Scanning Probe Microscopies
Outline
• Microscopies Length Scales.
• System requirements for scanning
probe microscopies.
– Piezo issues.
– Image processing.
• Near Field Scanning Optical Microscope
(NSOM).
• Scanning Tunneling Microscope (STM)
• Atomic Force Microscope (AFM).
Overview: Microscopy Techniques
Characteristics of Common Microscopy Techniques
Light Microscopy Scanning Electron
(LM)
Microscopy (SEM)
Scanning Probe
Microscopy (SPM)
Sample Operating
Environment
Ambient
Liquid
Vacuum
Vacuum
Ambient
Liquid
Vacuum
Depth of Field
Small
Large
Medium
Depth of Focus
Medium
Small
Small
Resolution (x,y)
1.0 micron
5 nm
0.1 - 3.0 nm
Resolution (z)
N/A
N/A
0.1 nm
Magnification
Range
1X - 10 3X
10X - 10 6X
5 x 10 2X - 10 8X
Sample
Preparation
Little
Freeze drying, coating
None
Sample
Requirements
Sample must not
Surface must not build up
be completely
charge and sample must be
transparent to light
vacuum compatible
wavelength used
Sample must not have
excessive variations in
surface height (~5 micron)
Basic Principles
&
Hardware
Principles of SPM
Images acquired from Park Scientific Instruments
Hardware Requirements
• Coarse positioning system for probe approach.
– Stepper motor.
• Fine positioning system for sample imaging.
– Piezo electric crystal.
• Probe position sensor
– Tuning fork (NSOM).
– Laser (AFM).
– Tunneling current (STM/AFM).
• Feedback controller for probe position.
– PI controller
• Data acquisition system and GUI.
Piezo Issues: Scanners
Calibration Grid
10µm squares
• piezocrystals
– high precision positioning
• non-linear response
– creep, hysteresis
– cross-coupling, resonances
Adapted from: http://www.park.com
• software/hardware
correction
Calibration: Piezo Nonlinearities
Intrinsic Nonlinearity
Creep
Hysteresis
Aging
Nonlinearity plots adapted from Park Scientific Instruments
Calibration: Piezo Nonlinearities
Cross Coupling
Piezo Nonlinear Effects Combined
Nonlinearity plots adapted from Park Scientific Instruments
NT-MDT TGX01 Calibration Grid
Software Solutions:
Calibration grating for lookup table
Modeling of PZT non-linear response
Corrections dependent on speed/direction
Error up to 10% when scan conditions change
Hardware Solutions:
External sensors monitor scanner’s position
Total nonlinearity less than 1%
Image Processing
• Piezo nonlinearities necessitate flattening.
– Line by line fit polynomial.
– Fit surface to plane.
– Fourier methods.
• Thresholding for counting particles.
– User set.
– Maximum entropy.
• Roughness.
• Facet orientation.
• Tip reconstruction.
• Polymer contours.
Near Field Scanning Optical
Microscopy
NSOM
NSOM
• Resolution of optical
microscope limited
to ~ 1/2λ.
• By passing light
through a hole <λ
and collecting
detected light an
image is
constructed.
http://physics.nist.gov/Divisions/Div844/facilities/nsom/nsom.html
NSOM
• Shear Force Mode.
– Tuning fork oscillates probe & amplitude reduction
on approach to surface is controlled.
• Transmission mode.
– Light passed through sample -> transparent sample.
• Reflection mode.
– Light reflected off the sample -> opaque sample.
• Luminescence mode.
– Light passed through sample and filtered.
NSOM
http://physics.nist.gov/Divisions/Div844/facilities/nsom/Fig5.jpg
DNA Imaged by Shear Force
NSOM
DNA plasmid pGem7zf+ (Promega) 3000 b. p. linearized with the SmaI
endonuclease deposited on freshly cleaved mica. DNA01 test sample was
measured by Solver P47H using the Shear Force head. Humidity - 1-10%.
http://www.ntmdt.com/Scan-gallery/SNOM/index.html
Scanning Tunneling Microscopy
STM
We have experienced a revolution in surface analysis since the advent of the STM...
In 1973, a Nobel Prize in Physics was given to Esak, Giaever and Josephson for their studies
on electron tunneling.
in 1978,
H. Rohrer - a staff researcher at IBM Zurich
G. Binnig - Graduate Student (Frankfurt) came to work with Rohrer
January, 1979 - Binnig did some calculations:
The Original
Hypothesis
The Revised
Hypothesis
r = 1000Å
tunneling current falls
off with distance
between the surface
and the tip
45Å
1Å
STM
•Calculations suggested that the tunneling current would only come
from a 45Å area because of the exponential decay with distance.
•March 18, 1981 - the first traces recorded on an x-y chart recorder
•Soon after, Binnig and Rohrer note atomic sized features (versus the
45Å resolution they thought they had!).
•Revised Hypothesis - small protuberances on the tip of their probe,
not the tip radius, led to enhanced spatial resolution
(Take note of the willingness to discard an unsatisfactory hypothesis and to develop a new
hypothesis to explain the results. This "willingness" led to a Nobel Prize, 1986.)
Appropriate to a Nobel Prize…
The central concept nucleated a world of variants:
• Scanning Tunneling Microscopy (STM)
• Atomic Force Microscopy (AFM)
• Friction/Lateral Force (FFM/LFM)
• Electromagnetic modes (EFM/MFM/ECFM)
• AC techniques
– Force modulation (FMAFM)
– Resonant drive (Tapping, Intermittant and Noncontact)
• Surface indenters, nanolithography
• Near-field optics (NSOM)
• Chemical sensing (SFM-CS)
• Also, NMR, thermal, optical, spectroscopy
STM
• 1981: Vacuum tunneling observed.
– (Binnig, Rohrer, Gerber, Weibel; IBM
Zurich)
• 1986: Nobel prize award to Binnig and
Rohrer.
• The STM can create an image of:
– the local electron density of states
– contains topographic information
– contains spectroscopic information
– contains tip shape information
STM Schematic
•
A simplification:
– Quantum tunneling between
two metallic electrodes,
separated by a vacuum region.
•
J α exp(-Ak0d)
– J = tunneling current
– d = distance, Å; A = area
– k0 = range of wavefunction out
of solid (average inverse decay
length)
k0
•
Adapted from: http://www.park.com
=
1/2 (φ1 + φ 2)
φ1 + φ 2 = work functions of two metals
For a general work function: the
tunneling current decreases about
an order of magnitude for every
angstrom ofseparation.
STM Scan Modes
Can be used to
track surfaces
that are not
atomically flat
Useful for
rapid scanning
(since only
the electronics
must respond -not the piezo)
based upon: Hansma and Tersoff, J. Appl. Phys. 61(2), A1, (1987)
Some STM Issues
• Density of states vs. true topography.
• Wire tips: platinum + rhodium/iridium, tungsten.
– mechanically cut, electrochemically etched, ion milled.
– tip shape is always convoluted into the image.
• Conductive sample required in most cases.
– can image insulators by working at very low tunneling
currents (≈1pA), and high bias (1-5 V).
• Imaging in conductive liquids (ie. saline) requires
tip coating with insulator.
• Interaction force decays by a factor of 2 slower
than the tunneling current.
Graphite Observed by STM
Graphite is one of the easiest materials to observe by STM
C
C
C
C
C
d = 2.4Å
C
The six carbons making up the ring are not equivalent
(3 of the carbons have neighboring atoms immediately below)
By STM, three carbons in each ring will appear lower -actually they are not -- they just have different electron densities
The tunneling current is proportional to the surface electric charge density (s.e.c.d.).
Normally, the s.e.c.d. is where the atoms are, not not always!
REMEMBER: STM measures electron density -- not topography
Highly ordered
pyrolytic graphite (HOPG)
a=2.46Å
STM and Surface Science
•
Pb on Cu(100)
•
Disordered surface alloy
found at low coverages of Pb.
•
Deposition of 3:8 Pb:Cu
surface atomic ratio results
in a well-ordered surface
structure.
•
Structural model:
– bright "walls" of the maze
are rows of Pb atoms
– rows of Cu atoms between
them (dark and
unresolved in STM
images).
•
Taken from:
http://www.iap.tuwien.ac
at/www/surface/STM_Gallery
/Pb_on_Cu.html
Gold Surfaces
imaged with STM
atomic resolution
300°C, 7 min.
sputtered
annealed
4.47x4.47x 0.5 nm
677x677x 60 nm
677x677x 20 nm
Heights of features are coded in a scale from the
lightest color to the darkest.
Slide by Thomas Boland
STM Fancy Stuff
•
D.M. Eigler, E.K. Schweizer. Positioning single
atoms with a scanning tunneling microscope. Nature
344, 524-526 (1990).
•
Xenon on Nickel (110)
Adapted from: http://www.almaden.ibm.com/st/
•
M.F. Crommie, C.P. Lutz, D.M. Eigler, E.J.
Heller. Waves on a metal surface and quantum
corrals. Surface Review and Letters 2 (1), 127-137
(1995).
•
Iron on Copper (111)
STM Spectroscopy
• Current vs. Position spectroscopy I(z).
– Determines tip quality.
– Work function calculations.
• Current vs. Voltage spectroscopy I(V).
– High vacuum study of semiconductors.
Atomic Force Microscopy
AFM
Atomic Force Microscopy
Split Photodiode
Detector
Laser
Feedback Control
Y
Sample
Z
X
Piezoelectric Drive
Cantilever
Force versus Displacement
Curves
Approach
E
Retract
F
D
B
Force
A
I
C
H
Position
G
A Cantilever approaches surface
(thermal noise)
B Snap to contact, tip is on
surface (Hamaker constant)
C “Attractive” work
D Constant compliance region
(Surface stiffness)
E Hysteresis (viscous damping)
F Retracting slope (surface
stiffness)
G “Adhesion” work (energy to
separate surfaces)
H Last bond(s) to break (“single
bond forces”)
I Cantilever is off surface
AFM Cantilevers
Small Cantilever
9-50µm long
3-5 µm wide
Traditional Cantilever
Image from Hansma Group - UCSB
Calibration: Tip Characterization
Adapted from Park Scientific Instruments
Tip Characterization using NT-M DT TGT01
Undefined tip shape results in:
Unknown degree of feature dilation
Lack of quantitative ability without
tip radius (R)
Tip characterization solutions:
Tip characterizer grids
Blind tip reconstruction (Villarubia, 1997)
Substitution with colloidal sphere
Tip Shape Artifacts
Cantilever Calibration
• Many methods
– large scale spring
– 1/2kbT=1/2k<x2>
– added mass Q shift
– k=kbT/<x2>
– thermal noise
– k=2w(πlfo)3(ρ3/E)1/2
(Å^2)
0.2
• Thermal Noise
displacement
0.4
K (N/m)
0.3
0.1
y = 2E -15x + 0.0882
2
R = 0.9379
0.2
k<P >
Linear (k<P >)
rms
0.1
0
0
2E +13
4E +13
6E +13
8E +13
1E +14
FR EQUENCY^3 (Hz^3)
0
1.0E+04
1.5E+04
Frequency (Hz)
2.0E+04
2.5E+04
1.2E +14
1.4E +14
SFM: Interaction Regimes
• Forces Between Tip and Surface
– Attractive Forces
• Van der Waals
• Electrostatic (hydrogen bonding)
• Quantum mechanical
• Hydrophobic
• Ion correlation
• Solvation (hydration force)
• Specific binding
– Repulsive Forces
• Quantum mechanical (Core-core)
• Van der Waals disjoining pressure
• Electrostatic
• Solvation (hydration force)
Image acquired from Park Scientific Instruments
• Entropic (steric forces, doublelayer forces)
– Dynamic Interactions
• Hydrodynamic forces
• Viscous forces
• Friction forces
Lateral Force Mode
Photodiode detection
• Measures torsional
deflections from forces
parallel to surface
• Frictional contrasts due to:
– Interaction with different
materials
– Change in height (artifact)
• Trace and retrace should
be monitored to distinguish
friction from height
• Semi-quantitative
Images acquired from Park Scientificmethod
Instruments and DI
– Torsional spring constant difficult
Scanning Force Microscopy:
Pulsed Force Mode
• Pulsed Force Mode Operation
– Simultaneous acquisition of
topography, qualitative
stiffness, semiquantitative
adhesion
– Piezo modulated 100 Hz - 2 kHz,
amplitude of 10 - 500 nm
– Complete force-distance cycle
at repetition rate
Topography
Stiffness
Adhesion
– Peak pickers and lock-in
amplifiers used to extract
essential data, resulting in
reduced data set
– Non-destructive imaging
technique used for polymers and
soft samples
Tapping/Intermittent Contact
(IC-AFM)
• Tip “taps” on sample (or hammers?)
• Little sample degradation
• Penetrates water layer
Magnetic AC Mode (MAC
Mode)
• Oscillation magnetically induced
• Frequency and amplitude
controlled
• Works well in fluids
• Lower interaction forces
Adapted from M olecular Imaging
The oscillating magnetic field below
the cantilever drives it directly.
DMPC liposomes in pH 7.0 buffer
From Molecular Imaging
FN on Mica 1mg/ml 1min in PBS, Imaged in
Air MAC mode, Scan Size 2000 nm x 2000
nm
Imaging in Water – increasing the Q
factor
Frequency spectrum of a typical
magnetically driven cantilever
Imaging DNA
Z range 3nm
Q factor = 3
3µm
3µm
Q factor = 300
Images courtesy Andy Round, University of Bristol
Phase Imaging
• With any of above modulated modes
• Phase lag varies in response to surface
mechanical properties
Topography
Phase
Two Phase structure in a polymer blend
Images from DI
Living Cell Imaging
Topographic image
Z range 2.5 µm
Simultaneous Phase Image
z-range 60 degrees
Images taken with an effective quality factor of 300.
Image is 32 x 32 microns
Images courtesy Rachel Owen, University of Bristol
Masking techniques for
measuring layer thicknesses
Apply mask
Apply coating
Remove mask
Masks from PDLA in acetone or polystyrene in toluene
PG Hartley et al (2000) Plasmas and Polymers
Plasma polymer thickness
DLVO Theory and Force
Curves
I nter actio n Energy
Double Layer Repulsion
Separation (nm)
Total
van der Waals Attraction
Scanning Force Microscopy:
DLVO Analysis of Force Curve Data
Derjaguin-Landau-Verway-Overbeek Theory:
FDLVO ( z ) = Fel ( z ) + FvdW ( z ) =
R = probe radius
k = Boltzmann constant
qi = component ionic valency
ε0 = permittivity of free space
Theory:
(EDL)
4&R%s%p
"e
$e$ 0
!
z
#d
+
! AR
6z
2
#D =
z = probe-sample separation
ci = component concentration
σs = surface charge density (sample)
σp = surface charge density (probe)
" 0" e kT
e 2 ! ci qi2
= Debye length
A = Hamaker constant
T = temperature
e = unit charge
εe = electrolyte dielectric permittivity
Ion cloud accumulates at surface to shield charge -> electric double layer
Electric double layers overlap as surfaces approach -> EDL force (Fel)
Van der Waals interaction provides strong attraction at small distances (FvdW)
Assumptions: Interaction of spherical probe with flat plane
Force curve is performed under aqueous conditions
Derjaguin approximation valid only if R>>z
Quantitation: Poisson-Boltzmann equation used to fit Fel(z)
Debye length describes thickness of EDL
Analysis typically performed by external software
Yields semi-quantitative model of the force curve
Colloid Probes
Solutions for DLVO theory
assume sphere on flat
One component of the
solution – Derjaguin
approximation is only valid
if R>>z
Courtesy of Pat Hartley, CSIRO M olecular Science, Australia
R = radius of sphere
z =SEM
tip-sample
Sphere radius measured by
or opticalseparation
microscopy
Polysaccharide Grafting
1. AApp
Poly(ethyleneimine)
Acetaldehyde
plasma polymer
H
NH
O H
C
2. Graft PEI
NH
NH
NH
O
C
NaCNBH3
Reductive Amination
Substrat
e
-
3. Graft CMD
(carboxymethyl dextran)
Vary COO- density
-
COO-
-
COO-
-
-
COO-
- - - - COOCOO- - COO-
Substrate
Substrat
e
Carbodiimide
EDC/NHS
Chemistry
EDC/NHS
Electrostatic interactions
Silica probe vs grafted polyethyleneimine
0.1
F/R (mN/m)
0.05
0.15M NaCl
0
-0.05
-0.1
0.0015M NaCl
-0.15
-0.2
0
10
20
30
Separation (nm)
40
50
60
Scanning Force Microscopy:
Hertzian Analysis of Force Curves
Hertzian Theory: Assumes elastic interaction between probe and sample.
Assumes no adhesion in the contact regime.
Used to determine sample stiffness.
Modeled here with a rigid conical tip (other geometries may be used).
z ! z0 = d ! d 0 +
k tan(" )
d ! d0
K
z = sample height
z0 = sample height at contact
k = cantilever spring constant
K = reduced elastic modulus
θ = opening angle between tip and surface
d = cantilever deflection
d0 = equilibrium deflection at contact
Range of analysis:
Domke & Radmacher, 1998
Necessary when soft samples are analyzed (z0 = ??)
Generates two equations for the two unknowns (K, z0)
Hertzian Analysis
Force Distance Curves
Indentation-Loading Force
Curves (Converted from
Force Distance Curves)
7.5
Silicon
Indentation
5.5
14
PPNIPA
M 37ºC
3.5
1.5
12
PPNIPAM
25ºC
-0.5
-20
-15
-10
-5
0
5
10
Indentation (nm)
Cantilever Deflection (nm)
9.5
37ºC
25ºC
10
8
6
4
2
Z-piezo Displacement (nm)
0
0.00
10
0.05
0.10
0.15
0.20
Indentation (nm)
Loading Force (nN)
37ºC
25ºC
1
0.01
0.1
Loading Force (nN)
Fit to
Hertz
Model
0.25
0.30
Tip Functionalization
Tip Functionalization Strategies:
• Alkylsilanes
• Alkanethiols
• RF-glow discharge
React with X
• LB film deposition
XXXXXXXXXXXXX
X
• Biomolecule physisorption
XXXXXXXX
X
• Sphere gluing
X X
Imaging contrasts due to:
X
• Hydrophobic/hydrophilic interactions
• Van der Waals forces
X
• Friction differences
X
X
• Specific recognition events
X
X X X
attach X labeled bead
PDMS Contamination on AFM tips
(Lo et. al, 1999)
NH2
NH2
Tip Functionalization
OH
DMSO
O
HO
overnight
Ethanolamine HCl
S
S
O
O
29
O
NH2
O
29
CHCl3
O
N
O
O
O
O
N
O
N
S
N
S
NEt3
O
SSIMS imaging of modified
AFM cantilevers
Biosensors and DNA arrays
1. Functionalize AFM tip with PNA
2. Look at force interactions with a
Alkane thiol monolayer
Biosensors and DNA arrays
3. Hybridize with target DNA
or mutant DNA
4. Re-measure surface forces
Analysis of Pull-off Forces
Analyze distribution of pull-off forces
a = PNA-modified tip
b = PNA/DNA modified tip
c = PNA/DNA mutant modified tip
Able to detect single base mismatch in the analyte DNA
Lioubashevski et al (2001) Langmuir ASAP
Single Molecule Force Microscopy
• Effects of Mechanical vs Chemical Stresses on proteins
Force
• Protein structure and unfolding – Titin module
Data courtesy of Dr. J. Clarke, S. Fowler and A. Steward of Cambridge University, UK.
Effects of Mutations on Protein
Stability
Point mutations
in IgG I27 module
All substitutions Pro
Mechanical stability at
A pulling rate of 0.6 nm/s
Li et al Nat. Struc. Biol (2000) 7 1117
Dip Pen Nanolithography
Image courtesy of Joseph Wei
Still More ….
Magnetic Force Microscopy (MFM)
Kelvin Probe Microscopy (SKM)
Electrostatic force microscopy (EEM)
Scanning Capacitance Microscopy (SCM)
Scanning Acoustical Microscopy
Scanning Calorimetry Microscopy
Magnetic forces
Surface Potential
Static charges
Capacitance
Acoustical waves
Thermal conductivity
Site
Link List
URL
Advanced Surface Microscopy
http://www.ai.com/asm
Digital Instruments
http://www.di.com/Biblio/biobib.html
Blaine's SPM Page
http://www.mcs.com/~wbstine/spm/spm.html
Burleigh
http://www.burleigh.com
Digital Instruments
http://www.di.com
JEOL
http://www.jeol.com
Molecular Imaging
http://www.molec.com
Nanosensors
http://www.nanosensors.com
NT-MDT
http://www.ntmdt.com
Olympus
http://www.olympus.co.jp/LineUp/Technical/Cantilever/levertopE.htm
Omicron
http://www.omicron-instruments.com/
A Practical Guide to Scanning Probe Microscopy
http://www.thermomicro.com/spmguide/contents.htm
RHK
http://www.rhk-tech.com/main/pages/inddiv.html
Scanning and Local Probe Technique Links
http://www.embl-heidelberg.de/~altmann/
ThermoMicroscopes
http://www.thermomicroscopes.com/
Witec
http://www.witec.de