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Atomic Force Microscopy


Title: Chemical Force Microscopy Author: rubylight Last modified by: user Created Date: 5/5/2005 2:17:06 PM Document presentation format: On-screen Show (4:3) – PowerPoint PPT presentation

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Title: Atomic Force Microscopy

Atomic Force Microscopy Chemical
Force Microscopy
  • Biological systems can only be fully understood
    if their structure is known
  • Structural Biology the science investigating
    the structure and
  • function of the components of living
  • Traditional methods
  • - X-ray crystallography, NMR Too
    complicated, limited
  • size (200 Kd, 40 Kd)
  • - Electron microscopy
  • - Impossible for observation under
    physiological conditions

AFM Atomic force microscope
  • High resolution type of scanning probe microscope
  • Invented by Binnig, Quate, Gerber in 1986
  • To determine the surface topography of native
    biomolecules at sub-nanometer resolution not only
    under physiological conditions, but while
    biological processes are at work.
  • One of the foremost tools for imaging,
    measuring, and manipulating matter at the
  • High signal-to-noise (S/N) ratio details
    topological information
  • is not restricted to crystalline specimens.
  • Utilize a sharp probe moving over the surface of
    a sample in a raster scan.
  • The probe is a tip on the end of a cantilever
    which bends in response to the
  • force between the tip and the sample surface

Principle of AFM
  • Scan an object point by point using a cantilever
  • Determine the forces between the tip and the
    sample based on a deflection of the cantilever
    according to Hooks law.
  • The cantilever obeys Hooks law for small
    displacement, and
  • the interaction force between the tip and
    the sample can be
  • determined.
  • Measure the deflection using a laser spot
    reflected from the top of the cantilever into an
    array of photodiodes.

Schematic of AFM using the light deflection mode
  • As the cantilever flexes, the light from the
    laser is reflected onto the photo-diode
  • Change in the bending of the cantilever is
  • The movement of the tip or sample is performed by
    an extremely precise positioning device made from
    piezo-electric ceramics, mostly in the form of a
    tube scanner.
  • The scanner moves the sample or the cantilever in
    x, y, and z direction at sub-angstrom resolution

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Force curve as a function of the distance between
the tip and the surface Van der waals force
Van der waals force f(r) -1/r6 1/r12
10-7 10-11 N
Feedback operation
  • If the tip were scanned at a constant height, the
    tip would collide with the surface, causing
  • A feedback mechanism is employed to adjust the
    tip-to sample distance to maintain a constant
    force between the tip and the sample
  • The sample is mounted on a piezoelectric tube
    that can move the sample in the z-direction for
    maintaining a constant force, and the x and y
    directions for scanning the sample.
  • Operation in two principle modes
  • - With feedback control the positioning
    piezo responds to any changes in force that are
    detected, and alter the tip-sample separation to
    restore the force to a predetermined value ?
    constant force mode ? a fairly faithful
    topographical image
  • - Without feedback control Constant height
    or deflection mode
  • - Useful for imaging very flat sample at
    high resolution
  • - A small amount of feedback-loop gain to
    avoid problems with thermal drift
  • or damaging the tip and/or

Imaging modes
  • Contact mode (Static mode)
  • Dynamic force mode
  • - Non-contact mode
  • - Intermittent contact mode (Tapping mode)
  • - Force modulation mode

Contact mode
  • The most common method
  • The tip and sample remain in close contact,
    namely in the repulsive regime of the
    inter-molecular force curve as the scanning
  • Repulsive force 110 nN
  • Deflection of cantilever with a low spring
  • Determine the reflection of laser from the top
    of the cantilever using a photodiode
  • Alter the tip-sample separation to restore the
    force to a predetermined value scanner
  • Image the surface by analyzing the changes in

Contact mode
  • Very sensitive to a small force
  • Measuring a displacement as small as 0.01nm
  • Image with high resolution
  • Damage of the sample and/or tip , cantilever
  • Large lateral forces on the sample as the tip is
  • effectively dragged across the surface
  • Combined effects from the capillary forces of the
    water contamination layer

Dynamic Modes
  • Distance between the tip and the sample 2 30
  • Attractive force 0.1 0.01 nN
  • Vibration of cantilever around its resonance
  • Due to a too small force, it is impossible to
    determine directly the deflection
  • of cantilever
  • Measure the changes in the frequency (fo) of
    cantilever caused by interaction
  • between the sample and cantilever
  • Oscillation of the cantilever mechanical,
    magnetic or piezoelectric in air.
  • Oscillation in liquid is driven acoustically

Non-contact mode
  • The tip remains at all times in the attractive
    part of the interaction curve, and scans above
    the surface with a relatively small amplitude.
  • The tip may jump into contact with the surface if
    the attractive forces exerted are greater than
    the spring constant of the cantilever.
  • Much stiffer cantilever is required
  • Resonant frequency 150 300 kHz
  • Almost unusable in liquid system as the damping
    of the small cantilever oscillation by water or
    other liquids is too large and the signal
  • Low resolution with a minimum value of around 1

Typical Characteristics
  • Resolution similar to contact mode
  • Removal of the lateral forces
  • ? No surface damage
  • Sharp cantilever with a high resonance frequency
  • large spring constant (more stiff

Dynamic modes
  • Resonant frequency of cantilever
  • feff 1/2p (keff / m)1/2
  • K eff the spring constant of the
    cantilever, m the mass of the cantilever
  • As the tip approaches the surface, the effective
    mass of the cantilever will change due to the
    attractive forces acting on the point.
    Accordingly, the resonant frequency of the
    cantilever, feff, will change.
  • Changes in the resonant frequency causes the
    variation in amplitude or the phase shift
  • ? Two modes of detection are possible
    amplitude or phase shift
  • By defining the set point in terms of the signal
    amplitude or phase shift, the feedback loop is

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Intermittent contact mode Tapping mode
  • The next most common mode
  • The cantilever moves rapidly with a large
    oscillation between the repulsive and attractive
    regimes of the force curve.
  • The maximum forces applied to the surface may be
    lower or higher than those experienced in the
    contact mode, but such forces are not applied
    constantly, lowering drag forces on the sample.
  • Stiff cantilever with resonant frequencies in the
    range of 200- 400 kHz ? To break free of water
    contamination damping problem
  • The problem of capillary forces is removed.
  • The phase shift is highly sensitive to the
    tip-sample interaction and generates information
    on the mechanical properties of the sample.
  • Phase shifting may occur via adhesion between the
    tip and the sample or by a viscoelastic response
    of the sample.

Force modulation mode
  • Combine the oscillation of the cantilever with
    scanning in the contact mode.
  • Low oscillation between 1 5 kHz
  • The information extracted concerns the mechanical
    and viscoelastic properties of the sample
  • Useful for imaging the sample containing
    composite materials.

  • Material Si, Si3N4
  • Stiffness
  • soft contact mode (thickness 0.6?)
  • stiff dynamic force (thickness 4?)
  • Spring Constant (k) 0.1 10 N/m
  • Resonance frequency 10100 kHz

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Artifacts related to tip size and shape
  • The sharpness of the scanning tip One of the
    most important factors affecting the resolution
  • Tip convolution
  • - Broadening Occur when the radius of the
    tip curvature is comparable or greater than
    the size of the feature to be imaged. As the tip
    scans over the surface, the sides of the tip
    make contact before the apex, and the microscope
    begins to respond to the feature Tip
  • - Compression The tip is over the feature
  • - Interaction forces Change in force
    interaction due to the chemical nature of the tip
  • - Aspect ratio when imaging steep sloped

Tip deconvolution effects
Observed width W (8dR)1/2
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Tip length 10 ?m Radius 1520 nm Tip length 10 ?m (last 2 ?m 71) Radius 410 nm Tip length 10 ?m Radius 2 nm
Images of AFM
Dynamic force mode
  • Contact mode

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AFM topographs of purple membrane from
Halobacterium salinariumPurple membrane consists
of 25 lipid and 75 bacteriorodopsin. The
light driven proton pump comprises 7
transmembrane a-helices that surround the
photoactive retinal
AFM images of the cytoplasmic surface of the
hexagonally packed intermediate layer of the
bacterium Deinoccocus radiodurans Protruding
protein cores
Dip pen-nanolithography using AFM
MHA 16-mercaptohexadecanoic acid Passivated by
11-mercaptoundecyl-tri(ethylene glycol)
Lee et al. Science, 295, 1702-1705 (2002)
Chemical Force Microscope
  • Force-Distance Analysis
  • When the tip is placed at a fixed point on the
    sample and move in the vertical direction to the
    surface and then retracted from the surface in
    place of scanning, the deflection of the
    cantilever can be measured as it moves.
  • The cantilever is in the repulsive, contact
    region of the cycle, and the adhesion
    interactions between the tip and the surface
  • The deflection of the cantilever will provide
    information on the mechanical properties of the
    material during the part of the approach and the

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(a) and (b) When the sample is hard and
incompressible, as would be seen with glass,
ceramics or metallic surfaces, the tip will
simply approach the surface, jump into contact
and then bend the retraction curve will be the
same. (c) For more compressible samples, the
curve will be expected to resemble that shown in
(c) and information on the mechanical properties
of the sample may be extracted .
Force versus Distance
  • Adhesion force Fadh

R size of the sphere (radius) W work of
Work of adhesion Dupre equation
- For a typical hydrocarbon, ?w 435, ?HC 108,
?HC/W 304 J/mol/A2
- For the 2.7 nN rupture force required to
separate the complementary DNA interface, we
calculate 1.6 10-4 J/m2 for the work of
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Preparation of chemical tips
SAMs (self-assembled monolayers)
CFM probe tip
Chemical force microscopy
CH3/CH3 1.00.4 nN CH3/COOH 0.3 0.2 nN
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Chemical force imaging Chemical sensitive
  • AFM probe tips are covered by particular chemical
    functional groups (-CH3, NH2, COOH or more exotic
    biological molecules)
  • Scanned over a sample to detect adhesion
    differences between the species on the tip and
    those on the surface of the sample
  • Chemical imaging of structures present on the
    surface due to differences in interactions
    between the tip and sample

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Small molecule DNA binding mode
  • Cell replication and gene expression specific
    DNA-protein interactions
  • Blocking of the processes by small molecules
    Therapeutic agents
  • Binding modes of small molecules Understanding
    of their functions and development of new drugs
  • Binding through interactions, groove binding, and
    covalent attachment
  • - Cisplatin ( cis-platinum diammine
    dichloride) the cross-linking anti-cancer drug
  • - Berenil the anti-trypanosomal minor
    groove binder
  • - Ethidium bromide the intercalating dye

Four Bases in DNA A,G,C,T
Pyrimidine (??? ??? ??? 6?? ?? ) thymine,
cytosine Purine (??? ??? ??? 6??? 5??? ?? ??)
adenine, guanine
Sugar-Phosphate Backbone of DNA
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DNA structure (Space-filling Model)
  • ?? ?? ???? ?? ?? 2?? ??
  • ? ??? 5 ?? 3 ?? ??? ??? ???
  • ?? ??? 5 ?? 3 ?? ???? ?? ???.
  • ?? ?? ???? ?? ?? ?? ??
  • ??? ????
  • ? 10? ?????? / ?? ??? 3.4 nm

  • The cooperativity of the overstretching
    transition is strongly dependent on the base
    stacking in the DNA double helix
  • Different binding modes of small molecules cause
    different perturbations in base stacking ? Unique
    force curve profile

Single molecule force spectroscopy
Mechanical property of DNA
  • - 50 pN the worm-like chain model
  • - 65-70 pN transition from B-DNA
  • to S-DNA ? Loss of the stacking interaction of
    DNA bases ? melting of the double helix ?
    breakage of hydrogen
  • bonds
  • Rotation of DNA molecule to alleviate torsional
    strain ? a nick in one of the DNA strands
  • 150 pN separation of double stranded DNA
  • - Relaxation trace does not resemble the
    extension trace ( melting hysteresis) ?
    forced-induced melting

Experimental conditions 10 mM Tris buffer (pH
8.0) containing 150 mM NaCl and 1 mM EDTA
A-, B-, and Z-form DNA
Left-handed double helix
Binding mode of small molecules with DNA
- Digested phage DNA 2130 nm long fragment -
6260 bp, 50 GC content
Cisplatin acts by crosslinking DNA in several
different ways, making it impossible for rapidly
dividing cells to duplicate their DNA for
mitosis. The damaged DNA sets off DNA repair
mechanisms, which activate apoptosis when repair
proves impossible. The chlorine undergoes slow
displacement with water molecules forming a
positively charged molecule which then
crosslinks the DNA.
Insertion of single dye molecule Increase of
the base pair rise by 3.4 A Unwinding of the
double helix by 26o
Berenil 1,3-bis(4'-amidinophenyl)triazene
Bind to the narrow minor groove of AT-rich
regions through hydrogen bonding via the
bis-amidinium groups at each molecule and van
der Waals interactions
The base pair unbinding forces G-C (20 3 pN),
A-T (9 3 pN)
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A Force-Based Protein Biochip Discrimination
between specific and non specific interactions
  • B. Surface contact (10 min) and biomolecular
  • Surface separation
  • D. Rupture of the weaker bond ? Cy3 remains
    connected to the stronger bond.
  • E. Fluorescence upon the bottom surface
  • ? No signal in non-binder and control spots

K. Blank et al. Proc. Natl. Acad. Sci. USA 100,
11356 (2003)
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Chemical force microscopy
  • Johnson-Kendall-Roberts (JKR) theory
  • JKR theory considers the effect of finite
    surface energy on the properties of the interface
  • For external load(Lext) and internal load(Lint)

friction coefficient
Chemical force microscopy
Chemical force microscopy
  • External load(Lext), internal load(Lint)? ??

friction coefficient
  • JKR theory? ?? contact area of interface (radius
    a)? elasticity (K 3.4 109J/m3 for
    polystyrene) ? ??? ??? ? ??

Dynamic force mode
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