3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C.

Ortiz, MIT-DMSE
I
LECTURE 5: AFM IMAGING
Outline :
LAST TIME : HRFS AND FORCE-DISTANCE CURVES .......................................................................... 2
ATOMIC FORCE MICROSCOPY : GENERAL COMPONENTS AND FUNCTIONS................................. 3
Deflection vs. Height Images ................................................................................................... 4
3D Plots and 2D Section Profiles ............................................................................................ 5
Normal Modes of Operation ..................................................................................................... 6
Other Modes of Operation........................................................................................................ 7
Factors Affecting Resolution .................................................................................................... 8
Tip Deconvolution .................................................................................................................... 9
Imaging of Cells ..................................................................................................................... 10
Imaging of DNA...................................................................................................................... 11
HRFS COMBINED WITH AFM : SPATIALLY SPECIFIC SURFACE INTERACTIONS........................... 12

Objectives: To review the basic principles, capabilities, and current state of the art uses of the
atomic force microscopy

Readings: Course Reader Document 12-15.

Multimedia : Watch Introduction to AFM by Asylum Research, Inc. (Quicktime Movie) for Lectures
4-5.

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3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C. Ortiz, MIT-DMSE

HIGH RESOLUTION FORCE SPECTROSCOPY (HRFS) EXPERIMENT : FORCE-

DISTANCE CURVES
approaching
(=cantilever deflection)

Force, F (nN)
A. G. repulsive
1=0
tip and sample tip and sample regime
out of contact out of contact

B.
attractive F. kc
interaction tip releases attractive
2<0
pulls tip down from surface regime
towards surface

E. 0
3<0
C. attractive force
tip jumps
to surface
keeps tip in
contact with
Tip-Sample Separation
surface Distance, D (nm)
4>0 D. - Conversion of raw data; sensor output, s (Volts) vs. z-
D. tip and sample piezo displacement/deflection, (nm) to Force, F, versus
tip and sample / z-piezo tip-sample separation distance, D :
/ z-piezo move in unison =s/m
move in unison
m= slope in constant compliance regime =s/ (V/nm)
retracting F=k
-Normal vs. Lateral Force Microscopy D= z
-zeroing the baseline

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3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C. Ortiz, MIT-DMSE

ATOMIC FORCE MICROSCOPY : GENERAL COMPONENTS AND FUNCTIONS

laser diode sensor output, , F -Piezo rasters or scans in the x-y
mirror A B direction across the sample surface
C D
cantilever position sensitive
spring which deflects as probe tip
scans sample surface photodetector
measures deflection of -Cantilever deflects () in response to
cantilever
an a topographical feature (hill or
probe tip valley)
senses surface
10-15 properties and causes ERROR =
cantilever to deflect
sample
actual signal - set point Feedback loop

computer -System continuously changes in

controls system
performs data acquisition, response to an experimental output
display, and analysis
(= cantilever deflection which is the
feedback parameter)
piezoelectric z feedback loop
scanner y controls z-sample -Computer adjusts the piezo z-
positions sample x
position
displacement to keep constant =
(x, y, z) with
accuracy
"setpoint"

Advantages : 1) Unlike electron microscopes, samples do not need to be coated
or stained, minimal damage, 2) Unlike electron microscopes, samples can be Error signal (actual signal-set point)
imaged in fluid environments (near-physiological conditions), 3) Unlike STM used to produce a topographical
samples do not need to be conductive, 4) Sub-nm resolutions have been (height) map in the z-direction of the
achieved on biological samples (detailed information on the molecular surface
conformation, spatial arrangement, structural dimensions, rate dependent
processes, etc.)

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3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C. Ortiz, MIT-DMSE

ATOMIC FORCE MICROSCOPY : DEFLECTION VS HEIGHT IMAGES

Two images removed due to copyright restrictions. See Tai and Ortiz, Nano Letters 2006.

Deflection Image: Height Image:

-Raw data output of cantilever deflection from
photodiode, very clear, the less feedback the clearer,
One can identify and measure high resolution (x/y)
spatial dimensions of structural features
-Processed data from z-piezo, less clear
compared to deflection image, maximize your
feedback system, can quantify the height of
structural features, in 2D image corresponds to
brightness

(e.g. images are a large residual nanoindent of bone using an instrumented indenter and Berkovich diamond probe
showing plastic deformation of mineralite nanogranular structure, K. Tai and C. Ortiz Nano Letters, 2006)

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3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C. Ortiz, MIT-DMSE

ATOMIC FORCE MICROSCOPY : 3D PLOTS AND 2D SECTION PROFILES

z (m)
7
1.5
6
Two images removed due to
copyright restrictions. See Tai and
5
1.0
Ortiz, Nano Letters 2006 4

Z[]
3
0.5 2

0 0
0 1 2 3 4 5 6 7
0 2 4 6 8 10
x X[m]
(m)
3D Height image
2D Height image
2D Section Profile
-Select linear region of plot and plot 2D section profile (height along line) z vs. x to get quantitative mathematical
functional form of topography. For example, we can see the profile of the deformation of indent plus form of plastically
deformed "pileup" regions" one can use these profiles in conjunction with modeling to extract out material properties
such as modulus, yield stress, anisotropy, and strain hardening behavior.

-In the next assignment, you will have to use a section profile on nanoparticles to estimate the probe tip radius.

(e.g. images are a large residual nanoindent of bone using an instrumented indenter and Berkovich diamond probe
showing plastic deformation of mineralite nanogranular structure, K. Tai and C. Ortiz Nano Letters, 2006)

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3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C. Ortiz, MIT-DMSE

ATOMIC FORCE MICROSCOPY IMAGING : NORMAL MODES OF OPERATION

Contact (DC and AC or Force Modulation) :

tip remains in contact with sample
feedback signal, c
potentially destructive due to high lateral
Force (x/y) forces
high resolution

Intermittent Contact (AC, Tapping) Mode :

Intermittent-contact tip is oscillated near its resonant frequency
and "taps" on and off the surface
feedback signal, oscillation amplitude or
phase
less destructive due to reduction of lateral
contact forces
Distance (tip-to-sample separation)
loss of spatial resolution

Noncontact (AC) Mode :

non-contact tip is oscillated near its resonant frequency
without touching the surface
feedback signal, oscillation amplitude
nondestructive
loss of spatial resolution
difficult in practice
Graph by MIT OCW.
Schematic after
Thermomicroscopes
"Introduction to AFM."

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3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C. Ortiz, MIT-DMSE

ATOMIC FORCE MICROSCOPY IMAGING : OTHER MODES OF OPERATION

I. Normal Force Microscopy

Contact DC and AC
Intermittant Contact/
(Force Modulation
Tapping / Lift (AC) Noncontact (NC)
Microscopy (FMM), Phase 1995
Imaging) Hansma, et al., 1991 Hansma, et al., 1994

II. Friction or Lateral

Force Microscopy (FFM/
LFM) Frisbie, et al., 1994
Courtesy of Veeco Instruments. Used with permission.
http://www.di.com/AppNotes/LatChem/LatChemMain.html
III. Force / Volume
Adhesion Microscopy
chemically
chemicallyfunctionalized X Radmacher, et al., 1994
X probe tip: X
X X=-OH,-CH3,-NH2
X
X Courtesy of Veeco Instruments. Used with permission.
X X
http://www.di.com/AppNotes/ForceVol/FV.array.html
X X
X X Surface Maps:
X X Topography & Roughness, Electrostatic Interactions, Friction
X X X X X X IV. Chemical Force Chemical, Adhesion , Hardness, Elasticity /Viscoelasticity
Microscopy (CFM) Dynamic Processes :
Frisbie, et al., 1994 Erosion, Degradation, Protein-DNA Interactions

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3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C. Ortiz, MIT-DMSE

ATOMIC FORCE MICROSCOPY IMAGING : FACTORS AFFECTING RESOLUTION

PIEZO AMPLIFIER, SENSOR AND SPECIMEN
CONTROL ELECTRONICS, DEFORMATION & ADHESION FORCE
Yang, et al. Ultramicroscopy 1993, 50, 157
MECHANICAL PARAMETERS THERMAL
Physik Instruments, Nanopositioning 1998
FLUCTUATIONS
Hoh, et al. Biophys. J. 1998, 75, 1076.

Force, F (nN)
D+D

d
-Z D 0
L+L
~
+Z L
voltage
applied Fadhesion
-X +Y +X
z 0
y
electrodes
x Distance, D (nm)
connecting
wires
polarization

PROBE TIP SHARPNESS

CANTILEVER Sheng, et al. J. Microscopy 1999, 196, 1.

THERMAL NOISE
Lindsay Scanning Tunneling Microscopy
and Spectroscopy 1993, 335. k
t
Shao, et al. Ultramicroscopy 1996, Image removed due to Image removed due to copyright restrictions.
66, 141. = copyright restrictions.
m 3-D model of sharp probe tip on a protein,
cantilever from Lieber et al, 2000 (http://cnst.rice.edu)
t(max) m

t(max) m

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3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C. Ortiz, MIT-DMSE

ATOMIC FORCE MICROSCOPY IMAGING : TIP DECONVOLUTION

-Imaging very sharp vertical surfaces is influenced by the sharpness of the tip. Only a tip with sufficient sharpness can
properly image a given z-gradient. Some gradients will be steeper or sharper than any tip can be expected to image
without artifact. False images are generated that reflect the self-image of the tip surface, rather than the object surface.
Mathematical methods of tip deconvolution can be employed for image restoration. The effectiveness of these methods
will depend on the specific characteristics of the sample and the probe tip.

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 3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C. Ortiz, MIT-DMSE

ATOMIC FORCE MICROSCOPY IMAGING OF CELLS

Contact mode image of human red blood cells - note cytoskeleton
is visible. blood obtained from Johathan Ashmore, Professor of Red Blood Cells
Physiology University College, London. A false color table has been Shao, et al., : http://www.people.virginia.edu/~js6s/zsfig/random.html
used here, as professorial blood is in fact blue. 15m scan courtesy
M. Miles and J. Ashmore, University of Bristol, U.K.

Courtesy of Mervyn
J. Miles. Used with
permission.

Courtesy of Manfred
Radmacher. Used with
permission.
Rat Embryo Fibroblast (*M. Stolz,C. Height image of endothelial cells taking in fluid using Contact Mode AFM. 65
Radmacher, et al., Cardiac Cells m scan courtesy J. Struckmeier, S. Hohlbauch, P. Fowler, Digital
Schoenenberger, M.E. Mller Institute,
http://www.physik3.gwdg.de/~radmacher/ Intruments/Veeco Metrology, Santa Barbara, USA.
Biozentrum, Basel Switzerland)

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 3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C. Ortiz, MIT-DMSE

ATOMIC FORCE MICROSCOPY IMAGING OF DNA

Tapping Mode image of nucleosomal DNA.
Courtesy of Yuri Lyubchenko. Used with permission.
Image of PtyrTlac supercoiled DNA. 750 nm scan
courtesy C. Tolksdorf, Digital Instruments/Veeco,
Santa Barbara, USA, and R. Schneider and G.
Muskhelishvili, Istitut fr Genetik und
Mikrobiologie, Germany.

Curtesy of Veeco Instruments and http://people.virginia.edu/~zs9q/zsfig/DNA.html

G. Muskhelishvili. Used with permission. Courtesy of Zhifeng Shao. Used with permission.

AFM image of short DNA fragment with RNA

The high resolution of the SPM is able to polymerase molecule bound to transcription
discern very subtle features such as these recognition site. 238 nm size. Courtesy
two linear dsDNA molecules overlapping of Bustamante Lab. Chemistry Department.
each other. 155nm scan. University of Oregon, Europe OR
Courtesy of Zhifeng Shao. Used with permission.
Courtesy of W. Blaine Stine. Used with permission. Courtesy of Prof. Carlos Bustamante.
Used with permission.

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3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07 Prof. C. Ortiz, MIT-DMSE

HRFS COMBINED WITH AFM : SPATIALLY SPECIFIC SURFACES INTERACTION

INFORMATION

0.4
1 m Position 1
Position 2 4
Position 3

Force/Radius (mN/m)
0.3 Position 4
Position 5
Grain 2 Grain 3 Position 6 3

Force (nN)
5 Position 7
6
4 0.2
3 7 2
2
1 0.1 1

0.0 0
Grain 1
0 5 10 15
Distance from Surface (nm)
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.

AFM can be combined with high resolution force spectroscopy and nanoindentation since cantilever probe tip can be
employed for both imaging and nanomechanical measurements nanomechanical measurements with positional
accuracy down to the nanoscale (Vandiver, et al. Biomaterials 26 (2005) 271283).

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