AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD...
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Transcript of AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD...
AFM Imaging In Liquids
W. Travis Johnson PhDAgilent TechnologiesNanomeasurements Division
10-10 10 -9 10-8 10-7 10-6 10-5 10-4 10-3 10-2 10-1 1MeterMillimeterMicronNanometer
Cell 30μm
Red BloodCell 5μm
Bacteria 1μmHuman Hair 75μm
Proteins10 nm
Virus 50nm
Optical Microscope
Angstrom
Near Field OpticalTEM
Si AtomSpacing 0.4nm
Ant 5mmDNA 2nm
SEM
Imaging Techniques: ScalesImaging Techniques: Scales
AFM
Z
XY
AFM Basic ConfigurationAFM Basic ConfigurationA B
C D
Liquid Cell and Perfusion (flow through)Cell
Intermolecular/Atomic Interactions Important in AFM
Ionic Interactions (charge-charge)• Si AFM probes (SiO-)• SiN AFM probes (SiO- and NH3
+)VDW interactions (“electrostatic” between uncharged molecs)• Induced Dipole (aka Dispersion or London Forces; temp., induced &
fluctuating dipoles)• Dipole-Dipole (perm dipoles)• VDW repulsion (VDW radius)
– Atoms near end of probe vs atoms further awayHydrophobic (nonpolar; decay exp with distance)Surface tension (meniscus forces, capillary; in air)Can Modify Ionic, VDW, Hydrophobic Properties• Perform Chemistry on AFM probe (coat with hydrophobic or charged
silanes)
Distance (tip position)
Dipole-DipoleAttractions
VDWRepulsion
contact
non-contact
VDW Interactions: AFM Probe VDW Interactions: AFM Probe -- SampleSampleCantilever
Deflection in
Force (N) or
Voltage (V)
Adhesion greatly exaggerated
WDW radius
Additional Forces in Liquid
H Bonding (RO…..H, RS…..H, RN…..H)Ionic Interactions Change (salt bridges)Hydrodynamic Forces• Viscous damping
– Viscosity & Probe Geometry– Increase in Effective Mass– Res Freq Reduction & Peak Broadening
• Compression Repulsive Force– Viscosity in Confined Areas (Viscoelasticity)
Liquid Lowers Q Factor• AFM Probe Sensitivity & Responsiveness• Q100 Air ~ Q2-3 Liquid• Low Q Damage Soft Samples• Q Further Decreases as Tip SurfaceThermal Fluctuations (< noise)Oscillatory Forces (Fliquid) • Smooth Surfaces (liquid order, packing)• Rough Surfaces (liquid asymmetry)• Can Cause Artifacts• Size of the Liquid Molecules Liquid New Freqs Arise for Dynamic (AC) Imaging Modes
Oscillatory Forces & Viscosity
AFM tip immersed in liquid. In high res imaging, force between tip-sample (Fimage) should be small. Solvent forces (Fliquid) also interact between tip and sample.
AFM tip surface roughness can disrupt tip-liquid-surface interface. Roughness introduces disorder, reduces packing and helps to minimize oscillatory forces. Smaller solvent molecules also increase averaging.
O’Shea Jpn. J. Appl. Phys. 40 (2001) 4309
Force curve showing increase in viscosity of confined liquid between tip and sample. As tip approaches surface, viscosity increases.
Immobilization Chemistry is Critical for Samples in Immobilization Chemistry is Critical for Samples in Liquid (e.g., Biological Samples)Liquid (e.g., Biological Samples)
SiOH
O O SiSi Si NH2
OEtEtO
OEt
EtOH
SiO O SiSi
Si NH2
OEt
OEtO
OH OH
OH OH
++++++
SiO O SiSi
SiOEt
OEtO N H
OH O
H H
OO
OHOH
Glut-Mica
micaAPTES
Glutaraldehyde
Covalent Protein Immobilization on
MicaProteins (lysine groups)
Electrostatic Attachment
Negatively charged mica
positively charged protein
NH2
NH2
NH2
NH2 NH2
NH2
Dynamic in x and yTip in contact or near contact with surfaceSmall vertical forceProbe exerts lateral forces on sampleDamage to soft samplesWeakly bound samples move which lowers lateral resolution
X
X
y
Topography (height) Deflection (error)
Data Channels (typical)Topography – Signal representing the voltage applied to Z piezo in order to maintain constant force between tip and sample.Deflection – Servo input or “error” signal which shows difference between setpoint and actual deflection. Shows edges clearly
Contact Mode Imaging
200 nm
Deflection Images of Live BCE CellDeflection Images of Live BCE Cell
100 um25 um
HIGH FORCELOW FORCE
Piezoelectric transducer shakes cantilever holder at or near resonant frequency of cantileverDynamic in x, y, & zIntermittent contactInteraction with sample reduces oscillation amplitude. Reduction in amplitude used as feedback signalSoft surfaces stiffened by viscoelastic response Impact forces predominately verticalLarge vertical force, small lateral forceHigher lateral resolution than contact mode
AC Mode Imaging
XX
yz
~ V
Short Range Interactions (Attraction/Repulsion) Short Range Interactions (Attraction/Repulsion) Effect Resonance FrequencyEffect Resonance Frequency
Attractive
Repulsive
Attractive gradient is equivalent to additional spring tension on tip, which reduces
resonance frequency of cantilever
Repulsive gradient is equivalent to additional spring
compression on tip, which increases resonance frequency
of cantilever
ΔF
ΔF
AttractiveRepulsive
Phase ImagingPhase ImagingDrive Signal Detected Signal
Before contact
Contact with hard material dampens amplitude but has little effect on phase
Contact with soft material effects phase and amplitude
Phase lag related to material stiffness
Topography image of block copolymer
Phase image ofblock copolymer
500nm
500nm
AC AFM Data Channels (typical)• Topography – Derived from voltage applied to Z piezo needed to
keep oscillation amplitude constant
• Amplitude – Error signal from photo detector that shows the change in amplitude
• Phase – Phase lag of cantilever response with respect to drive signal
AC Mode images of inner surfaceof blood vessel in buffer
AmplitudeTopography Phase
Image courtesy of Nanotechnology Center of Tsinghua University
AC AFM in LiquidAC AFM in LiquidAAC Mode
MAC Mode®
•A solenoid applies an AC field to piezoelectric transducer•Transducer-shakes the cantilever holder •Reduction of amplitude signals contact
Forest of Peaks in Liquids
•Cantilever is coated with magnetic film•Magnetic field drives cantilever ONLY
Cleaner Oscillation in Liquids
Human Rhino Virus 2 (HRV2)Human Rhino Virus 2 (HRV2)20 nm70 nm300 nm
Kienberger et al., Single Mol. 2 (2001) 99
HRV2
Absorbed on Mica/NiCl
10 nm
0 1.5 nm
Protein Loops
In situ Experiment:In situ Experiment:RNA Release from HRV2RNA Release from HRV2
pH= 4.1
2 hours
300 nm 300 nm
Kienberger et al., J. Virol. 78 (2004) 3203900 nm
Thank YouThank You
Questions ?Questions ?