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 systems.
• 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 subnanometer 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
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 Hook’s law.
• The cantilever obeys Hook’s 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
measured
• The movement of the tip or sample is
performed by an extremely precise
positioning device made from piezoelectric ceramics, mostly in the form of a
tube scanner.
• The scanner moves the sample or the
cantilever in x, y, and z direction at subangstrom resolution
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 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.
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 intermolecular force curve as the scanning
proceeds
• Repulsive force : 1~10 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
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 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
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
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)
Dynamic modes
•
Resonant frequency of cantilever
feff = 1/2π (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.
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.
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 : 10~100 kHz
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
Tip deconvolution effects
Observed width W = (8dR)1/2
Resolution
VEECO
TESPA®
VEECO
TESPA-HAR®
NANOWORLD
SuperSharpSilicon®
Tip length : 10 m
Radius : 15~20 nm
Tip length :10 m
(last 2 m 7:1)
Radius : 4~10 nm
Tip length :10 m
Radius : 2 nm
Images of AFM
Contact mode
Dynamic force mode
Chloroplast ATP synthase is
revealed to be composed of 14
subunits
AFM topographs of purple membrane from Halobacterium salinarium
Purple 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
Lee et al. Science, 295, 1702-1705 (2002)
MHA : 16-mercaptohexadecanoic acid
Passivated by 11-mercaptoundecyl-tri(ethylene glycol)
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.
(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 adhesion
Work of adhesion : Dupre equation
- For a typical hydrocarbon, γw= 435, γHC = 108, γHC/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.
Preparation of chemical tips
SAMs (self-assembled monolayers)
CFM probe tip
Chemical force microscopy
CH3/CH3 : 1.0+0.4 nN
CH3/COOH : 0.3 + 0.2 nN
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
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 (질소와 탄소로 구성된 6각형 고리 ) : thymine, cytosine
Purine (질소와 탄소로 구성된 6각형과 5각형의 이중 고리) : adenine, guanine
Sugar-Phosphate Backbone of DNA
DNA structure (Space-filling Model)
• 서로 반대 방향으로 되어 있는 2개의 사슬
한 가닥은 5’ 에서 3’ 으로 위에서 아래로 달리고
다른 가닥은 5’ 에서 3’ 으로 아래에서 위로 달린다.
• 오른 나사 방향으로 꼬인 이중 나선 구조
• 염기간 상보결합
• 약 10개 뉴클레오티드 / 나선 한바퀴 : 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
Z-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)
A Force-Based Protein Biochip
Discrimination between specific and non specific interactions
Top Surface (PDMS):
DNA force sensor (14 pN) – Ligand/Protein – Cy3
B. Surface contact (10 min) and
biomolecular interactions
C. 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
Bottom Surface (Glass): Protein Microarray
K. Blank et al. Proc. Natl. Acad. Sci. USA 100, 11356 (2003)
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
where
또는
Chemical force microscopy
• External load(Lext), internal load(Lint)에 대해
: friction coefficient
JKR theory에 의해 contact area of interface
(radius a)는 elasticity (K = 3.4 * 109J/m3 for
polystyrene) 의 함수로 나타낼 수 있다

Dynamic force mode