3.052 Nanomechanics of Materials and Biomaterials Tuesday 02/27/07
Prof. C. Ortiz, MIT-DMSE
I
LECTURE 6: AFM IMAGING II :
ARTIFACTS AND APPLICATIONS
Outline :
LAST TIME : BASIC PRINCIPLES OF ATOMIC FORCE MICROSCOPY ................................................ 2
FACTORS AFFECTING RESOLUTION .................................................................................................... 3
Tip Deconvolution .................................................................................................................... 4
IMAGING OF CELLS ................................................................................................................................. 5
IMAGING OF DNA .................................................................................................................................... 6
HRFS COMBINED WITH AFM : SPATIALLY SPECIFIC SURFACE INTERACTIONS............................. 7
Objectives: To review artifacts present in atomic force microscopy imaging and current state of the
art uses.
Readings: Course Reader Document 12-15.
Multimedia : Listen to "Structured Water" Podcast corresponding to journal article : Higgens, et al.
Biophys. J. 2006 91, 2532.
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3.052 Nanomechanics of Materials and Biomaterials Tuesday 02/27/07
Prof. C. Ortiz, MIT-DMSE
ATOMIC FORCE MICROSCOPY : GENERAL COMPONENTS AND FUNCTIONS
laser diode
mirror
A B
C D
cantilever
sensor output, δ, F
-Basic principles
position sensitive
photodetector
• spring which deflects as probe tip
scans sample surface
δ
probe tip
• senses surface
properties and causes
cantilever to deflect
≈10°-15°
-Deflection vs. Height images
• measures deflection of
cantilever
-2D Section Profiles
ERROR =
actual signal - set point
sample
-3D Images
-Normal modes of operation (Contact
AC/DC, Tapping AC, Noncontact AC)
computer
• controls system
• performs data acquisition,
display, and analysis
piezoelectric z
y
scanner
• positions sample
x
feedback loop
• controls z-sample
position
-Other modes of operation (LFM,
CFM, Force/Volume)
-DEMOs
(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.)
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3.052 Nanomechanics of Materials and Biomaterials Tuesday 02/27/07
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. kt
Shao, et al. Ultramicroscopy 1996,
=
66, 141.
Image removed due to
copyright restrictions.
m
cantilever
Fadhesion
0
z
electrodes
0
δt(max)
δt(max)
m
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
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3.052 Nanomechanics of Materials and Biomaterials Tuesday 02/27/07
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.
probe tip
probe tip
scan direction
square nanoobject
2D Height Profile
θ
θ
z
w/2
substrate
x
height would
theoretically be
accurate
2D Height
Profile
r*
tan( θ) =
x
→ x = wtan( θ)
w
w
+ wtan( θ)
2
r* 1
= + tan( θ)
w 2
-Deep feature
-Depth underestimated
-Need high aspect ratio probe tip.
r* =
-tip broadening-width is overestimated → mention pset
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3.052 Nanomechanics of Materials and Biomaterials Tuesday 02/27/07
Prof. C. Ortiz, MIT-DMSE
AFM IMAGING OF BIOLOGICAL MACROMOLECULES: DNA
Image of PtyrTlac supercoiled DNA. 750 nm scan
courtesy C. Tolksdorf, Digital Instruments/Veeco,
Tapping Mode image of nucleosomal DNA.
Courtesy of Yuri Lyubchenko. Used with permission. Santa Barbara, USA, and R. Schneider and G.
Muskhelishvili, Istitut für Genetik und
Mikrobiologie, Germany.
Courtesy of Veeco Instruments and
G. Muskhelishvili. Used with permission. Courtesy of Zhifeng Shao. Used with permission.
http://people.virginia.edu/~zs9q/zsfig/DNA.html
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.
Courtesy of W. Blaine Stine. Used with permission.
AFM image of short DNA fragment with RNA
polymerase molecule bound to transcription
recognition site. 238nm scan size. Courtesy
of Bustamante Lab, Chemistry Department,
University of Oregon, Eugene OR
Courtesy of Prof. Carlos
Bustamante. Used with permission.
Courtesy of Zhifeng Shao. Used with permission.
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3.052 Nanomechanics of Materials and Biomaterials Tuesday 02/27/07
Prof. C. Ortiz, MIT-DMSE
HRFS COMBINED WITH AFM : SPATIALLY SPECIFIC SURFACES INTERACTION
INFORMATION
Courtesy Elsevier, Inc., http://www.sciencedirect.com. Used with permission.
(Vandiver, et al. Biomaterials 26 (2005) 271–283).
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3.052 Nanomechanics of Materials and Biomaterials Tuesday 02/27/07
Prof. C. Ortiz, MIT-DMSE
SUPPORTED LIPID BILAYERS
http://faculty.virginia.edu/tamm/pages/project_support.html
Courtesy of Lukas K. Tamm and the Biophysical Society. Used with permission.
See Wagner, M., and L. K. Tamm. Biophysical Journal 79 (2000): 1400-1414.
http://en.wikipedia.org/wiki/Image:Cell_membrane_detailed_diagram.svg
DPPC
Higgens, et al. Biophys. J. 2006 91, 2532.
DOPC
Courtesy of the Biophysical Society. Used with permission.
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3.052 Nanomechanics of Materials and Biomaterials Tuesday 02/27/07
Prof. C. Ortiz, MIT-DMSE
NANOMECHANICS OF SUPPORTED LIPID BILAYERS
Higgens, et al. Biophys. J. 2006 91, 2532.
Courtesy of the Biophysical Society. Used with permission.
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