1. AFM Basics
Xinyong Chen

2. Outline How AFM works Force measurements with AFM Scanning
Feedback control
Contact mode and tapping mode
Force measurements with AFM
How AFM measures forces
Calibrations
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3. How AFM works
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4. How AFM works
Direct mechanical contact between the probe and the sampler surface
Essential difference from traditional microscopy
How AFM “feels” the surface topography?
Optical level detection
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5. Optical level detection
Voltage
Difference
Between
Top & Bottom
Photodiodes
With this “split photodiode”, any slight vertical movement of
the reflection spot position is detected by checking the
difference between the “top” and the “bottom” photodiode
dutputs (the “T-B signal”).
(Click for the next)
Top-Bottom Signal (V)
or Deflection (nm)
or Force (nN)
Let’s split the photodiode into two – the “top” and the “bottom”.
Assume that the optical reflection spot originally locates in the
exactly middle of this split photodiode, resulting in the exactly
same voltage output from the two photodiodes. So, the difference
between the “top” (T) and the “bottom” (B) is zero.
(Click for the next)
During scanning, the sample surface may
lift the cantilever up, resulting in corresponding
move up of the optical reflection spot on the
photodiode. However, this single photodiode
couldn’t detect small position change of the spot.
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Quad photodiode to detect
Both vertical and horizontal
Movements of the light spot.
Photodiode
Laser
Scanner
Cantilever + Sharp probe
Photodiode
Laser
Scanner
Cantilever + Sharp probe
Photodiode
Laser
Scanner
Cantilever + Sharp probe
Photodiode
Laser
Scanner
Cantilever + Sharp probe
Photodiode
Laser
Scanner
Cantilever + Sharp probe
Photodiode
Laser
Scanner
Cantilever + Sharp probe
Photodiode
Laser
Scanner
Cantilever + Sharp probe
Photodiode
Laser
Scanner
Cantilever + Sharp probe
Photodiode
Laser
Scanner
Cantilever + Sharp probe
Photodiode
Laser
Scanner
Cantilever + Sharp probe
Photodiode
Laser
Scanner
Cantilever + Sharp probe
Photodiode
Laser
Scanner
Cantilever + Sharp probe
Photodiode
Laser
Scanner
Photodiode
Laser
Scanner
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6. How AFM works
Direct mechanical contact between the probe and the sampler surface
Essential difference from traditional microscopy
How AFM “feels” the surface topography?
Optical level detection
Constant-height scan versus Constant-force scan
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7. Constant-height scan Click for the Next
Click on graph to play animation (internet connection required)

8. Constant-height scan Advantages: Disadvantages:
Simple structure (no feedback control)
Fast response
Disadvantages:
Limited vertical range (cantilever bending and detector dynamic range)
Varied force
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9. Constant-force scan Click for the Next
Click on graph to play animation (internet connection required)

10. Optical level detection in constant-force mode
In constant-force mode, whenever the
sample surface topography would result in
the cantilever deflection change, the other
end of cantilever would be accordingly
adjusted so that the cantilever deflection angle,
and hence the contact force, would keep
constant.
Photodiode
Laser
Z scanner
Cantilever + Sharp probe
Photodiode
Laser
Z scanner
Cantilever + Sharp probe
Photodiode
Laser
Z scanner
Cantilever + Sharp probe
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11. Feedback control in constant-force mode
In constant-force mode, the cantilever’s
vertical position is adjusted by an
electronic feedback loop, with the T-B
signal as the input and the vertical
scanner voltage as the output.
P.I.D. Control
Horizontal
Vertical
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12. Constant-force scan vs. constant-height scan
Constant-height mode
Constant-force mode
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Click on graph to play animation (internet connection required)

13. Constant-force scan vs. constant-height scan
Advantages:
Large vertical range
Constant force (can be optimized to the minimum)
Disadvantages:
Requires feedback control
Slow response
Constant-height
Advantages:
Simple structure (no feedback control)
Fast response
Disadvantages:
Limited vertical range (cantilever bending and detector dynamic range)
Varied force
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14. How AFM works
Direct mechanical contact between the probe and the sampler surface
Essential difference from traditional microscopy
How AFM “feels” the surface topography?
Optical level detection
Constant-height scan and constant-force scan
Feedback control in constant-force scan
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15. Sample swept by AFM probes
The constant AFM probe contact
with the sample surface may cause
damage of the sample, typically shown
as “sweeping”. One of the techniques
to avoid such a problem is the
“tapping mode”.
1 mm
Self-assembly of octadecyl phosphonic acid (ODPA) on single crystal alumina surface imaged in ethanol with tapping mode. The central 1 mm × 1 mm area was previously scanned in contact mode with heavy loading force.
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16. Tapping mode AFM Click for the Next Click on graph to play animation

17. Feedback control in tapping mode
In tapping mode, the system uses
the same feedback control as that
used in constant-force contact mode.
However, it usually uses the cantilever’s
oscillation amplitude (the “AC” signal)
instead of its DC component (the
“Deflection”) as the input signal.
P.I.D. Control
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18. Tapping mode AFM PLA/PSA blend on Si imaged in air Height Phase 1 mm
In addition to the normal topographic image, tapping mode AFM
can also provide simultaneously a “phase image” map, which
results from variation in interactions between the AFM probe and
the various sample surfaces.
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19. How AFM works
Direct mechanical contact between the probe and the sampler surface
Essential difference from traditional microscopy
How AFM “feels” the surface topography?
Optical level detection
Constant-height scan and constant-force scan
Feedback control in constant-force scan
Contact mode and tapping mode
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the Next

20. Dimension AFM
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21. MultiMode AFM
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22. AFM Tips
80 – 320 mm
20 mm
35 mm
125 mm
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23. AFM sample preparation
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24. AFM in liquid environment
One extraordinary feature of AFM is to work in liquid environment. A key point
for liquid AFM is a transparent solid (usually glass) surface, which, together with
the solid sample surface, retains the liquid environment whilst maintains stable
optical paths for the laser beams. An optional O-ring can be used to form a sealed
liquid cell. Otherwise, the system can also work in an “open cell” fashion.
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25. Liquid AFM Images Click for the Next 70 nm
t=0 min 12 19 20 22 41 45 48 56 60
Effect of DNase I enzyme on G4-DNA (0.5:1) complex, the complex was immediately adsorbed onto mica and imaged until stable images were obtained, then the DNase I was introduced.
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Nucleic Acids Research, 2003, Vol. 31, No

26. Outline How AFM works Force measurements with AFM
Scanning and feedback control
Contact mode and tapping mode
Force measurements with AFM
How AFM measures forces
Calibrations
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27. Force measurements with AFMB C D
(A+B)-(C+D)
A+B+C+D
Defl=
P.I.D. Control
When an AFM works in force measurement
mode, the feedback loop is temporarily
“cut off”. The cantilever deflection (the
“T-B signal”) is then recorded while the
AFM probe is vertically “ramped”
towards/backwards the sample surface.
(Click step-by-step to see how this is
done.)
Z Displacement
Deflection
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28. Experimental Force Curves
Contact slope to study hardness
Adhesion to study intermolecular interactions
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29. Calibration of force measurements
Slope = DD / DZ (V/nm)
The Hooke’s law
F = -kx
Detector sensitivity
S = Inverse of the contact slope measured on a hard surface (nm/V)
Spring constant (N/m)
Property of the cantilever and provided by the manufacturer
Large variation due to difficulty in cantilever thickness control
Should (and can) be experimentally measured for accuracy requirement
Thermal fluctuation
Resonance + geometry
Mass adding + resonance
Standard with known spring constant
etc.
T-B Signal
Z Displacement (nm) x
(V)
Deflection (nm) DD
Force (nN) DZ x
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30. Humidity affects the adhesion
AFM probe
Salbutamol
Measurement of particle-particle interaction
Lactose
1µm
Force (nN)
200
400
600
800
1000
1200
<10%
22%
44%
65%
‘Nanoscale’ contact
‘Macroscale’ contact
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31. Environmental AFM Both temperature and humidity can be controlled
in this environmental
chamber.
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32. Intermolecular interactions
MFP is specially designed for force measurement
purpose
MFP
Schematic of the force–extension characteristics of DNA: at 65 pN the molecule is overstretched to about 1.7 times its contour length, at 150 pN the double strand is separated into two single strands, one of which remains attached between tip and surface.
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33. Adhesion Force Imaging
Height
Adhesion
pH 7
Albumin
Polystyrene PS
Albumin Si
5 mm
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34. Adhesion and Hardness Imaging
Height
Adhesion
Stiffness
1 mm
PLMA/PmMl6 blend on Si imaged in water
PLMA: poly (lauryl methacrylate)
PmMl6: 2-methacryloyloxyethyl phosphorylcholine-co-lauryl methacrylate (1:6)
Simultaneous Height, Adhesion and Stiffness maps are obtained
with “Pulsed-Force” AFM technique.
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35. Conclusions How AFM works Force measurements with AFM
Constant-height and constant-force scans (contact mode)
Feedback control in constant-force mode
Contact mode and tapping mode
Force measurements with AFM
Force curves: contact part to measure hardness and adhesion to measure intermolecular interactions
Calibrations:
Detector sensitivity (nm/V) = Inverse of contact slope on a hard surface => Convert the measured T-B signal (V) to cantilever deflection (nm)
Spring constant (N/m) => Convert the cantilever deflection to force (N) [F=-kx]
End