HIGH-RESOLUTION IMAGING WITH FORCES
(ATOMIC FORCE MICROSCOPY)
Krystyn J. Van Vliet
krystyn@mit.edu
3.052 Spring 2003
March 4, 2003
Review: Typical HRFS output on stiff
substrate compression
no
interaction
CONVERTED DATA
repulsive
regime
jump-to-contact
0
adhesion
0
z-Piezo Deflection, z (nm)
kc
attractive
regime
0
Tip-Sample Separation
Distance, D (nm)
Force, F (nN)
Photodiode
Sensor Output, s (V)
RAW DATA
substrate
Review: Typical HRFS output on stiff
nm)
repulsive
regime
kc
attractive
regime
0
Tip-Sample Separation
Distance, D (nm)
Force, F (nN)
no
action
CONVERTED DATA
substrate
Review: Typical HRFS output on stiff
substrate compression
no
interaction
CONVERTED DATA
repulsive
regime
jump-to-contact
0
adhesion
0
z-Piezo Deflection, z (nm)
kc
attractive
regime
0
Tip-Sample Separation
Distance, D (nm)
Force, F (nN)
Photodiode
Sensor Output, s (V)
RAW DATA
substrate
Review: Experimental Aspects of
Force Spectroscopy
Conversion of raw data in a high-resolution force spectroscopy experiment :
• sensor output, s  transducer displacement, d  force, F
F=kd
d = s/m
• z-piezo deflection, z  tip-sample separation distance, D
D=zd
Typical force spectroscopy data for a weak cantilever on stiff substrate (ksample>> kcantilever) :
RAW DATA
substrate compression
D
no
interaction
D
jump-to-contact
B/C
0
E
F
adhesion
CONVERTED DATA
D
D
repulsive
regime
A
B/C
G
kc
E
attractive
regime
A
G
F
0
0
z-Piezo Deflection, z (nm)
Tip-Sample Separation
Distance, D (nm)
*Note: For an adhesive interaction
Force, F (nN)
cantilever undeflected, zero force (set F=0)
• B/C: attractive interaction pulls tip down to
surface and tip jumps to contact, cantilever
exhibits mechanical instability
• D: contact, constant compliance regime,
no sample indentation, tip and sample move
in unison (Ds/Dz=1)
RETRACT :(*sample and tip move apart)
• D: repulsive contact, constant compliance
regime, tip deflected up
• E: attractive force (adhesion) keep tip
attached to surface, tip deflected down
• F: tip pulls off from surface, cantilever
instability
• G: same as region A
Photodiode
Sensor Output, s (V)
APPROACH : (*sample and tip come together)
• A: tip and sample out of contact, no interaction,
Atomic Force Microscopy Imaging
• BASIC PRINCIPLES :
 piezo rasters or scans in x/y direction across sample surface
 cantilever deflects in response to a topographical feature
 computer adjusts the z-piezo distance to keep
the cantilever deflection constant and equal to the setpoint value
“feedback loop” : system continuously changes in response to an
experimental output (cantilever deflection)
ERROR SIGNAL = actual signal- set point
(*used to produce 2D topographical image in contact mode)
Atomic Force Microscopy:
General components and functions
cantilever
AFM : Normal Force Spectroscopy Modes of Operation
*AC=dynamic(tip is driven to oscillate), DC=static(no external oscillation on tip)
Contact (DC and AC) :
Force Modulation
Intermittent Contact : Tapping (AC)
Non-Contact (AC)
AFM : Contact Mode
Output:
“Isoforce” Height
Feedback Error:
Deflection
http://www.physik3.gwdg.de/~radmacher/publications/osteoblasts.html
AFM : Tapping Mode
Output:
“Isoamplitude” Height
Feedback Error:
Amplitude
Additional Feedback:
Phase
Evaporated gold surface
AFM : Normal Force Spectroscopy Modes of Operation
AFM : First high resolution images
LAYERED HARD CRYSTALLINE SOLID MATERIALS
Highly Oriented Pyrolytic Graphite (HOPG)
SAMPLE
graphite
molybdenum sulfide
boron nitride
gold
sodium chloride (001)
lithium flouride
(1014) cleavage plane of a
calcite (CaCO3) crystal
REFERENCE
Binnig, et al., Europhys. Lett. 3, 1281 (1987)
Albrecht, et al., J. Vac. Sci. Tech. A 6 271 (1988)
Manne, et al., Appl. Phys. Lett. 56 1758 (1990)
Meyer, et al., Appl. Phys. Lett. 56 2100 (1990)
Meyer, et al., Z. Phys.B. 79 3 (1990)
Ohnesorge, et al., Science 260 1451 (1993)
(http://www.energosystems.ru/fgallery.htm)
http://stm2.nrl.navy.mil/how-afm/how-afm.html
http://www.physics.sfasu.edu/afm/afm.htm
AFM: Tip Functionalization
1. Gold coating
Purpose:
Methods:
TOP VIEW
TOP VIEW
Si3N4
cantilever
ONE-TIME Au
COATING :
100 nm
heterogeneous,
rougher larger
polydomain
microstructure
SIDE VIEW
INTERVAL Au
COATING :
Si chip
homogeneous,
smoother smaller
polydomain
microstructure
100 nm
SIDE VIEW
AFM: Tip Functionalization
2. Chemical coating
http://www.di.com/AppNotes/LatChem/LatChemMain.html
Purpose:
microfabricated
Si3N4 probe tip
Methods:
• Molecular Elasticity of Individual Polymer Chains
• Protein Folding
• DNA Interatomic Bonds
• Receptor-Ligand Interactions
• Covalent Bonds
• Colloidal forces
Applications: • Van der Waals forces
• Hydration forces
• Hydrophobic forces
• Surface Adhesion
• Nanoindentation
• Electrostatic DLVO forces
• Cell Adhesion
• Steric Forces of Polymer Brushes
+
-
+
synthetic polymers
-
-
-
-
+
-
proteins
polyelectrolytes
+
- -
antibodies
-
-
+
+
self-assembling
monolayer
ligands
AFM: Tip Functionalization
(c)
(c)
colloidal particle
(a) Single Cell Dictyostelium Discoideum
(d)
nanotube with
individual ligand
(b) E. Coli Bacteria
(a) Benoit, M.; Gabriel, D.; Gerisch, G.; Gaub, H. E. Nature Cell. Bio 2000, 2 (6), 313.
(b) Ong, Y-L.; Razatos, A.; Georgiou, G.; Sharma, M. K. Langmuir 1999, 15, 2719.
(c) J . Seog, Ortiz/ Grodzinsky Labs 2001
(d) Wong S.S.; Joselevich E.; Woolley, A.T.; Cheung, C. L.; Lieber, C. M. Nature 1998, 394 (6688), 52.
AFM: Applications of modes
Timeline:
Contact DC and AC (Force Modulation Microscopy (FMM), Phase Imaging):
Hansma, et al., 1991
Intermittant Contact/Tapping / Lift (AC):
Hansma, et al., 1994
Noncontact (NC)
1995
I. Normal Force Microscopy
II. Friction or Lateral Force Microscopy (FFM/ LFM) Frisbie, et al., 1994
III. Force / Volume Adhesion Microscopy Radmacher, et al., 1994
http://www.di.com/AppNotes/ForceVol/FV.array.html
X
X
X
X
X
X
XX
X XX XX
X
X
X
X
X
X
X
X=-OH,-CH3, -NH2
IV. Chemical Force Microscopy (CFM) Frisbie, et al., 1994
Surface Maps:
Topography & Roughness, Electrostatic
Interactions, Friction
Chemical, Adhesion , Hardness,
Elasticity /Viscoelasticity
Dynamic Processes :
Erosion, Degradation, Protein-DNA
Interactions
AFM: Resolution factors/Artifact sources
Physik Instruments, Nanopositioning 1998
SPECIMEN
DEFORMATION &
THERMAL
FLUCTUATIONS
Hoh, et al. Biophys. J. 1998, 75, 1076.
D+DD
d
-Z D
L+DL
~
+Z
L
voltage
applied
-X
+Y
+X
ADHESION FORCE
Yang, et al. Ultramicroscopy 1993, 50, 157
Force, F (nN)
PIEZO AMPLIFIER, SENSOR AND
CONTROL ELECTRONICS,
MECHANICAL PARAMETERS
y
Distance, D (nm)
x
connecting
wires
polarization
PROBE TIP SHARPNESS
CANTILEVER
THERMAL NOISE
Lindsay Scanning Tunneling Microscopy
and Spectroscopy 1993, 335.
kt
Shao, et al. Ultramicroscopy 1996,
=
66, 141.
cantilever
Sheng, et al. J. Microscopy 1999, 196, 1.
m
dt(max)
dt(max)
Fadhesion
0
z
electrodes
0
m
m
(*http://cnst.rice.edu/pics.html
Lieber, et al., 2000)
AFM: Advantages as tool to assess biological responses
Biological Applications: AFM Images of Cells
Contact mode image of human red blood cells - note cytoskeleton is
visible. blood obtained from Johathan Ashmore, Professor of Physiology
University College, London. A false color table has been used here, as
professorial blood is in fact blue. 15µm scan courtesy M. Miles and J.
Ashmore, University of Bristol, U.K.
Red Blood Cells
Shao, et al., : http://www.people.virginia.edu/~js6s/zsfig/random.html
Rat Embryo Fibroblast(*M. Stolz,C.
Radmacher, et al., Cardiac Cells
Schoenenberger, M.E. Müller Institute,
http://www.physik3.gwdg.de/~radmacher/
Biozentrum, Basel Switzerland)
Height image of endothelial cells taking in fluid using Contact
Mode AFM. 65 µm scan courtesy J. Struckmeier, S. Hohlbauch, P.
Fowler, Digital Intruments/Veeco Metrology, Santa Barbara, USA.
Biological Applications: Manipulation of Living Cells
• rest cantilever on top of cell and monitor cantilever deflection up and down = beating of cell
d>0
d=0
d<0
rest
position
cardiac cell
• I. confluent layer of cells : beat regularly in terms of frequency and amplitude, enormous
stability of pulsing, cell are synchronized and coupled together : diverse pulse shapes due to
macroscopic moving centers of contraction and relaxation
• II. individual cell : sequences of high mechanical activity alternate with times of quietness,
irregular beating which often last for minutes, active sequences were irregular in frequency and
amplitude
• III. group of cells: “pulse mapping”
Biological Applications: AFM Images of DNA
TappingMode image of nucleosomal DNA was the highlight Image of PtyrTlac supercoiled DNA. 750 nm
of the "Practical Course on Atomic Force Microscopy in
scan courtesy C. Tolksdorf, Digital
Biology," held at the Biozentrum in Basel, Switzerland, July Instruments/Veeco, Santa Barbara, USA, and R.
1998. Image courtesy of Y. Lyubchenko.
Schneider and G. Muskhelishvili, Istitut für
Genetik und Mikrobiologie, Germany.
http://www.people.virginia.edu/~js6s/zsfig/DNA.html
AFM image of short DNA fragment with RNA
The high resolution of the SPM is able to discern
polymerase molecule bound to transcription
very subtle features such as these two linear dsDNA
recognition site. 238nm scan size. Courtesy of
molecules overlapping each other. 155nm scan. Bustamante Lab, Chemistry Department, University
Image courtesy of W. Blaine Stine
of Oregon, Eugene OR
AFM: From Nano to MicroStructures
Human hair (C. Ortiz)
Eggshell