INTRODUCTION

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.
The first AFM was made by meticulously gluing a tiny shard of diamond onto one end of a
tiny strip of gold foil. In the fall of 1985 Gerd Binnig and Christoph Gerber used the
cantilever to examine insulating surfaces. A small hook at the end of the cantilever was
pressed against the surface while the sample was scanned beneath the tip. The force between
tip and sample was measured by tracking the deflection of the cantilever. This was done by
monitoring the tunneling current tot a second tip positioned above the cantilever. They could
delineate lateral features as small as 300 . The force microscope emerged in this way. In
fact, without the breakthrough in tip manufacture, the AFM probably would have remained a
curiosity in many research groups. It was Albrecht, a fresh graduate student, who fabricated
the first silicon microcantilever and measured the atomic structure of boron nitride. Today the
tip-cantilever assembly typically is microfabricated from Si or Si3N4. The era of AFM came
finally when the Zurich group released the image of a silicon (111) 7X7 pattern. The world of
surface science knew that a new tool for surface microscope was at hand. After several years
the microcantilevers have been perfected, and the instrument has been embraced by scientists
and technologists.
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
between tip and cantilever changes the tunneling current by an order of magnitude. It is
straightforward to measure deflections smaller than 0.01 . 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. An expanded view
of the image at left, and a legend describing its parts is found here.
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 Tappingmode shows
a prosperous future to image the micro-world.
Operation

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 Si3N4 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.
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.
AFM Resolution

The concept of resolution in AFM is different from radiation based microscopies because
AFM imaging is a three dimensional imaging technique. The ability to distinguish two
separate points on an image is the standard by which lateral resolution is usually defined.
There is clearly an important distinction between images resolved by wave optics and
scanning probe techniques. The former is limited by diffraction, and later primarily by apical
probe geometry and sample geometry. Usually the width of a DNA molecule is loosely used
as a measure of resolution, because it has a known diameter of 2.0 nm in the B form. Some of
the best values for AFM imaging are 3.0 nm quoted form DNA in propanol. Unfortunately,

this definition of resolution can be misleading because the sample height clearly effects this
value.
Indeed, many authors have seen that it is the radius of curvature that significantly influences
the resolving ability of the AFM. Images of DNA made by the sharper tip have shown
dramatic improvements in resolution widths (see the two pictures below). Even greater
improvements in resolution have been attained with Tappingmode but contact imaging still is
capable of high resolution imaging. For brief discussion on resolution see the article by
Keller in Physics Today (October, 1995).
Vibration Isolation
In order to obtain good AFM results, the vibration isolation platform is needed. The vibration
isolation consists of a large mass attached to bungy cords firmly anchored to the building.
(Notice that the resonance frequency only depends on the stretch of the bungy cord).
Damping of the oscillation is believed to result from rubbing of the rubber fibres inside of the
bungy cord against the outside lining material. Between the low resonance frequency of the
bungy cord system and the high resonance frequency of the microscope hardware itself (> 10
kHz ), the AFM effectively comprises a band pass filter. This allows the microscopists to
safely image their samples in the intermediate range of about 1 - 100 Hz and obtain atomic
resolution
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
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). Look here for a summary.
Contact 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. The force on the tip is repulsive with a mean
value of 10 -9 N. This force is set by pushing the cantilever against the sample surface with a
piezoelectric positioning element. In contact mode AFM the deflection of the cantilever is
sensed and compared in a DC feedback amplifier to some desired value of deflection. If the
measured deflection is different from the desired value the feedback amplifier applies a
voltage to the piezo to raise or lower the sample relative to the cantilever to restore the
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. A few instruments operate in UHV but the majority operate in
ambient atmosphere, or in liquids. Problems with contact mode are caused by excessive
tracking forces applied by the probe to the sample. The effects can be reduced by minimizing
tracking force of the probe on the sample, but there are practical limits to the magnitude of
the force that can be controlled by the user during operation in ambient environments. Under
ambient conditions, sample surfaces are covered by a layer of adsorbed gases consisting
primarily of water vapor and nitrogen which is 10-30 monolayers thick . When the probe
touches this contaminant layer, a meniscus forms and the cantilever is pulled by surface
tension toward the sample surface. The magnitude of the force depends on the details of the
probe geometry, but is typically on the order of 100 nanoNewtons. This meniscus force and
other attractive forces may be neutralized by operating with the probe and part or all of the
sample totally immersed in liquid. 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.
In addition, a large class of samples, including semiconductors and insulators, can trap
electrostatic charge(partially dissipated and screened in liquid). This charge can contribute to
additional substantial attractive forces between the probe and sample. All of these forces
combine to define a minimum normal force that can be controllably applied by the probe to
the sample. This normal force creates a substantial frictional force as the probe scans over the
sample. In practice, it appears that these frictional forces are far more destructive than the
normal force and can damage the sample, dull the cantilever probe and distort the resulting

data. Also many samples such as semiconductor wafers can not practically be immersed in
liquid. An attempt to avoid these problem is the Non-contact Mode.
Non-contact Mode
A new era in imaging was opened when microscopists introduced a system for implementing
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.
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.
During tapping mode operation, the cantilever oscillation amplitude is maintained constant by
a feedback loop. Selection of the optimal oscillation frequency is software-assisted and the
force on the sample is automatically set and maintained at the lowest possible level. 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 tipsample separation to maintain a 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.
Tapping mode operation in fluid has the same advantages as in the air or vacuum. However
imaging in a fluid medium tends to damp the cantilever's normal resonant frequency. In this
case, the entire fluid cell can be oscillated to drive the cantilever into oscillation. This is
different from the tapping or non-contact operation in air or vacuum where the cantilever
itself is oscillating. When an appropriate frequency is selected (usually in the range of 5,000
to 40,000 cycles per second), the amplitude of the cantilever will decrease when the tip
begins to tap the sample, similar to TappingMode operation in air. Alternatively, the very soft
cantilevers can be used to get the good results in fluid. The spring constant is typically 0.1
N/m compared to the tapping mode in air where the cantilever may be in the range of 1-100
N/m.
Summary
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.
Non-contact imaging generally provides low resolution and can also be hampered by the
contaminant layer which can interfere with oscillation.
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. In many
cases, phase imaging complements lateral force microscopy (LFM) and force modulation
techniques, often providing additional information more rapidly and with higher resolution.
Phase imaging is as fast and easy to use as TappingMode AFM -- with all its benefits for
imaging soft, adhesive, easily damaged or loosely bound samples -- and is readily
implemented on any MultiMode or Dimension Series SPM with NanoScope III controller
equipped with an Extender Electronics Module.
In TappingMode AFM, the cantilever is excited into resonance oscillation with a
piezoelectric driver. The oscillation amplitude is used as a feedback signal to measure
topographic variations of the sample. In phase imaging, the phase lag of the cantilever
oscillation, relative to the signal sent to the cantilever's piezo driver, is simultaneously
monitored by the Extender Electronics Module and recorded by the NanoScope III SPM
controller. The phase lag is very sensitive to variations in material properties such as
adhesion and viscoelasticity.

Once the SPM is engaged in TappingMode, phase imaging is enabled simply by displaying a
second image and selecting the Phase data type in the NanoScope software. Both the
TappingMode topography and phase images are viewed side-by-side in real time. The
resolution of phase imaging is comparable to the full resolution of TappingMode AFM. Phase
imaging can also act as a real-time contrast enhancement technique. 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. It can be turned on while using TappingMode AFM with no cost in speed or
resolution, and all NanoScope users are encouraged to add it to their SPM repertoire. Phase
imaging can complement force modulation and LFM methods, often with superior image
detail, and can in some cases provide information not revealed by these or other SPM
techniques. The rapidly growing list of phase imaging applications includes characterizing
the components of composite materials, mapping of surface friction and adhesion, and
identification of surface contamination. Phase imaging promises to play an important role in
the ongoing study of material properties at the nanometer scale.
Force Curves
1.Force Curve Measurements
In addition to these topographic measurements, the AFM can also provide much more
information. The AFM can also record the amount of force felt by the cantilever as the probe
tip is brought close to - and even indented into - a sample surface and then pulled away. This
technique can be used to measure the long range attractive or repulsive forces between the
probe tip and the sample surface, elucidating local chemical and mechanical properties like
adhesion and elasticity, and even thickness of adsorbed molecular layers or bond rupture
lengths.
Force curves( force-versus-distance curve) typically show the deflection of the free end of the
AFM cantilever as the fixed end of the cantilever is brought vertically towards and then away
from the sample surface. Experimentally, this is done by applying a triangle-wave voltage
pattern to the electrodes for the z-axis scanner. This causes the scanner to expand and then
contract in the vertical direction, generating relative motion between the cantilever and
sample. The deflection of the free end of the cantilever is measured and plotted at many
points as the z-axis scanner extends the cantilever towards the surface and then retracts it
again. By controlling the amplitude and frequency of the triangle-wave voltage pattern, the
researcher can vary the distance and speed that the AFM cantilever tip travels during the
force measurement.
Similar measurements can be made with oscillating probe systems like TappingMode and
non-contact AFM. This sort of work is just beginning for oscillating probe systems, but
measurements of cantilever amplitude and/or phase versus separation can provide more
information about the details of magnetic and electric fields over surfaces and also provide
information about viscoelastic properties of sample surfaces.
2.Anatomy of a Force Curve
A:The cantilever starts not touching the surface. In this region, if the cantilever feels a longrange attractive (or repulsive) force it will deflect downwards (or upwards) before making
contact with the surface.

B: As the probe tip is brought very close to the surface, it may jump into contact if it feels
sufficient attractive force from the sample.
C: Once the tip is in contact with the surface, cantilever deflection will increase as the fixed
end of the cantilever is brought closer to the sample. If the cantilever is sufficiently stiff, the
probe tip may indent into the surface at this point. In this case, the slope or shape of the
contact part of the force curve can provide information about the elasticity of the sample
surface.
D:After loading the cantilever to a desired force value, the process is reversed. As the
cantilever is withdrawn, adhesion or bonds formed during contact with the surface may cause
the cantilever to adhere to the sample some distance past the initial contact point on the
approach curve (B).
E:A key measurement of the AFM force curve is the point at which the adhesion is broken
and the cantilever comes free from the surface. This can be used to measure the rupture force
required to break the bond or adhesion.
One of the first uses of force measurements was to improve the quality of AFM images by
monitoring and minimizing the attractive forces between the tip and sample. Force
measurements were also used to demonstrate similarly reduced capillary forces for samples in
vacuum and in reduced humidity environments.
3.Measuring Fundamental Forces
Concern with the fundamental interactions between surfaces extends across physics,
chemistry, materials science and a variety of other disciplines. With a force sensitivity on the
order of a few piconewtons (pN = 10 to -12 N), AFMs are excellent tools for probing these
fundamental force interactions. Force measurements in water revealed the benefits of AFM
imaging in this environment due to the lower tip-sample forces. Some of the most interesting
force measurements have also been performed with samples under liquids where the
environment can be quickly changed to adjust the concentration of various chemical
components. In liquids, electrostatic forces between dissolved ions and other charged groups
play an important role in determining the forces sensed by an AFM cantilever. The liquid
environment has become an important stage for fundamental force measurement because
researchers can control many of the details of the probe surface force interaction by adjusting
properties of the liquid. Experimentally, the electrostatic tip-sample forces depend strongly
on pH and salt concentration. In fact, it is often possible to adjust the pH or salt concentration
such that the attractive Van der Waals forces are effectively negated by repulsive electrostatic
forces. This has been an important discovery because it can allow tuning of the liquid
environment to minimize adhesive tip-sample forces that can damage the sample during
imaging.
Atomic Force Microscopy has made its mark on a wide variety of applications as a
topographic measurement and mapping tool. Now AFM force measurements are providing
information on atomic- and molecular-scale interactions as well as nano-scale adhesive and
elastic response. These measurements are beginning to revolutionize the way we
quantitatively observe and, indeed, think about our chemical, biological and physical world.
The vast majority of AFM force measurements have been performed with NanoScope
systems and we are actively adding functionality to make these measurements increasingly
accurate, repeatable and easier to perform.
Biological Applications of AFM

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. Biosamples 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.

Little sample preparation is required for bioimaging with the AFM. In most cases it is as
simple as spotting a few microliters of solution on mica or glass. Of course contaminations
that cover surface features have to be avoided or removed. First the substrate-adsorbate
should be rinsed with a large excess of buffer. The following procedures are dialysis,
centrifugation and homogenization. In order to get good contrast and to reduce mechanical
damage of the soft biological materials, the samples can be stabilized by adding covalent
cross-linking agents or certain cations that are able to link the constituents of the sample to
each other or to the substrate. Cooling can also stiffen the sample. Nevertheless, all these
methods have significant influence on the properties of biomolecules. The sharper tip now is
available commercially and really does help a lot.
One area of significant progress is the 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 ).
Cell 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. 30nm lipidic pore formation could also be resolved during calcium reduction.
AFM imaging of cells usually achieves a resolution of only 20-50 nm, not sufficient for

resolving membrane proteins but still suitable for imaging other surface features, such as
rearrangements of plasma membrane or movement of submembrane filament bundles. The
requirement for the imaging buffer is not restrictive, as long as the buffer does not severely
affect the integrity of the cells. EDTA should be avoided because cells will detach from the
substrate in the absence of divalent cations. Cultured cells normally adhere well to the
substrate and are not displaced by modest probe forces.
There has been recent success imaging individual proteins and other small molecules with the
AFM such as collogen. Smaller molecules that do not have a high affinity for common AFM
substrates have been successfully imaged by employing selective affinity binding procedures.
Thiol incorporation at both the 5' and 3' ends of short PCR products has been shown to confer
a high affinity for ultraflat gold substrates. A similar approach was used to immobilize
antibodies (IgG1) on treated mica. In this case, the low affinity that IgG molecules have for
mica was overcome by cloning a metal-chelating peptide into the carboxy terminus sequence
of the IgG's heavy chain. The recombinant sequence was transformed into cells that
expressed the complementary light chain. The purified IgG containing the metal-chelating
peptide was shown to bind in a regiospecific manner to nickel-treated mica. Covalent binding
of biological structures to derivatized glass substrates has also enabled high resolution
imaging of some samples that are not stable on untreated glass substrates. New approaches in
AFM have provided a solid foundation from which research is expanding into more complex
analyses. Higher resolution imaging of a variety of small molecules is improving at a rapid
pace.
The recent innovation, such as Digital Instruments BioScope system, which combines the
high resolution of AFM with the ease of use and familiarity of inverted optical microscopes,
has further added to the attractiveness of AFM for biological imaging. Bright-field,
flourescence and other optical techniques can be used to identify structures of interest while
the AFM simultaneously generates nanometer-resolved images of the sample surface.
Countless biological processes - DNA replication, protein synthesis, drug interaction, and
many others - are largely governed by intermolecular forces. 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. 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. Specifically, the AFM can
observe the elasticity and, in fact, the viscosity of samples ranging from live cells and
membranes to bone and cartilage.
AFM gives scientists a key tool to investigate the real world.