AFM application: Beyond Imaging
Probing Nano-Scale Forces
Probing Nano-Scale Forces with the Atomic Force Microscope
How did the AFM system measure the force between tip and sample
in contact mode?
The cantilever starts (point A) not touching the surface. In this
region, if the cantilever feels a long-range attractive (or
repulsive) force it will deflect downwards (or upwards) before
making contact with the surface. In the case shown, there is
minimal long range force, so this “noncontact”part of the force
curve shows no deflection.
As the probe tip is brought very close to the surface, it may jump
into contact (B) if it feels sufficient attractive force from the sample.
Once the tip is in contact with the surface, cantilever deflection will
increase (C) as the fixed end of the cantilever is brought closer to
the sample. During the process, adhesion or bond forms between tip
and 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 (C) can provide information
about the elasticity of the sample surface.
As the cantilever is withdrawn, adhesion or bonds formed during
contact with the surface may cause the tip to adhere to the sample
(section D) some distance past the initial contact point on the
approach curve (B).
A key measurement of the AFM force curve is the point (E) 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.
Details of force measurements
Calculating forces
Photodiode sensitivity measurement ( convert Volts
To cantilever deflection(nm)
VoltsDeflection
Scanner Z position
Hard surface
F=-kx
k: Spring constant of the cantilever; x: deflection of the
cantilever
Spring constant measurement
Spring constant measurement
Cleveland method: Rev. Sci. Instrum. 1993, 64, 403
This method is based upon measuring the resonance frequency
of the cantilever in air, as a function of applied mass.
When an end mass M is added, the resonant frequency is given by
1
v
2
k
M  m*
k
M
 m*
2
(2v)
Nondestructive Added Mass Spring Calibration with the MFP-3D™
http://www.asylumresearch.com/Applications/CantileverCal/
AddedMass.pdf
Some typical force curves
Beginnings — Controlling Surface Adhesion
Measurement of the forces between an
AFM tip and a mica surface in air and
water. The measurement in air shows a
large adhesive force due to capillary forces
from the liquid contaminant layer on the
surface.
Once the whole cantilever probe is
immersed in water, capillary forces are
mostly eliminated and adhesion is
substantially diminished.
From A.L. Weisenhorn, Appl. Phys. Lett.
1989, 54, 2651
Improve image quality
Sharpened tip
Imaging in vacuum
Imaging in some solvent to reduce the tip-sample adhesion
( for example, alcohol, remove organic contamination)
Ozone or UV treat the tip
Tip modification
Colloidal Forces
The forces between colloidal particles dominate the behavior of a
great variety of materials, including paints, paper, soils, clays and,
in some circumstances, biological cells.”
Colloid forces also play a critical role in the formation of ceramics
and composite materials. Even in Pharmacy, Lotion, cream (
TiO2 and PbO)
The AFM can measure forces on
colloidal particles. In this case, a
silica bead was glued to an AFM
cantilever.
Science 1994, 264, 415
Signature Feature from Bruker AFM
Peak Force Tapping
AFM Imaging Mode Revolution
Contact Mode
TappingMode
Force-Volume
PeakForce Tapping
First
introduced
1986 (original mode)
1992
~1994
2009
Tip-sample
interaction
Tip scans in constant contact
with the sample
Cantilever is oscillated at its
resonance, so the tip
intermittently contacts or
“taps” the sample
Whole probe is ramped
linearly up and down, so the
tip intermittently contacts the
sample
Whole probe is ramped
sinusoidally, so the tip
intermittently contacts or
“taps” the sample
Tip
oscillation
Not applicable
At cantilever resonance (typ.
10-1000’s kHz) with typ.
amplitude of 1-10’s nm
Far below cantilever
resonance (typ. <100 Hz) with
typical amplitude of 1001000’s nm
Below cantilever resonance
(typ. <10 kHz) with typical
amplitude of 10-100’s nm
Imaging
feedback
Constant force (cantilever
deflection)
Constant tapping amplitude
No feedback. Each curve
individually triggered.
Constant peak force
Sketch
Off surface
On surface
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F0
FSP
Afree
ASP
F0
F0
F0
F0
F0
Ftrig
F0
Fpeak
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17
Peak
Force
Peak
Force
Peak repulsive force can
be as small as tens of pN
•
Most Direct and Fastest way to conduct Quantitative Nanomechanical Mapping through force
curves, no sample dependent empirical parameters required.
•
Simultaneously obtain quantitative modulus, adhesion, dissipation, and deformation data while
imaging topography at high resolution
•
Direct force control keeps the imaging force low, which limits indentation depths to deliver nondestructive, high-resolution imaging
•
Material properties can be characterized over a very wide range to address samples in many
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Bruker Confidential
different research applications.
Capabilities Unlike Anything Else
PFQNM-Height
PFQNM-Adhesion
PFQNM-Modulus
PeakForce QNM can quantitatively and
unambiguously identify modulus and
adhesion variations. Phase imaging and
multifrequency imaging techniques cannot.
•
Comparison of the adhesion and phase
images clearly shows that the phase contrast
is primarily due to adhesion, whereas one
might more commonly assume that it
reflects modulus variations
•
Section plot illustrates ability to measure the
modulus across the polymer layers
Tapping-Height
Tapping-Phase
Multilayered polymer
film, 10 µm scans
Left: PeakForce QNM
Right: TappingMode
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Choose the Right Cantilever/Tip assemblies
Early detection of aging cartilage and
osteoarthritis in mice and patient samples
using atomic force microscopy
Local Nanomechanical Motion of the
Cell Wall of Saccharomyces cerevisiae
Science, 2004, 305, 1147
k=-F/x
k: the spring
constant of the cell surface
After the MDCK fixed on the surface, is it becoming
Softer or harder?
A
B
Effect of Dendrimer Generation on the Assembly and Mechanical
Properties of DNA/Phosphorus Dendrimer Multilayer
Microcapsules
Macromolecules 2006, 39, 5479-5483
Liu, G.-Y.
Langmuir 2006, 22, 8151-8155
Figure 1 (A) Schematic of the cell compression experiment. (B) Confocal micrograph
reveals the typical AFM probe position, that is, above the center of the designated cell.
(C) Confocal microscopy snapshots of a single cell under the compression cycle of AFM
force curve acquisition. Cell membrane deformation (blue boundaries) followed by the
flattening of the nucleus (see pink regions) was captured. At high deformation, the
Newton diffraction ring is clearly visible, which demonstrates the microscopic precision
in the alignment.
Figure 2 Typical force versus deformation curves for
living (green) and dead (blue) T cells. Insets show the two
types of cells upon the addition of 10 L of 4% trypan
blue solution, where dead cells turn blue under optical
microscopy.
Figure 4 (A) Least-squares
fitting of the force curves of
living cells according to eq 3.
(B) Least-squares fitting of the
force curves of dead cells
using eq 6. (C) Fitting of the
cell compression curves for
fixed cells using eq 6. The
corresponding cell
deformation mechanisms are
illustrated on the right column,
revealing a fluid-filled balloon
(A'), a soft and permeable
sphere (B'), and a hard and
permeable rubber ball model
(C'), respectively.
For Living cells
For Dead Cells and Fixed Cells
The bending of the membrane and cell interior compression (e.g.,
cytoskeleton and nuclei) are the main contributions to the forcedeformation correlation. The small bending of the spherical membrane
can be estimated following the example of living cells (see eq 1):
Is also very small
Stretching and breaking duplex DNA
by chemical force microscopy
Chemistry & Biology 1997, Vol4, 519
A schematic illustration of the chemical force microscopy setup. The sample is
mounted on a piezoceramic transducer (green) that can move the sample
vertically with subangstrom precision. The probe tip is attached to a flexible
cantilever (yellow) and the laser beam (red) is reflected off the cantilever back
onto a photodiode to monitor cantilever deflection.
The inset shows a cartoon representation of interactions between two
complementary strands immobilized on the tip and sample surfaces. Both the
tip and the sample are coated with gold; self-assembled monolayers of
lkanethiols (blue) are then formed on the gold surface. The DNA shown in the
inset corresponds to the relaxed B-DNA conformation.
Mixed monolayers of thiohexadecanol and
hexadecanethiol functionalized with oligonucleotides
were formed by spontaneous self-assembly from 3:l
H2O:EtOH solutions. The resulting hydroxylterminated
monolayer has a hydrophilic surface from which the DNA
strands protrude.
Trace I: noncomplementary pair in which the tip and surface
were functionalized with LS.
Trace II: complementary pair in which the tip was functionalized with LT
and the surface was functionalized with LS.
Force versus separation plots obtained from
individual approach withdrawal traces for interactions
between complementary DNA strands.
Sequence-dependent mechanics of
single DNA molecules
Gaub H. E. Nat. Struct. Biol.1999, 6 346
1 Force-induced melting transition in lDNA. a, Stretching (red) and relaxation
(blue) curve of a 1.5-mm-long segment of a
l-BstE II digested DNA molecule. The
superimposed black curve is a force curve
taken on ssDNA. Because the ssDNA
strand has a different contour length (~300
nm), the curves were scaled to the same
contour length before superposition. Inset:
low-force range of a relaxation curve of lDNA previously split in the melting
transition (blue curve). The black curve is a
fit according to the freely jointed chain
model with an additional segment elasticity
using the same parameters that have been
determined for ssDNA12 (Kuhn length 8 Å,
molecular spring constant per unit length
800 pN).
Superposition of four
extension traces of the same
molecule taken at different
pulling speeds (red, 3 mm s–1;
green, 1.5 mm s–1; blue, 0.7
mm s–1; black, 0.15 mm s–1).
Inset:
a sequence of three stretching (red) and relaxation (blue) cycles
performed each time on the same molecule (pulling speed: 0.25
mm s–1).
Partial melting of the double helix occurs during the B-S
transition (first trace) and is complete after the melting
transition (second and third trace). Reannealing of the two
single strands leads to the characteristic shape of the relaxation
traces.
Fig. 2 The mechanical compliance of DNA strongly depends on the
specific base pairing in the double helix.
a, For double-stranded poly(dG-dC) DNA the B-S transition occurs at
65 pN (see arrows), just as in l-DNA, while the melting transition is
raised to 300 pN, compared to 150 pN in l -DNA.
b, In duplex poly(dA-dT) DNA the force of the B-S transition is
reduced to 35 pN and the strands melt during this transition, so that
no distinct melting transition can be observed.
Direct measurement of base-pairing forces
The self-complementarily of poly(dG-dC) and poly(dA-dT)
allows for hairpin formation within the two DNA strands.
After force-induced melting of the double helix, base-pairing forces can be
directly measured in the strand attached to the tip.
Three repeated extension (red) and
relaxation (blue) curves of one poly(dGdC) molecule.
In the first extension/relaxation cycle, the
molecule is extended into the B-S
transition. This process is essentially
reversible.
In the second cycle the molecule is
extended partly into the melting transition,
which leads to a shortening of the B-S
plateau and the appearance of a new
plateau at FG-C = 20 pN in the relaxation
trace, caused by hairpin formation. This 20
pN plateau reappears as a hairpinunzipping plateau in the extension trace of
the third cycle. Here the molecule is
overstretched even more, resulting in
further growth of the 20 pN plateau at the
expense of the B-S plateau in the
relaxation trace.
For poly(dA-dT) DNA the zipping/unzipping force is FA-T = 9
pN. The two plots show the stretching (red) and relaxation
(blue) of a molecule that had already formed intrastrand
hairpins during a previous stretching. The first cycle shows
zipping and unzipping of the hairpins. In the second trace the
double strand is further converted into intrastrand hairpins.
The double helix starts to melt during the B-S transition.
Discriminating small molecule DNA binding modes
by single molecule force spectroscopy
FEBS Letters 510 (2002) 154
Drugs may interact with double stranded DNA via a variety of binding modes,
each mode giving rise to a specific pharmacological function.
This paper demonstrate the ability of single molecule force spectroscopy to
discriminate between different interaction modes by measuring the
mechanical properties of DNA and their modulation upon the binding of small
molecules.
Due to the unique topology of double stranded DNA and due to its base pair
stacking pattern, DNA undergoes several well-characterised structural
transitions upon stretching. In this paper they showed that small molecule
binding markedly affects these transitions in ways characteristic to the
binding mode and that these effects can be detected at the level of an
individual molecule.
.
Crosslinker:
cisplatin
Minor groove binder: Berenil
Intercalator: ethidium bromide
Homework: How to detect protein/DNA interaction?