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

download 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 10-2

If you can't read please download the document

  • date post

    31-Mar-2018
  • Category

    Documents

  • view

    214
  • download

    2

Embed Size (px)

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

  • 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 30m

    Red BloodCell 5m

    Bacteria 1mHuman Hair 75m

    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 BC 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.

    OShea 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 NH2NH2

  • 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

    F

    F

    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 transducerTransducer-shakes the cantilever holder Reduction of amplitude signals contact

    Forest of Peaks in Liquids

    Cantilever is coated with magnetic filmMagnetic 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 ?