Atomic Force Microscopy

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Atomic Force Microscopy Chemical
Force Microscopy
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• 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
  systems.
• Traditional methods
• - X-ray crystallography, NMR Too
  complicated, limited
• size (200 Kd, 40 Kd)
• - Electron microscopy
• - Impossible for observation under
  physiological conditions

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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
  nanoscale
• 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

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Principle of AFM

• Scan an object point by point using a cantilever
  tip
• 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.

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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
  measured
• 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
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Feedback operation

• If the tip were scanned at a constant height, the
  tip would collide with the surface, causing
  damage
• 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
  cantilever.

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Imaging modes

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

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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
  proceeds
• Repulsive force 110 nN
• Deflection of cantilever with a low spring
  constant
• 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
  z-direction

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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

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Dynamic Modes

• Distance between the tip and the sample 2 30
  nm
• Attractive force 0.1 0.01 nN
• Vibration of cantilever around its resonance
  frequency
• 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

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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
  disappears.
• Low resolution with a minimum value of around 1
  nm

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Typical Characteristics

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

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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
  engaged.

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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.

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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.

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Cantilever

• 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
  convolution.
• - 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
  features

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Tip deconvolution effects
Observed width W (8dR)1/2
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Resolution
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VEECO TESPA VEECO TESPA-HAR NANOWORLD SuperSharpSilicon

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
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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
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AFM images of the cytoplasmic surface of the
hexagonally packed intermediate layer of the
bacterium Deinoccocus radiodurans Protruding
protein cores
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Dip pen-nanolithography using AFM
MHA 16-mercaptohexadecanoic acid Passivated by
11-mercaptoundecyl-tri(ethylene glycol)
Lee et al. Science, 295, 1702-1705 (2002)
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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
  retraction.

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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 .
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Force versus Distance
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• Adhesion force Fadh

R size of the sphere (radius) W work of
adhesion
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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
adhesion.
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Preparation of chemical tips
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SAMs (self-assembled monolayers)
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CFM probe tip
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Chemical force microscopy
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CH3/CH3 1.00.4 nN CH3/COOH 0.3 0.2 nN
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Chemical force imaging Chemical sensitive
imaging

• 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

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Four Bases in DNA A,G,C,T
Pyrimidine (??? ??? ??? 6?? ?? ) thymine,
cytosine Purine (??? ??? ??? 6??? 5??? ?? ??)
adenine, guanine
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Sugar-Phosphate Backbone of DNA
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DNA structure (Space-filling Model)

• ?? ?? ???? ?? ?? 2?? ??
• ? ??? 5 ?? 3 ?? ??? ??? ???
• ?? ??? 5 ?? 3 ?? ???? ?? ???.
• ?? ?? ???? ?? ?? ?? ??
• ??? ????
• ? 10? ?????? / ?? ??? 3.4 nm

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• 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

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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
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A-, B-, and Z-form DNA
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Z-DNA
Left-handed double helix
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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.
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Insertion of single dye molecule Increase of
the base pair rise by 3.4 A Unwinding of the
double helix by 26o
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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
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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
  interactions
• 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
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Chemical force microscopy
where
??
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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) ? ??? ??? ? ??

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Dynamic force mode