BMFB 3723 Wk 3

asyadi

Atomic Force
Microscopy

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 Motivation
• Digitally image a topographical surface
• Determine the roughness of a surface sample or
to measure the thickness of a crystal growth
layer
• Image non-conducting surfaces such as proteins
and DNA
• Study the dynamic behavior of living and fixed
cells

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 History
• The Scanning Tunneling Microscope (STM) was
invented by G. Binnig and H. Rohrer, for which
they were awarded the Nobel Prize in 1984.
• A few years later, the first Atomic Force
Microscope (AFM) was developed by G. Binnig,
Ch. Gerber, and C. Quate at Stanford University
by gluing a tiny shard of diamond onto one end
of a tiny strip of gold foil.
• Currently, AFM is the most common form of
scanning probe microscopy.
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 How the AFM Works
• The AFM brings a probe in
close proximity to the
surface
• The force is detected by
the deflection of a spring,
van der Waals force curve
usually a cantilever (diving
board)
• Forces between the probe
tip and the sample are
sensed to control the
distance between the the
tip and the sample.
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 Atomic Force Microscope (AFM)

deflection
sensor

cantilever
feedback
probing tip
regulator

sample

high voltage
xy-piezo (lateral position) amplifier

z-piezo
(tip-sample distance)

Negative feedback keeps the force constant by adjusting the z-piezo such
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that the up-down bending angle of the thin cantilever remains constant.
 Atomic Force Microscopy
• Operates by measuring forces between sample and
probe tip. Force depends on nature of sample,
probe geometry, spring constant of probe, distance
between probe & sample, and any contamination on
surface.
• An atomically sharp tip is scanned over surface with
feedback mechanisms that enable the piezoelectric
scanners to maintain the tip at a constant height (to
obtain force info) above the sample surface.
• Tip is brought close enough to surface to detect
repulsive force between atoms in tip and sample.
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 Atomic Force Microscopy

• The key element of the AFM is its microscopic force sensor.

• The most common force sensor is a cantilever. The tip is mounted at the end of the
cantilever. Tips typically made of Si3N4 or Si .
• The force between the tip and sample generates elastic bending of the cantilever.
• The amount of bending is monitored and recorded by position-sensitive
photodiodes.
• The photodiodes receive the laser beam reflected from the rear side of the
cantilever, which has mirror-like property due to coating.
• The amount of deflection can be mathematically converted into the force on the
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according to the elastic properties of the cantilever.

 Atomic Force Microscopy
 Probe tip is mounted on cantilever.
Interatomic forces will induce
bending and can be detected by laser
beam.
 Surface topography of sample is
tracked by monitoring deflection of
the cantilever.
 Any small deflection of the cantilever
will tilt the laser beam and change its
striking position on the photodiodes.
 Photodiodes measures difference in
light intensities then converts it to
voltage.
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 AFM – Probe’s Tip

The geometry of the tip can generate artifacts. Sharpness

of the tip affects the mapping of the sample surfaces
unless the tip is sharper than the surface features being
mapped.

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 Two Modes
Repulsive (contact)
• At short probe-sample
distances, the forces are
repulsive.
Attractive Force (non-
contact)
• At large probe-sample
distances, the forces are
attractive.
The AFM cantilever can be
used to measure both
attractive force mode and
repulsive forces. 11
 Contact Mode
• Contact mode operates in the
repulsive regime of the van
der Waals curve
• Tip attached to cantilever with
low spring constant (lower
than effective spring constant
binding the atoms of the
sample together).
• In ambient conditions there is
also a capillary force exerted
by the thin water layer
van der Waals force curve
present (2-50 nm thick).
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 Non-Contact Mode
• Uses attractive forces to
interact surface with tip
• Operates within the van
der Waal radii of the atoms
• Oscillates cantilever near its
resonant frequency (~ 200
kHz) to improve sensitivity
• Advantages over contact: no
lateral forces, non-
van der Waals force curve
destructive/no contamination
to sample, etc.
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 Biological Applications: AFM Images of Cells
Contact mode image of human red blood cells - note cytoskeleton is
visible. blood obtained from Johathan Ashmore, Professor of Physiology Red Blood Cells
University College, London. A false color table has been used here, as Shao, et al., : http://www.people.virginia.edu/~js6s/zsfig/random.html
professorial blood is in fact blue. 15µm scan courtesy M. Miles and J.
Ashmore, University of Bristol, U.K.

Rat Embryo Fibroblast(*M. Stolz,C.

Height image of endothelial cells taking in fluid using Contact
Radmacher, et al., Cardiac Cells Schoenenberger, M.E. Müller Institute, Mode AFM. 65 µm scan courtesy J. Struckmeier, S. Hohlbauch, P.
http://www.physik3.gwdg.de/~radmacher/ Biozentrum, Basel Switzerland) Fowler, Digital Intruments/Veeco Metrology, Santa Barbara, USA.
 Biological Applications: AFM Images of DNA
TappingMode image of nucleosomal DNA was the highlight Image of PtyrTlac supercoiled DNA. 750 nm
of the "Practical Course on Atomic Force Microscopy in scan courtesy C. Tolksdorf, Digital
Biology," held at the Biozentrum in Basel, Switzerland, July Instruments/Veeco, Santa Barbara, USA, and R.
1998. Image courtesy of Y. Lyubchenko.
Schneider and G. Muskhelishvili, Istitut für
Genetik und Mikrobiologie, Germany.

http://www.people.virginia.edu/~js6s/zsfig/DNA.html

AFM image of short DNA fragment with RNA

The high resolution of the SPM is able to discern polymerase molecule bound to transcription
very subtle features such as these two linear dsDNA recognition site. 238nm scan size. Courtesy of
molecules overlapping each other. 155nm scan. Bustamante Lab, Chemistry Department, University
Image courtesy of W. Blaine Stine of Oregon, Eugene OR
 STM and AFM
 The probe tip is kept at a short
distance (not touching) from the
surface.
 An STM uses tunneling current to
examine material surface.
 The tunneling current flows through
an atomic-scale gap between a sharp
metallic tip and conducting surface
atoms.
 STM images indirectly by calculating
the quantum degree tunneling between
the probe and sample.
 The tip in AFM gently touches the surface.
 AFM does not measure the tunneling
current but only measures the small force
between the surface and the tip to generate
signals of surface topography.
 The AFM more widely used than the STM
because it is not restricted to electrically
conducting surface.
 AFM resolution is better than the STM.
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 AFM vs. STM
1) The probe makes a direct contact with the surface or
calculates the incipient chemical bonding in AFM. The
STM images indirectly by calculating the quantum degree
tunneling between the probe and sample.
2) The tip in AFM touches the surface gently touches the
surface whereas in STM, the tip is kept at a short distance
from the surface.
3) AFM resolution is better than the STM. This is why AFM is
widely used in nano-technology.
4) When STM is only applicable to conductors, the AFM is
applicable to both conductors and insulators.
5) The AFM suits well with liquid and gas environments
whereas STM operates only in high vacuum. 17
CASE STUDY

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