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.
1
3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07
Prof. C. Ortiz, MIT-DMSE
HIGH RESOLUTION FORCE SPECTROSCOPY (HRFS) EXPERIMENT : FORCEDISTANCE CURVES
(δ=cantilever deflection)
A.
tip and sample
out of contact
B.
attractive
interaction
pulls tip down
towards surface
C.
tip jumps
to surface
D.
tip and sample
/ z-piezo
move in unison
δ1=0
δ2<0
δ3<0
δ4>0
G.
tip and sample
out of contact
F.
tip releases
from surface
E.
attractive force
keeps tip in
contact with
surface
D.
tip and sample
/ z-piezo
move in unison
retracting
-Normal vs. Lateral Force Microscopy
repulsive
regime
kc
attractive
regime
Force, F (nN)
approaching
0
Tip-Sample Separation
Distance, D (nm)
- Conversion of raw data; sensor output, s (Volts) vs. zpiezo displacement/deflection, δ (nm) to Force, F, versus
tip-sample separation distance, D :
δ=s/m
m= slope in constant compliance regime =Δs/Δδ (V/nm)
F=kδ
D= z±δ
-zeroing the baseline
2
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
mirror
A B
C D
cantilever
sensor output, δ, F
↓
position sensitive
photodetector
• spring which deflects as probe tip
scans sample surface
δ
≈10°-15°
-Cantilever deflects (δ) in response to
an a topographical feature (hill or
valley)
• measures deflection of
cantilever
probe tip
• senses surface
properties and causes
cantilever to deflect
-Piezo rasters or scans in the x-y
direction across the sample surface
ERROR =
actual signal - set point
sample
computer
↓ Feedback loop
-System continuously changes in
response to an experimental output
(δ= cantilever deflection which is the
feedback parameter)
• controls system
• performs data acquisition,
display, and analysis
piezoelectric z
y
scanner
• positions sample
x
feedback loop
• controls z-sample
position
(x, y, z) with Å
accuracy
Advantages : 1) Unlike electron microscopes, samples do not need to be coated
or stained, minimal damage, 2) Unlike electron microscopes, samples can be
imaged in fluid environments (near-physiological conditions), 3) Unlike STM
samples do not need to be conductive, 4) Sub-nm resolutions have been
achieved on biological samples (detailed information on the molecular
conformation, spatial arrangement, structural dimensions, rate dependent
processes, etc.)
-Computer adjusts the piezo zdisplacement to keep δ constant =
"setpoint"
↓
Error signal (actual signal-set point)
used to produce a topographical
(height) map in the z-direction of the
surface
3
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:
-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
Height Image:
-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)
4
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 (μm)
1.5
7
6
Two images removed due to
copyright restrictions. See Tai and
Ortiz, Nano Letters 2006
5
Z[Å]
1.0
4
3
0.5 2
1
0
0
0
0
3D Height image
1
2
2
4
3
x X[µm]
(μm)
4
6
5
8
6
7
10
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)
5
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, δc
● potentially destructive due to high lateral
(x/y) forces
● high resolution
Force
Intermittent-contact
contact
Distance (tip-to-sample separation)
non-contact
Graph by MIT OCW.
Schematic after
Thermomicroscopes
"Introduction to AFM."
Intermittent Contact (AC, Tapping) Mode :
● 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
forces
● loss of spatial resolution
Noncontact (AC) Mode :
● tip is oscillated near its resonant frequency
without touching the surface
● feedback signal, oscillation amplitude
● nondestructive
● loss of spatial resolution
● difficult in practice
6
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)
1995
Microscopy (FMM), Phase
Hansma,
et
al.,
1994
Imaging) Hansma, et al., 1991
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
probe tip:
X=-OH,-CH3,-NH2
X
X
X
X
X
X
X
X X X X X X
X
X
X
X
X
X
Radmacher, et al., 1994
X
Courtesy of Veeco Instruments. Used with permission.
http://www.di.com/AppNotes/ForceVol/FV.array.html
Surface Maps:
IV. Chemical Force
Microscopy (CFM)
Frisbie, et al., 1994
Topography & Roughness, Electrostatic Interactions, Friction
Chemical, Adhesion , Hardness, Elasticity /Viscoelasticity
Dynamic Processes :
Erosion, Degradation, Protein-DNA Interactions
7
3.052 Nanomechanics of Materials and Biomaterials Thursday 02/22/07
Prof. C. Ortiz, MIT-DMSE
Hoh, et al. Biophys. J. 1998, 75, 1076.
D+ΔD
d
-Z D
L+ΔL
~
+Z
L
voltage
applied
-X
+Y
+X
Force, F (nN)
ATOMIC FORCE MICROSCOPY IMAGING : FACTORS AFFECTING RESOLUTION
SPECIMEN
PIEZO AMPLIFIER, SENSOR AND
ADHESION FORCE
DEFORMATION &
CONTROL ELECTRONICS,
Yang, et al. Ultramicroscopy 1993, 50, 157
MECHANICAL PARAMETERS
THERMAL
Physik Instruments, Nanopositioning 1998
FLUCTUATIONS
y
Distance, D (nm)
x
connecting
wires
polarization
PROBE TIP SHARPNESS
CANTILEVER
THERMAL NOISE
Sheng, et al. J. Microscopy 1999, 196, 1.
Lindsay Scanning Tunneling Microscopy
and Spectroscopy 1993, 335. k
t
Shao, et al. Ultramicroscopy 1996,
=
66, 141.
Image removed due to
copyright restrictions.
m
cantilever
Fadhesion
0
z
electrodes
0
δt(max)
δt(max)
Image removed due to copyright restrictions.
3-D model of sharp probe tip on a protein,
from Lieber et al, 2000 (http://cnst.rice.edu)
m
m
8
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.
9
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
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
Courtesy of Mervyn
J. Miles. Used with
permission.
Courtesy of Manfred
Radmacher. Used with
permission.
Radmacher, et al., Cardiac Cells
http://www.physik3.gwdg.de/~radmacher/
Rat Embryo Fibroblast (*M. Stolz,C.
Schoenenberger, M.E. Müller Institute,
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.
10
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 für Genetik und
Mikrobiologie, Germany.
Curtesy of Veeco Instruments and
G. Muskhelishvili. Used with permission.
The high resolution of the SPM is able to
discern very subtle features such as these
two linear dsDNA molecules overlapping
each other. 155nm scan.
http://people.virginia.edu/~zs9q/zsfig/DNA.html
Courtesy of Zhifeng Shao. Used with permission.
AFM image of short DNA fragment with RNA
polymerase molecule bound to transcription
recognition site. 238 nm size. Courtesy
of Bustamante Lab. Chemistry Department.
University of Oregon, Europe OR
Courtesy of W. Blaine Stine. Used with permission.
Courtesy of Prof. Carlos Bustamante.
Courtesy of Zhifeng Shao. Used with permission.
Used with permission.
11
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 μm
Position 1
Position 2
Position 3
Position 4
Position 5
Position 6
Position 7
3
2
1
Grain 1
5
Force (nN)
4
Grain 3
6
7
3
0.2
2
0.1
1
0.0
0
0
5
10
Distance from Surface (nm)
Force/Radius (mN/m)
0.3
Grain 2
4
15
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) 271–283).
12