1

The principles of Atomic Force Microscopy (AFM)

Introduction:
Microscopes have historically been tools of great importance in biological science. The atomic force
microscopy (AFM) is one of a family of scanning probe microscopes which has grown steadily since
the invention of the scanning tunneling microscope by Binning and Rohrer in the early eighties for
which they received the Nobel Price for Physics in 1986.
There are some significant advantages of AFM as an imaging tool in biology and physics when
compared with complementary techniques such as electron microscopy. Not only does AFM achieve
molecular resolution but can be performed under fluids permitting samples to be imaged in near native
conditions. The fluid may be exchanged or modified during imaging and therefore there is the
potential for observing biological processes in real time, something which electron microscopy is not
currently able to achieve.
There have been many studies of biological materials using AFM in the few years since its conception.
Examples include nucleic acids and their complexes with proteins, two dimensional protein crystals
and individual isolated proteins, membranes and membrane bound proteins, and living cells. The
instrument is also capable of manipulating molecules and measuring the strength of molecular
interactions with pico-newton sensitivity.

Fig. 1: The AFM.

Adjustment of the microscope requires in this case coarse and fine approaches of fiber end and
cantilever on the one hand and of cantilever and sample on the other hand. This task is solved by a
combination of different piezoelectric actuators involving two concentric piezo tubes for fiber and
cantilever positioning, a motor driven by shear piezos for positioning of the probe with respect to the
sample, and an arrangement of three piezo tubes for scanning. Since the set-up does not contain any
elements which could only be adjusted manually, fine tuning of the interferometer or the probe-sample
seperation within a cryostat or UHV(ultrahigh vacuum)-chamber is straightfoward. The piezoelectric
walker can move stepwise over a few milimeters exhibiting a single-step precision of 100 nm or
less. Since the whole microscope head is made from non-magnetic components, it can be operated
under the influence of high magnetic fields.
Piezoelectric ceramics are a class of materials that expand or contract in the presence of a voltage
gradient or, conversely, create a voltage gradient when forced to expand or contract. Piezoceramics
make it possible to create three-dimensional positioning devices of high precision. Most scannedprobe microscopes use tube-shaped piezoceramics because they combine a simple one-piece

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construction with high stability and large scan range. Four electrodes cover the outer surface of the
tube, while a single electrode covers the inner surface. Application of voltages to one or more of the
electrodes causes the tube to bend or stretch, moving the sample in three dimensions.

Fig. 2: The tube.

If AFM should be performed on liquid / solid interfaces, the sample holder is substituted by an
electrochemical cell.

Fig. 3: An electrochemical cell.

For experiments where you
have to change the fluid during
scanning, there is also a cell
with two small plastic tubes for
controlled input and output of
fluids available. Another kind
of fluid cell can be heathen.

This device contains a couple of reference electrodes and is chemically largely inert. Sample,
cantilever and fiber end are all immersed in the liquid environment. A considerable strength of the
fiber interferometer is that no interference between light reflected off the cantilever on the one hand
and at the liquid / gas (or liquid / air) interface on the other hand affects the measurement. Ever more
complex are set-ups which additionally allow a variable sample temperature involving low and
elevated values. The most sophisticated approaches offer the option to additionally apply high
magnetic fields.
AFM can generally measure the vertical and horizontal deflection of the cantilever with picometer
resolution. To achieve this, most AFMs today use the optical lever, a device that achieves resolution
comparable to an interferometer while remaining inexpensive and easy to use. The optical lever
operates by reflecting a laser beam off the back of the cantilever. Angular deflection of the cantilever
causes a twofold larger angular deflection of the laser beam. The reflected laser beam strikes a
position-sensitive photodetector consisting of four side-by-side photodiodes. The difference between
the four photodiode signals indicates the position of the laser spot on the detector and thus the angular
deflection of the cantilever. If the tip is scanned over the sample surface then the deflection of the

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cantilever can be recorded as an image which represents the three dimensional shape of the sample
surface (deflection image).
But the AFM not only measures the force on the sample but also regulates it, allowing acquisition of
images at very low forces. The feedback loop consists of the tube scanner that controls the height of
the entire sample; the cantilever, which measures the local height of the sample; and a feedback circuit
that attempts to keep the cantilever deflection constant by adjusting the voltage applied to the scanner.
The faster the feedback loop can correct deviations of the cantilever deflection, the faster the AFM can
acquire images; therefore, a well-constructed feedback loop is essential to microscope performance.
c

Fig. 4a, b: The laser spot is focussed on the back of the

cantilever and the angle of the reflected laser is
detected by a PSD (photosensitive detector).
c: The principles of the feedback loop.

The tips:

*
E

*
D

*
*

Fig. 5:
a: A pyramidal tip.It can be
modified by chemical reactions.
b: The kind of cantilever which is used
in our lab. Each of the cantilever has
a characteristic spring constant, the
tip iself is positioned at the end of
the Vs and the bar (*). There are
magnetic and non-magnetic
cantilevers of this geometry
available. They are called (from the
left to the right) F (not shown), E, C,
D, B.

In order to detect local forces or closely related physical quantities the sharp probe scanning the
sample surface at same distance has to be liked to some sort of force sensor. A convenient way to
precisely measure forces is to convert them into deflections of a spring according to Hookes law:
z =

F
,
kc

where the deflection z is determinated by the acting force F and the spring constant kc.

Fig. 6:
a: Scheme of the tips spring constant.
b: Other kinds of tips.

Today a variety of cantilevers with different geometries (mainly bar- and V-shaped) and with
pyramidal as well as conical tips is commercially available.
The resonant frequency of a spring with spring constant kc and lumped effecteve mass m is given by
0 =

kc
.
m

Because of the Hooks law it is desirable to have a low spring constant in order to achieve maximum
force sensitivity. This is contadicted by three aspects:
The spring constant should be a maximum in order to achieve a maximum resonant
frequency, and thus, a minimum vibrational sensitivity and a maximum scan rate.
The ultimate sensitivity of the force measurement is restricted by thermal excitation of the
cantilever. The latter quantity can be determined from the equipartition theorem

(z )rms

k BT
,
kc

where (z )rms is the rms displacement amplitude of the end of the cantilever due to thermal
excitation.
If the cantilever is subject to a long-range attractive force, and this will almost always be the
case upon probe-sample approach, its position becomes unstable if the magnitude of the
force gradient equals the cantilevers spring constant. Thus, a certain minimum spring
constant is needed in order to approach the sample sufficiently closely without a jump to
contact.

In order to estimate the order of magnitude which the spring constant of the cantilever could have, it is
straightforward to match kc to the respective constant of interatomic coupling in solids.
Taking m = 10-25 kg and 0 = 1013 Hz for atomic masses and vibrational frequencies are arrives at
kc = 10 N/m. Even smaller spring constants can be easily obtained by minimizing the cantilevers
mass. Commercial cantilevers have a typical spring constant in the range of 10-2 N/m kc 102 N/m,
typical resonant frequencies in the range of 1 kHz 0 500 kHz, a radius of curvature of the probing
tip as small as 10 nm, and are usually fabricated of Si, SiO2 or Si3N4.
If one again takes the above estimate for the interatomic couplig (c = 10 N/m) for a rough estimate of
the resulting deflection of a cantilever which is subject to an interatomic interaction, one finds that a
force of 1 nN causes a deflection of 1, while thermal rms raise amounts to above 20% of this value.
Thus, the task is to precisely measure cantilever deflections being smaller than 1.
The resolution of AFM depends mainly on the sharpness of the tip which can currently be
manufactured with an end radius of a few nanometers. A close enough inspection of any AFM tip
reveals that it is rounded off. Therefore force microscopists generally evaluate tips by determining

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their end radius. In combination with tip-sample interaction effects, this end radius generally limits
the resolution of AFM. As such, the development of sharper tips, p. e. nano-tubes, is currently a major
concern.
Atomic resolution is easily obtained on relatively robust and periodic samples. Soft samples
particularly biological samples provide a more difficult surface to image because the forces exerted
by the tip during imaging can cause deformation of the sample. The problem involved with imaging
soft samples have been overcome to a large extent by the introduction of tapping mode AFM
imaging. Instead of maintaining a constant tip-sample distance of a nanometer or so, the cantilever is
oscillated in a direction normal to the sample resulting in only intermittent contact with the surface.
This greatly reduces the lateral forces being applied in the plane of the sample which are responsible
for most of the damage as the tip is scanned. The AFM is capable of better than 1 nm lateral resolution
on ideal samples and of 0.01 nm resolution in height measurement.

Fig. 7: The principle of scanning:

protrusions appear wider, depressions narrower
than they are in reality.

If the interaction decay length << R, the gap s between sample and tip, s << R, the tip geometry
determines the resolution; in the most simple approximation we imagine a sphere of radius R to roll
over the surface S, and the path A of its center to be recorded. The non-linearity of this process is
evident: the image of a protrusion appears wider, but the image of a depression appears narrower than
the object. The practical resolution, however, is also determined by the sensitivity of the height
detector, i. e., the noise level.
Provided, that the mechanical and electronic stability of the microscope allows detection of e. g.
0.01 nm height differences, we estimate that the tip radius R needs to be smaller than 3 nm to resolve
spheres of 0.5 nm diameter.

Fig. 8: Variables that determine the

resolution of the scan.

FWHM = 2 d (R + d 4 )

Scan modes:
According to Hookes law, the cantilever which is raster-scanned across the sample surface exhibits a
locally varying deflection which directly represents the corrugation of the sample surface.

Fig. 9: The parts of an approach-retraction cycle of the tip.

The distance regimes are labeled on the figure:

The contact regime and
the non-contact regime.
In the contact regime, the cantilever is held less than a few angstroms from the sample surface, and the
interatomic force between the cantilever and sample is attractive (largely a result of the long-range van
der Waals interactions).

Fig. 10: The parts of a force-distance curve (see

later) show the interactions of the tip and
the sample.

Contact mode
In contact mode, also known as repulsive mode, an AFM tip makes soft physical contact with the
surface. The tip is attached to the end of a cantilever with a low spring constant, lower than the
effective spring constant holding the atoms of the sample together. As the scanner gently traces the tip
across the sample (or the sample under the tip), the contact force causes the cantilever to bend to
accommodate changes in topography.
At the right side of the curve of the figure above the atoms are seperated by a large distance. As the
atoms are gradually brought together, they first weakly attract each other. This attraction increases
until the atoms are so close together that their electron clouds begin to repol each other electrostatically. This electrostatic repulsion progressively weakens the attractive force as the interatomic
seperation continues to decrease. The force goes to zero when the distance between the atoms reaches
a couple of angstroms, about the length of a chemical bound. When the total van der Waals force
becomes positive (repulsive), the atoms are in contact. The slope of the van der Waals curve is very
steep in the repulsive or contact regime. As a result, the repulsive van der Waals force balances almost
any force that attempts to push the atoms closer together. In AFM this means that when the cantilever
pushes the tip against the sample, the cantilever bends rather than forcing the tip atoms closer to the
sample atoms. Even if you design a very stiff cantilever, the interatomic seperation between the tip and
sample atoms is unlikely to decrease much, the sample surface is likely to deform.

Fig. 11: Soft samples are easier deformed than hard

samples if the tip-sample ineraction is permanent.

In addition to the repulsive van der Waals force, two other forces are generally present at contact
mode: a capillary force exerted by the thin water layer often present in an ambient environment and
the force exerted by the cantilever itself. The capillary force arises when water wicks its way around
and applying a strong attractive force (about 10-8 N) that holds the tip in contact with the surface. The
magnitude of the capillary force depends upon the tip-to-sample seperation.
Fig. 12:
There are two
kinds of tip-sample
interactions which
almost always
influence the scan:
van de Waals
forces (left) and
capillary forces
(right).

The force exerted by the cantilever is like the force of a compressing spring. The magnitude and sign
(repulsive or attractive) of the cantilever force depends upon the deflection of the cantilever and upon
its spring constant. As long as the tip is in contact with the surface, the capillary force should be
constant because the distance between the tip and the sample is virtually incompressible. It is also
assumed that the water layer is reasonably homogeneous. The variable force of the contact AFM is the
force exerted by the cantilever. The total force that the tip exerts on the sample is that of the capillary
plus cantilever forces, and must be balanced by the repulsive van der Waals force for contact. The
magnitude of the total force exerted on the sample varies from 10-8 N (with the cantilever pulling
award the sample almost as hard as the water is pulling down the tip), to the more typical operating
force of 10-7 to 10-6 N.
Once the AFM has detected the cantilever deflection, it can generate the topographic data set by
operating in one of two modes:
The constant-height mode and
the constant-force mode.

Fig. 13: The difference between the two modes:

In constant-force mode the cantilever deflection is constant (left) whereas in constant-height mode
the z-value is the constant held parameter (right).

In constant-force mode, the spatial variation of the cantilever deflection can be used directly to
generate the topographic data set because the height of the scanner is fixed as it scans.
In constant-force mode, the deflection of the cantilever can be used as input to a feedback circuit that
moves the scanner up and down in z, responding to the topography be keeping the cantilever
deflection constant. In this case, the image in generated from the scanner motion with the cantilever
deflection held constant, the total force applied to the sample is constant. In constant-force mode, the

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speed of scanning is limited by the response time of the feedback circuit, because total force exerted
on the sample by the tip is well controlled. Constant-force mode is generally preferred at most
applications.
Constant-height mode is often used for taking atomic-scale images of atomically flat where the
cantilever deflections and thus variations in applied force are small. Constant-height mode is essential
for recording real-time images of changing surfaces, where high scan speed is essential. The typical
magnitude of lateral forces is in the range of 10-10-10-8 N for forces which do not cause permanent
perturbations of the sample surface.
While topographic imaging uses the up-and-down deflection of the cantilever, friction imaging (or
lateral force imaging or lateral deflection imaging) uses torsional deflection. If the scanner moves
the sample perpendicular to the long axis of the cantilever, friction between the tip and samples causes
the cantilever to twist.
Lateral deflections of the cantilever usually arise from two sources:
Changes in friction and
changes in slope.
In the first case, the tip may experience different frictions as it traverses inhomogeneities causing the
cantilever to twist more or less strongly. In the second case, the cantilever may twist when it reachs
steep slope.
To seperate one effect from the other, normal topographical images should be collected
simultaneously.

Fig. 14: The two sources of lateral deflections:

a: Changes in friction.
b: Changes in slope.

AFM uses a position-sensitive photodetector (PSPD) to detect the deflection of the cantilever and the
cantilevers twist. The reflected laser beam is detected by a PSPD which is segmented into four
quadrants.

f
d

Fig. 15 a-c (see page 8) and d-f:

A photodetector recognises the reflected
laser spot. There are several differences
between atomic and frictional force
microscopy.

If the light beam moves between the upper and lower pairs of segments the deflection of the cantilever
can be deduced from a proper treatment of all individual photocurrents:

I vertical = (I upper _ left + I upper _ right ) (I lower _ left + I lower _ right )

I vertical is related to the deflection of the laser beam which is given by
y =

d
z ,
e

where 1 is the length of the cantilever and d that of the light path subsequent to reflection. Usually the
laser beam has a Gaussian intensity profile with a characteristic spot diameter increasing propotional
to the distance of between cantilever and photodetector. The photocurrent generated in the detector is
proportional to the flux density j of the photons hitting the photodiode:

I vertical ydj .
Since j 1 d 2 shows that I vertical is independent of the seperation d between cantilever and
detector. This is, however, valid only as long as the deflection y is small compared with spot
diameter of the laser beam. For larger deflections nonlinearities in I vertical (y) result. The
independence of the force sensors sensitivity of the seperation of photodetector and cantilever allows
the realization of very compact beam-deflection schemes.
The twisting results in a horizontal deflection of the laser spot on the surface of the photodetector:

I horizontal = (I upper _ left + I lower _ left ) (I upper _ right + I lower _ right ) .

In standard topographical imaging the occurance of lateral forces acting on the probing tip is often
unwanted because the forces affect the images usually in a complex way. Triangular cantilevers,
which are commercially available as standard products were originally intended to minimize the
torsion effects.

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Fig. 16: Images, made in contact mode (lateral force microscopy mode).
a: Polyacetate.
b: Latex Spheres.

Non-contact mode
When lifting the probe by at least one nanometer from the sample surface, only long-range interactions
remain. The relevant forces result in general from van der Waals interactions, electro- and
magnetostatic interactions, and, under ambient conditions, often from the formation of liquid
capillaries. Information of the atomic or nanoscale surface structure gets completely lost.
While van der Waals forces are relatively small and capillary forces can be avoided by either choosing
a sufficiently large working distance or by working on clean surfaces, electro- and magnetostatic
interactions can yield relatively strong forces. This provides important information about the electrical
or magnetic charge distribution in the near-surface regime of the sample. Since these charge
distributions can be manifold, the lateral variation as well as the range of the resulting interactions are
very different on different samples. Near-field operation means in this context that only charges in
probe and sample within a certain volume around the probe apex contribute to contrast formation. In
other words, if the static interaction is modeled in terms of a multipole expansion of the charge
distribution, one usually finds monopole, dipole and higher contributions which all have to be taken
into account up to a certain degrees. Thus, for the magnetostatic interaction it is very frequently found
that the resulting forces are not simply dipole forces but that the monopole term dominates contrast
formation.
Magnetic force microscopy images the spatial variation of magnetic forces on a sample surface. The
tip is coated with a ferromagnetic thin layer. Changes in the resonant frequency of ther cantilever are
detected.

Magnetic force microscopy can be used to image naturally occurring and deliberately written domain
structures in magnetic materials. An image taken with a magnetic tip contains information about both,
the topography and the magnetic properties of a surface. Which effect dominates depends upon the
distance of the tip from the surface, because the interatomic magnetic force persists for greater tip-tosample seperations than the van der Waals force. If the tip is close to the surface, the image will be
predominantly topographic. As you increase the seperation between the tip and the sample, magnetic
effects become apparent. Collecting a series of images at different tip heights is one way to seperate
magnetic from topographic effects.
a

Fig. 17:
a: Principle of magnetic force
microscopy.
b: A magnetic disc.

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Electrical charges can be present in probe and sample either permanently or can be introduced by
applying an electrical potential between probe and sample. Coulomb forces have been measured on a
variety of samples. Electrostatic force microscopy applies a voltage between the tip and the sample
while the cantilever hovers above the surface, not touching it.
Electrostatic force microscopy plots the locally charged domains of the sample surface, similar to how
magnetic force microscopy plots the magnetic domains of the sample surface. The magnitude of the
deflection, proportional to the charge density, can be measured with the standard beam-bounce system.
a

Fig. 18:
a: Electrostatic force microscopy.
b: A SRAM sample.

If a potential difference V is externally applied between probe and sample, the resulting Coulomb
force is given by

FC = (d )V 2 .
defends on the geometry of the probe, on the local geometrical configuration of the sample surface,
on the dielectric environment and explicitly on the probe-sample spacing d. If the probe-sample
arrangement is modeled to a first order by a simple parallel-plate capacitor with an electrode area A
involving a dielectric medium of relative constant r, one would find

(d ) = 0 r A (2d 2 ) .
FC exhibits a square dependence in the local electrical potential which opens the possibility to measure
potential variations across sample surfaces. Due to the dependence of on r also dielectric properties
of a sample surface can be measured. It has been demonstrated that it is very useful to apply a
potential difference also for nonelectrical measurements in order to seperate topographical influences
from the long-range interactions of interest.
Today, non-contact force microscopy usually involves a sinosoidal excitation of the cantilever with a
frequency close to its main resonant frequeny. If magnetic interactions are involved, it is possible to
externally modulate the oscillation (if the cantilever is coated ferromagnetically). The non-contact
mode of operation involving an oscillating cantilever is frequently also called AC mode (or dynamic
mode).
Fig. 19: MAC mode
(magnetic AC mode).
a: Each cantilever has
its characteristic
resonance frequency.
This can be controlled
by a special function
of the AFM. The AFM
b
always needs the
correct frequency
to work well, so all AFM-parameter have to been set
on the needed values.
b: An externally induced periodically changing
magnetic field moves the ferromagnetic coated
cantilever.

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In contrast to the detection of a quasistatic force the response of the cantilever in the dynamic mode is
more complex. If the cantilever is excited sinosoidally at its clamped end with a frequency and an
amplitude 0, the probing tip likewise oscillates sinosoidally with a certain amplitude exhibiting a
certain phase shift with respect to the driving signal.
The deflection sensor monitors the motion of the probing tip. The equation of motion, the solution of
which is monitored by the deflection sensor, is given by

2 d 0 d
2
+ 0 (d d 0 ) = 0 0 cos(t ) ,
+
2
Q t
t
where d0 is the probe-sample distance at zero oscillation amplitude and d(t) the momentum probesample seperation. Q is apart from the intrinsic properties of the cantilever, which are the lumped
effective mass and the resonant frequency, determined by the damping factor :

Q=

m 0
.
2

introduces the influence of the environmental medium which could be ambient air, a liquid, or ultrahigh vacuum (UHV). Q ranges from values below 100 for liquids, air or other gases at an appropriate
pressure to more than 100 000 in UHV. The steady-state solution is

d (t ) = d 0 + cos(t + )
for the forced oscillator and the amplitude of the probes oscillation is given by

0 0 2
2

2 2

+ 4
2

The phase shift between this oscillation and the excitation signal amounts to

= arctan

02
2

This simplified formalism is based on the assumption that the oscillation amplitude is sufficiently
small in comparison with the length of the cantilevers. All these results describe only free cantilever
oscillations, e. g. oscillations at the absence of any probe-sample interactions. This means, d0 is still so
large that no influence of the sample on the probes oscillation can be detected. If d0 is now decreased
such that a force F affects the motion of the cantilever, then a term F / m has to be added. In order to
consider almost all interactions which could be interesting in force microscopy, one has to assume

d
F = F d, ,
t
which accounts, apart from static interactions, also for dynamic forecast (e. g. hydrodynamic effects).
Since F describes in the various types, i. e. of very different dependence on the probe-sample spacing,
the d(t) curves monitored by the deflection sensor usually represent enharmonic oscillations. If F(d)
can be substituted by a first-order Taylor approximation for 0 << d0, then the force microscope detects
the compliance or vertical component of the force gradient F / z. On the basis of this approximation
the cantilever behaves under the influence of the probe-sample interaction as if it would have the
modified spring constant

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cF = kc

F
,
z

where kc is the intrinsic spring constant. An attractive probe-sample interaction with F / z > 0 will
effectively soften the cantilever spring, while a repulsive interaction with F / z > 0 will make it
effectively stiffer. The change of the apparent spring constant will modify the cantilevers resonant
frequency to
= 0 1

1 F
.
k c z

Provided that F / z << kc, the shift in resonant frequency is given by

1 F
.
2k c z

A modification of the resonant frequency will result in a change of the probes oscillation amplitude
and of the phaseshift between probe oscillation and driving signal.
, and are experimentally accessible quantities which can be used to map the lateral variation of
F / z. Phase and amplitude additionally contain information about the damping coeffizient . Thus, a
local variation of this quantity can be seperated from the local variation of the compliance by
measuring the frequency shift and the change in amplitude or the phase shift.
The most commonly used detection method which is generally referred to as slope detection
involves driving the cantilever at a fixed frequency slightly to resonance. A change in F / z gives
rise to a shift in the resonant frequency in the amplitude of the cantilever vibration. is
obvisiously a maximum at that point of the amplitude-versus-frequency curve where the slope is a
maximum. The sensitivity is ultimately determined by thermal noise. The minimum detectable
compliance is given by
1
F
=

z min rms

2 k B T
,
0Q

where rms is the rms amplitude of the driven cantilever vibration and is the measurement bandwidth.
High Q values can be obtained by the reduction of air damping in vacuum (<10-3 mbar). It might thus
appear advantageous to maximize sensitivity by obtaining the highest possible Q. With slope
detection, increasing the Q restricts the bandwidth of the system. If F / z changes during scanning,
the vibration amplitude settles on a new steady-state value after a sufficient length of time given by

2Q

Thus, for a high-Q cantilever in vacuum (Q = 50 000) and a typical resonant frequency of 50 kHz the
maximum available bandwidth would be only 0.5 Hz which is usuable for most applications. The
dynamic range of the system would be similarly restricted. Because of these restrictions it is not useful
to try to increase sensitivity by raising the Q to such high values. If the experiments have to be
performed in vacuum, e. g. to prevent sample contamination, it may not be possible to obtain low
enough Q for an acceptable bandwidth and dynamic range. Therefore, slope detection is unsuitable for
most vacuum applications.
An alternative to slope detection is frequency modulation. In the frequency modulation detection
system a high-Q cantilever vibrating on resonance serves as the frequency-determining component of
an oscillator. Changes in F / z cause instantaneous changes in the oscillator frequency which are

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detected by an frequency modulation demodulator. The cantilever is kept oscillating at its resonant
frequency utilizing a positive feedback. The vibration amplitude is likewise maintained at a constant
level.
In contrast to slope detection, Q and are absolutely independent in frequency modulation detection.
Q depends only on the damping of the cantilever and is set only by the characteristics of the
frequency modulation demodulator. Therefore the frequency modulation detection method allows the
sensitivity to be greatly increased by using a very high Q without sacrificing bandwidth or dynamic
range. In addition, cantilevers used for non-contact AFM must be stiffer than those used for contact
AFM because soft cantilevers can be pulled into contact with the sample surface. The small force
values in the non-contact AFM and the greater stiffness of the cantilevers used for non-contact AFM
are both factors that make the non-contact AFM signal small, and therefore difficult to measure. The
resonant frequency of a cantilever varies as the square root of its spring constant.

Intermittent-contact mode

Tapping mode: a piezo is moved by an acustic signal.

MAC mode: a spool produces a magnetic field.
In the case of rigid samples, contact and non-contact images may look the same. If a few molecules of
condensed water are lying on the surface of a rigid sample, for instance, the images may look quite
different. An AFM operating in contact mode will penetrate the liquid layer to image the underlying
surface, whereas in non-contact mode an AFM will image the surface of the liquid layer.
Contact-mode force microscopy relies on the existence of long-range interactions between probe and
sample. If no static magnetic and electric fields are involved the only common long-range interactions
are in any case van der Waals forces and, in the presence of liquids, capillary forces. Van der Waals
forces are weak and carry only limited information on the surface structure. Thus, non-contact mode
force microscopy is on the one hand largely nondestructive but yields on the other hand only a lateral
resolution of the order of the probe-sampling spacing. In contrast, contact-mode force microscopy has
the potential of high spatial resolution. But it also involves the potential for surface perturbation.
A way to combine the positive aspects of both modes of operation is given if one oscillates the probe
such that there is only an intermittent contact between probe and sample during each oscillation
period. This can be realized if the average probe position d0 is sufficiently far away from the sample
surface. At the same time the driving amplitude 0 is chosing sufficiently large to establish the
intermittent contact. As a consequence, the small-amplitude approximation is no longer valid and the
tip experiences the full variation of the probe-sample interaction potential. The intermittent-contact
mode, which is generally considered as a special variant of the dynamic modes, does thus obviously
not probe a simple force gradient. The repulsive forces during intermittent contact lower the rms
oscillation amplitude which yields a highly surface-sensitive signal that can be used for feedback
support.
The important point is that the energy transferred from the oscillating probe to the sample surface is in
the intermittent-contact mode very much lower than that in the standard contact-mode of operation.
This makes the technique especially interesting for the analysis of delicate soft-matter samples. It has
also been found that intermittent-contact mode is more effective than non-contact AFM for imaging
larger scan sizes that may include greater variation in sample topography.
The intensity of the intermittent-contact mode can be controlled by appropriately setting the freevibration amplitude as well as the drop in amplitude which is kept constant during scanning. Under
ambient conditions, amplitudes as large as 10-100 nm are frequently used for cantilevers with resonant
frequencies of 100 kHz or more. Under liquid immersions the amplitude and its drop can be set much
smaller.

15

Fig. 20: The intermittent-contact mode.

Explaination of three types of tapping mode data:

Height data:
The vertical position of the probe tip is monitored by noting changes in the length of the z-axis
on the xyz-scanning piezo tube. Input voltage to the scanning piezo tube is proportional to the
length of the tube. The change in the z-axis is plottet as a topographical map of the sample
surface. Height data is good measure of the height of surface features but does not show
distinct edges of these features.
Phase data:
This type of imaging monitors the change in phase offset, or phase angle, of the input drive
signal [to the drive piezo] with respect to the phase offset of the oscillating cantilever. The
phase of the drive signal is compared to the phase of the cantilever response signal on the
photodiode detector. The phase offset between the two signals is defined as zero for the
cantilever oscillating freely in air. As the probe tip engages the sample surface, the phase
offset of the oscillating cantilever changes by some angle with respect to the phase offset of
the input drive signal. As regions of differing elasticity are encountered on the sample surface,
the phase angle between the two signals changes. These changes in phase offset are due to
differing amounts of damping experienced by the probe tip as it rasters across the sample
surface. These differences are plotted as the so-called phase image.
Amplitude data:
The amplitude of the cantilever is monitored by the photodiode detector. The rms value of the
laser signal on the y-axis of the detector is recorded for each of the 512 segments on a given
raster of the probe tip. These values are plotted as an amplitude map of the sample surface.
Amplitude images tend to show edges of surface features well.

16

Fig. 21: Images (see also page 15).

Other AFM techniques:

Conductive AFM:
Conductive AFM is used for collecting simultaneous topography imaging and current imaging.
Standard conductive AFM operates in contact AFM mode. Variation in surface conductivity can be
distinguished by this mode.
b

Fig. 22:
a: The principle of conductive AFM.
b: An sample.

Conductive AFM operates in contact AFM mode by using a conductive AFM tip. The contact tip is
scanned in contact with the sample surface. Just like contact AFM, the z-feedback loop uses the DC
cantilever defending a signal ton maintain a constant force between the tip and the sample to generate
the topography image. At the same time, a DC bias is applied to the tip. The sample is held at ground
potential. The built-in preamplified scanner head measures the current passing through the tip and
sample.

Scanning capacitance microscopy:

Scanning capacitance microscopy images spatial variations in capacitance. Like electronic force
microscopy, scanning capacitance microscopy induces a voltage between the tip and the sample. The
cantilever operates in non-contact, constant-height mode. A special circuit monitors the capacitance
between the tip and the sample. Since the capacitance depends on the dielectric constant of the
medium between the tip and the sample, scanning capacitance microscopy studies can image
variations in the thickness of a dielectric material as a semiconductor substrate. This microscopy can
also be used to visualize sub-surface charge-carrier distributions.
a
b

Fig. 23a: Principle of scanning capacitance microscopy.

b: A depletion transistor with source and gate.

17
A metallic tip is used to image a semiconductor with its native oxide sample in contact-mode AFM.
The tip-sample junction is therefore basically a MOS junction. As we apply a DC bias on the sample, a
depletion layer at the semiconductor-insulator interface will be created. This depletion layer increases
the oxide thickness of the MOS structure and therefore modify the capacitance of the junction.
Because the measured signal is very small, a lock-in technique has to improve the signal to noise ratio.
The actual measured signal is then dC / dV.

Scanning thermal microscopy

Scanning thermal microscopy measures the thermal conductivity of the sample surface. It allows
simultaneously acquisition of both topographic and thermal conductivity data.
A cantilever composed of two different metals or a thermal element made up of two metal wires is
used. The materials of the cantilever respond differently to changes in thermal conductivity, and make
the cantilever to deflect. The system generates an image, which is a map of the thermal conductivity
and the changes in the deflection of the cantilever. A topographic non-contact image can be generated
from changes in the cantilevers ampitude of vibration. Thus, topographic information can be
seperated from local variations of the samples thermal properties, and the two types of images can be
collected simultaneously.
a

Fig. 24:
a: A thermal cantilever.
b: A hot spot.

A second type of thermal cantilever uses a Wollastan wire as the probe.

This incorporates a resistive thermal element at the end of the cantilever. The arms of the cantilever
are made of silver wire. The resistive element at the end which forms the thermal probe, is made from
platinum or a platinum / rhodium alloy. The advantage of this design is that it may be used in one of
two modes allowing thermal imaging of sample temperature and thermal conductivity.
The scanning thermal microscopy can be used in two different operating modes, allowing thermal
imaging of sample temperature and thermal conductivity.
In the temperature contrast mode, the thermal probe is used as a resistance thermometer. Temperature
is monitored using a bridge circuit to measure the probe resistance. The probe current is constant and
the temperature changes on the sample surface.
In the conductivity contrast mode, the thermal probe is kept at a constant temperature. The probe
temperature remains constant by a feedback loop that keeps the probe resistance constant. Changes in
sample the conductivity affect the heat flow between the self-heating probe and the sample. This heat
now is monitored by thermal conductivity, by measuring the voltage necessary to keep the probe at a
constant temperature.

Nanolithography
Normally an AFM is used to image a surface without damaging it in any way. But an AFM can also be
used to modify the surface deliberately, but applying either excessive force or heigh pulses with an
AFM. Not only scientific literature, but also newspapers and magazines have shown exact surfaces
that have been modified atom by atom. This technique is known as nanolithography.

18

Fig. 25: Nanolithography.

a: PSI written as SiO2 on Si.
b: The oxide has been etched away
to give a depression.

The most common artifacts to watch for:

Double tip:

Fig. 26: Double tip.

The double image is caused by two areas of the AFM tip interacting with the sample, and in this case,
with something spherical being adsorbed onto the tip.
After letting it scan for a little while, the contamination can be removed (this may happen when
working with soft organic material like bacteria or polysaccharides). But if the image quality does not
improve after a few scans, the tip should be replaced.

Repeated geometry in image:

Fig. 27: Repeated geometry.

The background of this image shows a repeating, triangular pattern. This is probably the shape of the
tip. If this happens, the tip should be replaced again.

19
Set point is too low (for tapping mode only):
If the setpoint is too low, you will see features in the image that do not really exist (p. e. rings or
patches of noise).

Set point is too high (for tapping mode only):

Fig. 28: Streaks, caused by a too high set point

in tapping mode.

If the set point is too high, you will not be able accurately track the sample surface, and you will
probably see streaks as in this image. Adjusting the gains will also help improve your images, but if
the set point is not adjusted properly, then continuing to increase the gains will only make your image
noisier, not clearer.

Drive frequency is too high (for tapping mode only):

The topography of p. e. a bacterial surface appears to be damaged or to show holes. These holes are
artifacts and can be eliminated by decreasing the drive frequency, or by disengaging and repeating the
auto-tune of the cantilever.

Other errors:

Scanner produces loud warning sound:

That means, it is oversteered. The power has to be reduced immediately.
There is only noise seen on the screen:
Change the set point till a clear picture is seen (perhaps you have to use the stepper motor).
Signal is drifting:
Perhaps the AFM has not reached ist optimal working temperature yet, so wait a little bit. If
the drift is still seen after a few minutes or if it starts suddenly during the scan, it could be a
sign that there is an air bubble in the fluid cell. Withdrawel and remove it.
The set point is reached immediately, but there is only noise on the screen:
Look if the set point is set to zero during the approach and try it again.

Force distance cycles

Force microscopy is not only capable of providing information about the lateral variation of probesample interactions across a sample surface. One can also systematically determine the dependence of
the interaction on the probe-sample distance, i. e. the range of the interaction at a given location with
respect to the sample surface.

20

Fig. 29 a-c: The relationship between a forcedistance curve and the cantilevers deflection.
d: An antibody or a molecul can be mounted
on the tip via PEG (a crosslinker).

At the beginning, the probe is far away from the surface, so there is no interaction between the two. As
the probe-sample seperation is reduced beyond a certain point, forces between atoms on the two
surfaces begin to act, causing the flexible cantilever to bend toward the sample in the case of attractive
forces (van der Waals and electrostatic), or away from the sample in the case of repulsive forces
(electrostatic). This causes a nonlinear d(z) variation in
d = z z .
If the vertical component of the force gradient, F / z, at a sufficiently small probe-sample spacing d
exceeds the cantilevers spring constant kc, the instability causes a jump of the probe to contact.
[Contact may be defined as the point when depulsion is first detectable.]
A local minimum in the force curve indicates maximum attraction. Further decrease of the probesample spacing increases the loading force and the contact radius.
The attractive forces can cause the probe to snap to the surface earlier (from a greater distance) than
the expected time of contact in the absence of such forces. The jump-to contact observed in approach
curves limits the range of data that may be obtained on the approach cycle and also the gentleness of
the tip approach. When the jump-to contact is caused by electrostatic attraction, it can be minimized
by operating in electrolytes that screen these interactions. Once in contact with the surface, the probe

21
will experience an ever-increasing repulsive force (and the cantilever will bend away from the surface)
on the electron orbitals of the atoms in the probe and the sample will begin to overlay. In this region of
the force curve, referred to as the contact region, there may be elastic and / or plastic (reversible and /
or irreversible) deformations of either or both the probe and the sample.
There is a distinction between the force versus piezo z-displacement and the deflection of the
cantilever (with soft samples, sample deformation needs to be taken into account). At zero force, the
points in both types of curves coincide; the maximum difference between the two plots is at the
maximum force.
Upon withdrawing the probe, the motion of the cantilever is reversed. The not-loading force
continuously decreases. After reversibly reaching the origin of the force curve, which corresponds to
the origin of the z(z) curve, bending of the cantilever is again towards the sample surface. In further
trying to seperate probe and sample, one finds that they adhere to each other (because of adhesion
forces, created during contact and / or hydrophobic and solvation forces). This phenomenon causes an
extended motion of the cantilever with z = z. At a certain point of retraction the net force gradient
again becomes equal to the cantilevers spring constant and a jump out of contact occurs. From then
the probe does not experience any interaction with the sample and the cantilever is in its equilibrium
position.
Of special interest to us is the adhesion force that is estimated from the deflection of the cantilever
right before the jump-off contact point.
The cantilever deflections occuring as a result of interatom-interactions are converted into forces using
Hookes law again:
F = -kcd ,
where F is the force acting on the cantilever, kc is the spring constant of the cantilever, and d is its
deflection. There is considerable spread in the values of cantilever force constants and independent
calibration is essential.
AFM force curves are rich in information. They allow (among other things) measurements of the
strength of individual hydrogen bonds and of different kinds of covalent bonds.
Althought the dynamic mode presents several important advantages over the contact mode, the
interpretation of contact force curves is rather straightfoward, whereas curves recorded in the dynamic
mode are more difficult to interpret in terms of forces. The contact force curve is reproduced by
integration of the oscillation amplitude curves when the forces are conservative, i. e. the process is
reversible:

F ( z ) = k 0 1dz + C .
A

(z)

A0 is the undamped amplitude (~ 5 nm), A(z) is the amplitude at a distance z, and k is the spring
constant of the cantilever.
Comparison of contact and magnetic AC curves may be extremely useful in the interpretation of the
data, because, in the event that they reflect reversible, purely elastic processes, the two are simply
related. Thus, the pair of curves recorded simultaneously serves to distinguish elastic from dissipative
processes.
Tips are often modified to carry specific chemical groups or molecules, which allows assessing the
weak van der Waals, electrostatic and hydration forces acting between the molecules on the tip and
those on the surface. A thin wetting film of liquid (water) covers both the probe and the sample
when the AFM is operated in air. This thin film creates capillary forces that may have a dramatic
effect on the force curves. The overall magnitude of the capillary force can be large enough to obscure
the weak van der Waals forces. In view of this, experiments aimed at quantitation of interaction forces

22
should be performed in liquids, in vacuum or under dry nitrogen. Aqueous solution operation is
desirable with biological molecules because it preserves their native structure.
The major parameter affecting bond rupture is the applied loading rate (the change of force with time
dF / dt). Loading rate is also given as the product kcv of the tip velocity v and the spring constant kc.
The probability of thermal rupturing of the bond increases as exp(-[EB-FxB] / kBT), where EB is the
potential energy barrier of bond disruption, E is the applied force in the bond direction, xB is the
stretched distance in this direction, kB is the Boltzman constant, and T is the temperature. This
description of loading rate applies if the loading force is transmitted by a compliant polymer, the
loading rate at some site (the bond to be ruptured) within that polymer also depends on the compliance
and the extension of the polymer.

Intermolecular interactions:
As mentioned above, it is possible to use the capability of AFM to sense interaction forces between
atoms at the end of the probe and in the specimen to study the weak, non-covalent, usually short-range
forces involved in molecular recognition reactions. In order to be able to do so, the partners in the
molecular recognition have to be immobilized onto the surface and the probe. Probes containing
certain chemical functionalities can be used for mapping the spatial arrangement of chemical groups
on a surface, sometimes termed as chemical force microscopy. It was demonstrated that probes
functionalized with CH3 or COOH groups can specifically interact with similar groups on the
surface, with the spatial pattern of interaction reproducing the spatial distribution of functional groups
on the surface.
Measuring intermolecular interactions between partners in molecular recognition reactions utilizes as
analogous approach. It immobilizes each of the partners on either the probe or the surface, and then
takes the probe through approach / retraction cycles. The magnitude of the cantilever deflection at the
jump-off contact peak is taken to reflect the rupture force needed to break the molecular interactions
holding the two partners in close contact; breaking of the interaction bands restores the cantilever to its
neutral non-contact, non-deflected position.
It must be noted that the specific intermolecular interactions are registered against the background
probe / surface interactions, which may be as high as (or even higher than) the biologically interactions
of interest. This requires careful choice of specificity controls. These generally include the use of nonfunctionalized probes or surfaces, blocking the interactions between the immobilized molecular
partners with free ligands in the medium or changing the ph or the salt concentration in the medium.

Receptor / ligand interactions:

A lot of experimental effort has gone inot studying receptor / ligand interactions, on the
example of the small ligand biotin interacting with the closely related proteins streptavidin or
avidin. This ligand / receptor pair has been used as a model system because of its unusually
high affinity and the availability of structural and thermodynamic data. The biotin /
streptavidin system has been also utilized to study the dependence of measured interaction
forces on the loading rate. Bond strength progresses through three dynamic regimes of loading
rate: a slow-loading, a fast-loading and an ultrafast-loading regime. In each of these regimes,
the dependence of the bond strength on the loading rate is different. Thus, in order to expose
the energy landscape that governs bond strength, molecular adhesion forces must be examined
over an enormous span of time scales.
But for nine different fluorescein-anti-fluorescein antibody pairs the measured unbinding
forces correlated well with the respective thermal off-rates in solution; the dependence of the
force on the logarithm of the loading rates was linear over six orders of magnitude of the latter
parameter. Perhaps these differences could be attributed to certain atypical structural features
of the avidin / biotin pair.

23

Protein / protein interactions

The second populated class of intermolecular force measurements involved protein / protein
interactions, including the highly specific antigen / antibody interactions.

Interactions between complementary strands of DNA

The interactions between the two strands of a double-helical DNA molecule may also be
classified as intermolecular interactions. With increasing applied force, the final separation of
the complementary strands proceeded after DNA stretched to a stable form of approximately
twice the length of the B-form. This structural transition of double-stranded DNA from the Bform to the socalled S-(stretched)form was first identified in long DNA molecules in
experiments using optical fibers and optical tweezers. Stretching poly(dG-dC) and
poly(dA-dT) allowed the expected sequence dependence of melting to be directly
demonstrated, which base pair unbinding force for G-C of ~ 20 pN and that for A-T of ~ 9 pN.

Intramolecular structural transitions in polysaccharides, DNA and multi-domain

proteins
The AFM has proven particularly useful in studying intramolecular interactions, e. g. the abovementioned DNA stretching that precedes its melting. A major research effort has focused on the
unfolding of multi-domain protein molecules or of individual protein domains.

Fig. 30: The characteristing saw-tooth pattern of the

unfolding process of a multi-domain protein.

At small extensions (relative to the chain length), polymers generate a restoring force that is mainly
entropic in origin. If force is applied to a polymer chain, an opposing force is created as a result of the
reduction in entropy. The behavior of DNA, however, deviates from entropic elasticity at relatively
high forces: applications of forces above a certain threshold level to DNA leads to conformational
changes beyond simple staightening of the chain and results in extensions beyond the contour length.
DNA undergoes a stretching B- to S-transition above 65 pN. A well-expressed deviation from entropic
elasticity in the high-force stretching regime has also been observed for certain polysaccharides. This
deviation was attributed to either twisting and bending of bond angles, or to chain-boat transitions of
the glucopyranose ring of the stretched polysaccharide.

24
Stretching of multi-domain proteins presents an even more complicated case, where the forceextension curves are strings of successive enthalpic and entropic portions, reflecting the unfolding of
individual domains in the multi-domain polypeptide chain, followed by stretching of the unfolded
domain. As such proteins are elongated as a result of the initial application of force, they undergo a
typical entropic stretching at the beginning. At a certain force, one of the folded domains unfolds,
adding significant length to the chain and relaxing the stress on the cantilever, which returns to its nondeflected state. The denatured portion of the polypeptide chain can now undergo entropic stretching ,
behaving like a typical polymer chain. Further extension creates forces high enough to unfold a second
domain, which is then stretched entropically, etc. The unfolding and stretching of each individual
domain creates an individual peak in the force curve, leading to the characteristic saw-tooth pattern.
By analysing many protein unfolding events such as these under a range of experimental conditions it
is possible to determine a number of key parameters such as the folding and unfolding rate constants
in the absence of the applied force field, the size and position of energy barriers that control these rate
constants and make comparisons on the mechanical unfolding process with more traditional chemical
methods. In addition, the fact that these experiments are carried out on single molecules allows a direct
comparison with computer simulations of protein dynamics which invariably only deal with a single
molecule.

Measuring the viscoelastic properties of biological structures and macromolecules

It is clear that whenever the effective stiffness of the cantilever and the biological sample on the
surface are comparable, and the probe is pushed into the sample, the sample undergoes measureable
indentation of the local surface at the point of contact of the tip. When the stress (deformation force)
and the strain (the amount of deformation) are linearly related, the deformation of the material is
elastic, and the material will regain its original form upon relaxation. The depth of indentation can be
used to measure local elasticity.
b

Fig. 31a: The amplitude of cantilever oscillation varies according to the mechanical
properties of the sample surface.
b: Latex spheres.

The behaviour of soft biological samples, shows another type of deviation from theat of a hard surface,
this time on the retraction curve. Whereas the lift-off occurs quickly on hard surfaces, it may be
considerably slowed down in the case of soft biological samples. The lift-off speed may be used to
estimate the viscosity of the sample.

Measuring interactions between cells

Recently, force-distance curves have been also utilized to identify cell partners that interact
specifically in certain biological reactions. By functionalizing AFM tips with whole cells of a given
type and studying their interactions with monolayers of other cell types, it was possible to identify p. e.
the cell type in the uterine epithelium that interacts specifically with cells in the embryo during
implantation. The technique introduced in this work offers novel approaches to the study of cell-cell
interactions that are essential in many of the biological processes taking place in multicellular
organisms.

25
Force maps
Laterally resolved force curves can be recorded during raster scanning in the x-y-direction. Individual
curves can be assembled into a three-dimensional force volume. An alternative approach to producing
spatially resolved force measurements is to create isoforce images across the sample, by assiguing
each point of the surface a seperation distance at which a certain force is measured. Collecting series
of isoforce images at different forces can, in principle, reconstruct the same force volume as the one
created by collecting individual laterally resolved force curves. The wealth of information in the force
volume can be used to produce sample / surface maps reflecting different properties of the surface:
adhesion, viscosity, elasticity, electrostatic interactions, etc.
A much faster approach to the point by point force curve measurement mentioned above has been to
use the binding of a tethered antibody-modified tip to the substrate of the antibody to generate an
effective force volume image. In this method, the image is distorted by enlarged widths and increased
hights due to stretching of the antibody tether when the antibody binds the target antigen. This method
permits location of the antigens, but quantitative force measurements require that a conventional force
curve be acquired once the target is formed in the image.

Possible errors

Fig. 33:
There is a strong adhesion between tip and
sample. Change the cantilever.

Fig. 34:
Such a slope is a sign for a smooth deflection
of the cantilever. Normally thats because there
is something on the tip. Change the cantilever.

Fig. 35:
The laser spot is not focused carefully on the
cantilever. Do it again.