Using metallic knobs on the Laser Alignment Station, move
the cantilever tip to the center (Fig. 3c,d).
8. Using the beam’s knobs on the right of the AFM head, align
the laser beam that will be on the top of the cantilever tip. Try
to get the maximum signal sum (a well-aligned sum is around
6.0 V) (Fig. 3c,d).
9. Using the vertical (V) and horizontal (H) detector’s wheels on
the left of the AFM head, align the detector by moving the
mirror so that it will reflect the laser beam in the best possible
way to the detector (Fig. 3c). The laser is seen in the Setup
window as a red spot on a black field crossed with X and Y lines.
Manipulate the V and H wheels in such a way that the red spot
will move to the center (V and H deflection should be close to
0 V) (Fig. 3c).
10. Put a clean slide on the microscope stage, stabilize it with
magnetic clamps, and focus on the slide (see Note 9).
11. Move the AFM head on the microscope stage (Fig. 3e).
12. Go to Navigate and move the cantilever close to the slide.
13. Focus on the cantilever using the objective camera.
Atomic Force Microscopy to Study Cell Wall Mechanics in Plants
357