AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD...

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AFM Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division

Transcript of AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD...

Page 1: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

AFM Imaging In Liquids

W. Travis Johnson PhDAgilent TechnologiesNanomeasurements Division

Page 2: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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

Page 3: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

Z

XY

AFM Basic ConfigurationAFM Basic ConfigurationA B

C D

Page 4: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

Liquid Cell and Perfusion (flow through)Cell

Page 5: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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)

Page 6: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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

Page 7: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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

Page 8: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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.

Page 9: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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

Page 10: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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

Page 11: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

200 nm

Deflection Images of Live BCE CellDeflection Images of Live BCE Cell

100 um25 um

HIGH FORCELOW FORCE

Page 12: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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

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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

Page 14: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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

Page 15: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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

Page 16: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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

Page 17: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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

Page 18: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

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

Page 19: AFM Imaging In Liquids - Chemical Analysis, Life … Imaging In Liquids W. Travis Johnson PhD Agilent Technologies Nanomeasurements Division 10-10 10-9 10-8 10-7 10-6 10-5 10-4 10-3

Thank YouThank You

Questions ?Questions ?