14. Move the cantilever to the center using the knobs on the
microscope stage (Fig. 3e).
15. Go to Navigate and click on Sample Load Position. This moves
the AFM head up so that the manipulation of the AFM head
will not cause the damage of the cantilever.
16. Take the AFM head out, place the sapphire sample on the
microscope stage, and stabilize it with magnetic clamps.
17. Using the objective camera, move the sapphire sample to the
center, and if possible focus on the surface of the sample.
18. Move the AFM head back over the sample (Fig. 3e).
19. In the Navigate window, adjust the focus on the cantilever by
moving the AFM head down. Look at the objective camera to
control the distance of the cantilever from the sample. When
the cantilever is approaching the surface of the sample, the
shadow of the cantilever should be seen, it should then appear
sharp in the center. Decrease the speed when the cantilever is
close to the surface.
20. In the Check parameters window, set the Scan size to 0 nm.
21. Go to Engage to approach the sapphire probe. The cantilever
will move toward the sample in an automated way. The time it
takes to approach the sample depends on how far the cantilever
is from the surface.
22. Click Ramp, which will stop the scan and withdraw the tip.
23. In Ramp, adjust the parameters in Ramp Parameter List (Follow PeakForce QNM User Guide, Bruker for details) and click
on the Ramp Single.
24. A force curve will appear. Mark the elevated part of the curve
on the right (Fig. 2b, part of the curve within the region
highlighted in pale pink).
25. Click on Update Sensitivity.
26. Set Realtime Channel Sensitivities window will appear with the
Deflection Error sensitivity (nm/V) measured. Clicking OK will
update the deflection sensitivity.
27. Go to Withdraw to lift the cantilever.
28. In Calibrate and then in Thermal Tune, calibrate the spring
constant using thermal tuning (it is recommended for cantilevers with a spring constant 1 N/m).
29. Select Thermal Tune Range in kHz (look at the cantilever
specifications written on the box to know the range).
30. In Thermal Tune window, select Lorentzian (Air), temperature (usually 21.0
C), and press Acquire Data.
31. A peak will appear on the field presenting the Power over
Frequency. Place two lines around the peak.
Atomic Force Microscopy to Study Cell Wall Mechanics in Plants
359
microscope stage (Fig. 3e).
15. Go to Navigate and click on Sample Load Position. This moves
the AFM head up so that the manipulation of the AFM head
will not cause the damage of the cantilever.
16. Take the AFM head out, place the sapphire sample on the
microscope stage, and stabilize it with magnetic clamps.
17. Using the objective camera, move the sapphire sample to the
center, and if possible focus on the surface of the sample.
18. Move the AFM head back over the sample (Fig. 3e).
19. In the Navigate window, adjust the focus on the cantilever by
moving the AFM head down. Look at the objective camera to
control the distance of the cantilever from the sample. When
the cantilever is approaching the surface of the sample, the
shadow of the cantilever should be seen, it should then appear
sharp in the center. Decrease the speed when the cantilever is
close to the surface.
20. In the Check parameters window, set the Scan size to 0 nm.
21. Go to Engage to approach the sapphire probe. The cantilever
will move toward the sample in an automated way. The time it
takes to approach the sample depends on how far the cantilever
is from the surface.
22. Click Ramp, which will stop the scan and withdraw the tip.
23. In Ramp, adjust the parameters in Ramp Parameter List (Follow PeakForce QNM User Guide, Bruker for details) and click
on the Ramp Single.
24. A force curve will appear. Mark the elevated part of the curve
on the right (Fig. 2b, part of the curve within the region
highlighted in pale pink).
25. Click on Update Sensitivity.
26. Set Realtime Channel Sensitivities window will appear with the
Deflection Error sensitivity (nm/V) measured. Clicking OK will
update the deflection sensitivity.
27. Go to Withdraw to lift the cantilever.
28. In Calibrate and then in Thermal Tune, calibrate the spring
constant using thermal tuning (it is recommended for cantilevers with a spring constant 1 N/m).
29. Select Thermal Tune Range in kHz (look at the cantilever
specifications written on the box to know the range).
30. In Thermal Tune window, select Lorentzian (Air), temperature (usually 21.0
C), and press Acquire Data.
31. A peak will appear on the field presenting the Power over
Frequency. Place two lines around the peak.
Atomic Force Microscopy to Study Cell Wall Mechanics in Plants
359