Linear (k
)
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