ATOMIC FORCE MICROSCOPY

The Atomic Force Microscope (AFM ) is being used to solve processing and materials
problems in a wide range of technologies affecting the electronics, telecommunications,
biological, chemical, automotive, aerospace, and energy industries. The materials being
investigating include thin and thick film coatings, ceramics, composites, glasses, synthetic and
biological membranes, metals, polymers, and semiconductors. The AFM is being applied to
studies of phenomena such as abrasion, adhesion, cleaning, corrosion, etching, friction,
lubrication, plating, and polishing. By using AFM one can not only image the surface in atomic
resolution but also measure the force at nano-Newton scale. The publications related to the
AFM are growing speedily since its birth.

A. PRINCIPLE

The principles on how the AFM works are very simple. An atomically sharp tip is
scanned over a surface with feedback mechanisms that enable the piezo-electric scanners to
maintain the tip at a constant force (to obtain height information), or height (to obtain force
information) above the sample surface. Tips are typically made from Si 3N4 or Si, and extended
down from the end of a cantilever. The nanoscope AFM head employs an optical detection
system in which the tip is attached to the underside of a reflective cantilever. A diode laser is
focused onto the back of a reflective cantilever. As the tip scans the surface of the sample,
moving up and down with the contour of the surface, the laser beam is deflected off the
attached cantilever into a dual element photodiode. The photodetector measures the difference
in light intensities between the upper and lower photodetectors, and then converts to voltage.
Feedback from the photodiode difference signal, through software control from the computer,
enables the tip to maintain either a constant force or constant height above the sample. In the
constant force mode the piezo-electric transducer monitors real time height deviation. In the
constant height mode the deflection force on the sample is recorded. The latter mode of
operation requires calibration parameters of the scanning tip to be inserted in the sensitivity of
the AFM head during force calibration of the microscope.

1. Laser

2. Mirror

3. Photo detector

4. Amplifier

5. Register

6. Sample

7. Probe

8. Cantilever
 The force between the tip and the sample surface is very small, usually less than 10-9 N.
How to monitor such small forces is another story. The detection system does not measure
force directly. It senses the deflection of the microcantilever. The detecting systems for
monitoring the deflection fall into several categories. The first device introduced by Binnig
was a tunneling tip placed above the metallized surface of the cantilever. This is a sensitive
system where a change in spacing of 1 Å between tip and cantilever changes the tunneling
current by an order of magnitude. It is straightforward to measure deflections smaller than 0.01
Å. Subsequent systems were based on the optical techniques. The interferometer is the most
sensitive of the optical methods, but it is somewhat more complicated than the beam-bounce
method which was introduced by Meyer and Amer. The beam-bounce method is now widely
used as a result of the excellent work by Alexander and colleagues. In this system an optical
beam is reflected from the mirrored surface on the back side of the cantilever onto a position-
sensitive photodetector. In this arrangement a small deflection of the cantilever will tilt the
reflected beam and change the position of beam on the photodetector. A third optical system
introduced by Sarid uses the cantilever as one of the mirrors in the cavity of a diode laser.
Motion of the cantilever has a strong effect on the laser output, and this is exploited as a
motion detector. According to the interaction of the tip and the sample surface, the AFM can
be classified as repulsive or Contact mode and attractive or Noncontact mode. Now the
Tapping mode shows a prosperous future to image the micro-world

Some AFM's can accept full 200 mm wafers. The primary purpose of these instruments
is to quantitatively measure surface roughness with a nominal 5 nm lateral and 0.01nm vertical
resolution on all types of samples. Depending on the AFM design, scanners are used to
translate either the sample under the cantilever or the cantilever over the sample. By scanning
in either way, the local height of the sample is measured. Three dimensional topographical
maps of the surface are then constructed by plotting the local sample height versus horizontal
probe tip position.

Figure 2. Principle of the AFM. (a) A fine stylus is mounted on a cantilever spring and
scanned over the surface. At sufficiently small forces the corrugations of the scanning lines
represent the surface topography of the sample. (b) The vertical deflection of the cantilever is
detected by reflecting a laser beam onto a 2-segment photodiode. The photodiode signal is
used to drive a servo system which controls the movement of the piezo xyz-translator. In this
manner the applied force between the stylus and the sample can be kept constant within some
tens of a piconewton. The imaging process can be performed in a liquid cell filled with buffer
solution. This ensures that the biomolecules remain hydrated. (c) Atomic resolution of a mica
surface recorded in aqueous solution. The distance between adjacent protrusions is 5.4 Å.
B. AFM Instrumentation
An AFM is constructed using piezoelectric materials, a force transducer and feedback
control as illustrated in Figure. The force transducer measures a force between the probe and
surface; the feedback controller keeps the force constant by controlling the expansion of the Z
piezoelectric transducer. Then, the X-Y piezo electric ceramics are used to scan the probe
across the surface in a raster-like pattern. By monitoring the voltage on the Z ceramic, an
image of the surface is measured.

FIGURE: Block diagram showing the components in an AFM. The image is created by
monitoring the voltage that drives the Z piezoelectric ceramic.
There are three basic concepts that you must be familiar with to understand the
operation of an AFM. These are piezoelectric transducers, force transducers, and feedback
control.

Piezoelectric Transducers
Piezoelectric materials are electromechanical transducers that convert electrical
potential into mechanical motion. Piezoelectric materials are naturally occurring and may be
crystalline, amorphous or even polymeric. When a potential is applied across two opposite
sides of the piezoelectric, it changes geometry. The magnitude of the dimensional changes
depends on the material, the geometry of the device, and the magnitude of the applied voltage.
Piezoelectric materials are available in a number of sizes and shapes. Typically, the expansion
coefficient for a single piezo shape is on the order of 0.1 nm per applied volt. Thus, if the
voltage used to excite the piezo material is 2 Volts, then the material will expand
approximately 0.2 nm, or the diameter of a single hydrogen atom. Piezoelectric materials are
used for controlling the motion of the probe as it is scanned across a surface in an AFM.

Force Transducers
The force between a probe and a surface is measured with a force transducer. When the
probe comes into contact with the surface, the voltage output from the transducer increases. It
is important that the output of the transducer be monotonic and increase as a greater force is
applied between the probe and surface. Force transducers may be constructed that measure
forces as low as 10 picoNewtons between a probe and a surface. There are several types of
force sensors that may be used in an AFM
Feedback Control
Feedback control is used in AFM for maintaining a fixed relationship, or force, between
the probe and the surface. The feedback control operates by measuring the force between the
surface and probe, then controlling a piezoelectric ceramic that establishes the relative position
of the probe and surface. Feedback control is used in many applications,

C. Sample Preparation
Sample preparation for an AFM is reasonably simple. There are a few basic rules that
must be followed to adequately prepare a sample for AFM scanning. The rules are:
a) Sample must be adhered to the surface: If the sample has material adhered to the surface,
the material must be rigidly mounted to the surface. If the material is not rigidly adhered two
problems can occur. First, the probe can push the material to the edge of the scan range. When
this occurs, the image appears as though there is nothing on the surface and only the substrate
is observed. Second, the probe can pick up material from the surface because the material has a
greater affinity for the probe than the surface. In this case the images often have streaks in
them. The streaks are created by material moving on and off the probe, i.e. the probe geometry
is changed by the material from the surface.
b) Sample must be clean: AFM imaging requires that the probe move directly across the
sample’s surface topography. If the surface is dirty with a thick contamination layer, the probe
needs to penetrate through the contamination layer to reach the surface.
c) Sample dimensions must be realistic: The AFM can image a large variety of samples;
however, there are a few constraints. Features on the sample’s surface must be smaller than the
dynamic range of the Z ceramic. Typically this is less than 10 microns. If the features on the
surface are larger than 10 microns, then the Z piezo will not be able to move the probe over the
features. Second, the probe must be able to directly access the features. As an example, if the
sample has a 10 nm diameter hole, and the probe is 40 nm in diameter, the probe will not reach
into the hole.

D. The Common AFM Modes

Many AFM modes have appeared for special purpose while the technique of AFM is
becoming mature. Here I only specify the three commonly used techniques: Contact Mode
(left), Non-contact Mode (middle) and Tapping Mode (right).

Contact Mode

In contact AFM electrostatic and/or surface tension forces from the adsorbed gas layer
pull the scanning tip toward the surface. It can damage samples and distort image data.
Therefore, contact mode imaging is heavily influenced by frictional and adhesive forces
compared to non-contact or tapping mode.

The contact mode where the tip scans the sample in close contact with the surface is the
common mode used in the force microscope. In contact mode AFM the deflection of the
cantilever is sensed and compared in a DC feedback amplifier to some desired value of
deflection. The voltage that the feedback amplifier applies to the piezo is a measure of the
height of features on the sample surface. It is displayed as a function of the lateral position of
the sample. There are many advantages to operate AFM with the sample and cantilever
immersed in a fluid. These advantages include the elimination of capillary forces, the reduction
of Van der Waals' forces and the ability to study technologically or biologically important
processes at liquid solid interfaces. However there are also some disadvantages involved in
working in liquids. These range from nuisances such as leaks to more fundamental problems
such as sample damage on hydrated and vulnerable biological samples. An attempt to avoid
these problems is the Non-contact Mode.

Non-contact Mode

The non-contact mode which is used in situations where tip contact might alter the
sample in subtle ways. In this mode the tip hovers 50 - 150 Angstrom above the sample
surface. Attractive Van der Waals forces acting between the tip and the sample are detected,
and topographic images are constructed by scanning the tip above the surface. Unfortunately
the attractive forces from the sample are substantially weaker than the forces used by contact
mode. Therefore the tip must be given a small oscillation so that AC detection methods can be
used to detect the small forces between the tip and the sample by measuring the change in
amplitude, phase, or frequency of the oscillating cantilever in response to force gradients from
the sample. For highest resolution, it is necessary to measure force gradients from Van der
Waals forces which may extend only a nanometer from the sample surface. In general, the fluid
contaminant layer is substantially thicker than the range of the Van der Waals force gradient
and therefore, attempts to image the true surface with non-contact AFM fail as the oscillating
probe becomes trapped in the fluid layer or hovers beyond the effective range of the forces it
attempts to measure.

Non-contact imaging generally provides low resolution and can also be hampered by
the contaminant layer which can interfere with oscillation

Tapping Mode

Tapping mode is a key advance in AFM. This potent technique allows high resolution
topographic imaging of sample surfaces that are easily damaged, loosely hold to their substrate,
or difficult to image by other AFM techniques. Tapping mode overcomes problems associated
with friction, adhesion, electrostatic forces, and other difficulties that an plague conventional
AFM scanning methods by alternately placing the tip in contact with the surface to provide
high resolution and then lifting the tip off the surface to avoid dragging the tip across the
surface. Tapping mode imaging is implemented in ambient air by oscillating the cantilever
assembly at or near the cantilever's resonant frequency using a piezoelectric crystal. The piezo
motion causes the cantilever to oscillate with a high amplitude (typically greater than 20nm)
when the tip is not in contact with the surface. The oscillating tip is then moved toward the
surface until it begins to lightly touch, or tap the surface. During scanning, the vertically
oscillating tip alternately contacts the surface and lifts off, generally at a frequency of 50,000 to
500,000 cycles per second. As the oscillating cantilever begins to intermittently contact the
surface, the cantilever oscillation is necessarily reduced due to energy loss caused by the tip
contacting the surface. The reduction in oscillation amplitude is used to identify and measure
surface features.
 When the tip passes over a bump in the surface, the cantilever has less room to oscillate
and the amplitude of oscillation decreases. Conversely, when the tip passes over a depression,
the cantilever has more room to oscillate and the amplitude increases (approaching the
maximum free air amplitude). The oscillation amplitude of the tip is measured by the detector
and input to the NanoScope III controller electronics. The digital feedback loop then adjusts
the tip-sample separation to maintain constant amplitude and force on the sample.

When the tip contacts the surface, the high frequency (50k - 500k Hz) makes the
surfaces stiff (viscoelastic), and the tip-sample adhesion forces is greatly reduced.
TappingMode inherently prevents the tip from sticking to the surface and causing damage
during scanning. Unlike contact and non-contact modes, when the tip contacts the surface, it
has sufficient oscillation amplitude to overcome the tip-sample adhesion forces. Also, the
surface material is not pulled sideways by shear forces since the applied force is always
vertical. Another advantage of the TappingMode technique is its large, linear operating range.
This makes the vertical feedback system highly stable, allowing routine reproducible sample
measurements..

TappingMode AFM was developed as a method to achieve high resolution without

inducing destructive frictional forces both in air and fluid. With the TappingMode technique,
the very soft and fragile samples can be imaged successfully. Also, incorporated with Phase
Imaging, the tapping mode AFM can be used to analyze the components of the membrane.

Phase Imaging: Beyond Topography

Phase Imaging is a powerful extension of Tapping Mode Atomic Force Microscopy

(AFM) that provides nanometer-scale information about surface structure often not revealed by
other SPM techniques. By mapping the phase of the cantilever oscillation during the
TappingMode scan, phase imaging goes beyond simple topographical mapping to detect
variations in composition, adhesion, friction, viscoelasticity, and perhaps other properties.
Applications include identification of contaminants, mapping of different components in
composite materials, and differentiating regions of high and low surface adhesion or hardness.

Because phase imaging highlights edges and is not affected by large-scale height
differences, it provides for clearer observation of fine features, such as grain edges, which can
be obscured by rough topography.

Phase imaging is a powerful tool for mapping variations in sample properties at very
high resolution.

E. Comparison of AFM and other imaging techniques

1. AFM versus STM:

It's interesting to compare AFM and its precursor -- Scanning Tunneling Microscope. In some
cases, the resolution of STM is better than AFM because of the exponential dependence of the
tunneling current on distance. The force-distance dependence in AFM is much more complex
when characteristics such as tip shape and contact force are considered. STM is generally
applicable only to conducting samples while AFM is applied to both conductors and insulators.
In terms of versatility, needless to say, the AFM wins. Furthermore, the AFM offers the
advantage that the writing voltage and tip-to-substrate spacing can be controlled independently,
whereas with STM the two parameters are integrally linked.

2. AFM versus SEM:

Compared with Scanning Electron Microscope, AFM provides extraordinary topographic

contrast direct height measurements and unobscured views of surface features (no coating is
necessary).

3. AFM versus TEM:

Compared with Transmission Electron Microscopes, three dimensional AFM images are
obtained without expensive sample preparation and yield far more complete information than
the two dimensional profiles available from cross-sectioned samples.

4. AFM versus Optical Microscope:

Compared with Optical Interferometric Microscope (optical profiles), the AFM provides
unambiguous measurement of step heights, independent of reflectivity differences between
materials.

F. Biological Applications of AFM

1. One of the advantages of AFM is that it can image the non-conducting surfaces. So it was
immediately extended to the biological systems, such as analyzing the crystals of amino acids
and organic monolayers. Applications of AFM in the biosciences include: DNA and RNA
analysis; Protein-nucleic acid complexes; Chromosomes; Cellular membranes; Proteins
and peptides; Molecular crystals; Polymers and biomaterials; Ligand-receptor binding.
Bio-samples have been investigated on lysine-coated glass and mica substrate, and in
buffer solution. By using phase imaging technique one can distinguish the different
components of the cell membranes.

2. Imaging of nucleic acids: The ability to generate nanometer-resolved images of unmodified

nucleic acids has broad biological applications. Chromosome mapping, transcription,
translation and small molecule-DNA interactions such as intercalating mutagens, provide
exciting topics for high-resolution studies. The first highly reproducible AFM images of DNA
were obtained only in 1991. Four major advances that have enabled clear resolution of nucleic
acids are: Control of the local imaging environment including sample modification;
TappingMode scanning techniques; Improved AFM probes (such as standard silicon nitride
probes modified by electron beam deposition and Oxide Sharpened NanoProbes) and
Compatible substrates (such as salinized mica and carbon coated mica).
3. Cell dynamic behavior study: Biologists have applied the AFM's unique capabilities to
study the dynamic behavior of living and fixed cells such as red and white blood cells,
bacteria, platelets, cardiac myocytes, living renal epithelial cells, and glial cells. For
example, plasma membrane in migrating epithelial cells has been imaged in real time. The
dynamic membrane invagination process was observed in the presence of calcium and when
calcium levels were reduced the process was prevented.

4. Quantify the molecular interaction in biological systems: AFM has the ability to measure
forces in the nanonewton range. This makes it possible to quantify the molecular interaction in
biological systems such as a variety of important ligand-receptor interactions such as - DNA
replication, protein synthesis, drug interaction, and many others - are largely governed by
intermolecular forces.

5. Another application of AFM force measurements is to image or quantify electrical surface

charge. The dynamics of many biological systems depends on the electrical properties of the
sample surface. In addition to measuring binding forces and electrostatic forces, the AFM can
also probe the micromechanical properties of biological samples.

6. The AFM can observe the elasticity and, in fact, the viscosities of samples ranging from live
cells and membranes to bone and cartilage.

7. Provides new possibilities for the characterization of both surface structure and
mechanical properties of freshly excised articular cartilage under physiological conditions

8. The extracellular and periplasmic surface topographies of 2-D crystalline lipid-protein

arrays of the E. coli outer membrane protein OmpF have been determined with the AFM.

9. Identification by AFM of individual amino acid residues at protein surfaces appears now
to be feasible.