turgid plant cells requires a softer cantilever with bigger and round
tip (nondestructive), e.g., a cantilever with a stiffness of 0.1 N/m is
used to measure stiffness in the cell walls of meristematic cells in the
shoot apical meristem (SAM) [22]. However, using too soft cantilevers can cause strong deformations and uncontrolled vibrations
during the indentation. The study of harder materials involves a
stiffer cantilever with a smaller and sharper tip, but the cantilever
should not be too stiff as it can go too deep and damage the sample.
Before the indentation, the cantilever deflection sensitivity and the
spring constant must be calibrated. The spring constant is
measured using the thermal tuning method [23, 24]. For cantilevers with a spring constant >100 N/m, the deflection sensitivity is
calibrated against the very hard sapphire, which serves as the maximum stiffness reference (sapphire stiffness is 470 GPa) [25]. The
AFM tapping mode relies on direct contact between the tip and the
surface. Each single tapping cycle consists of the following stages:
before the approach to the surface (the cantilever is in a relaxed
position) (Fig. 1b), at the contact point (the cantilever is pressing
on the surface being deflected upward) (Fig. 1c), and after the
retraction from the surface (the cantilever is deflected downward
being hold by the adhesive forces) (Fig. 1d). The cantilever moves
along different zones of the sample. Due to local differences in the
topography and/or mechanical properties of the sample, the
tapping on different regions gives different responses of the cantilever (motion, bending, and generated forces), e.g., a stiffer material will cause stronger deflection of the tip. The laser beam reflects
the response of the cantilever (Fig. 1a) and sends it to the detector
and electronics. The deflection of the cantilever is converted into
force curves, which can be presented as a function of force over
time (Fig. 2a) or force over height (distance between tip and
surface) (Fig. 2b). The results of the indentation are stored as an
image, in which every pixel is represented by an individual forceindentation curve. The force curves within the regions of interest
(ROIs) can be extracted and processed to calculate different physical parameters such as the apparent elastic modulus. This allows to
select and compare different ROIs in the image. Note that the
acquired force curves should resemble the reference curves
(Fig. 2a,b), and any deviation in the force curve morphology may
indicate a problem with the sample (e.g., the sample is too sticky
and the tip is not able to withdraw off the sample) or with the
cantilever tip (e.g., it is too big for the measurement or damaged).
The AFM resolution greatly exceeds the optical diffraction
limit and it is similar to the resolution of the electron microscope
[26]. The major advantages of AFM compared to other techniques
are that it does not require sample preparation; it can be operated in
ambient or liquid environment; and it reconstructs the
3D-topography of the sample [27]. The major limitations of
AFM are the restricted scan area (max. 150 μm
2 ) and height
352
Mateusz Majda
tip (nondestructive), e.g., a cantilever with a stiffness of 0.1 N/m is
used to measure stiffness in the cell walls of meristematic cells in the
shoot apical meristem (SAM) [22]. However, using too soft cantilevers can cause strong deformations and uncontrolled vibrations
during the indentation. The study of harder materials involves a
stiffer cantilever with a smaller and sharper tip, but the cantilever
should not be too stiff as it can go too deep and damage the sample.
Before the indentation, the cantilever deflection sensitivity and the
spring constant must be calibrated. The spring constant is
measured using the thermal tuning method [23, 24]. For cantilevers with a spring constant >100 N/m, the deflection sensitivity is
calibrated against the very hard sapphire, which serves as the maximum stiffness reference (sapphire stiffness is 470 GPa) [25]. The
AFM tapping mode relies on direct contact between the tip and the
surface. Each single tapping cycle consists of the following stages:
before the approach to the surface (the cantilever is in a relaxed
position) (Fig. 1b), at the contact point (the cantilever is pressing
on the surface being deflected upward) (Fig. 1c), and after the
retraction from the surface (the cantilever is deflected downward
being hold by the adhesive forces) (Fig. 1d). The cantilever moves
along different zones of the sample. Due to local differences in the
topography and/or mechanical properties of the sample, the
tapping on different regions gives different responses of the cantilever (motion, bending, and generated forces), e.g., a stiffer material will cause stronger deflection of the tip. The laser beam reflects
the response of the cantilever (Fig. 1a) and sends it to the detector
and electronics. The deflection of the cantilever is converted into
force curves, which can be presented as a function of force over
time (Fig. 2a) or force over height (distance between tip and
surface) (Fig. 2b). The results of the indentation are stored as an
image, in which every pixel is represented by an individual forceindentation curve. The force curves within the regions of interest
(ROIs) can be extracted and processed to calculate different physical parameters such as the apparent elastic modulus. This allows to
select and compare different ROIs in the image. Note that the
acquired force curves should resemble the reference curves
(Fig. 2a,b), and any deviation in the force curve morphology may
indicate a problem with the sample (e.g., the sample is too sticky
and the tip is not able to withdraw off the sample) or with the
cantilever tip (e.g., it is too big for the measurement or damaged).
The AFM resolution greatly exceeds the optical diffraction
limit and it is similar to the resolution of the electron microscope
[26]. The major advantages of AFM compared to other techniques
are that it does not require sample preparation; it can be operated in
ambient or liquid environment; and it reconstructs the
3D-topography of the sample [27]. The major limitations of
AFM are the restricted scan area (max. 150 μm
2 ) and height
352
Mateusz Majda