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

Transcription

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

2 Imaging Techniques: Scales Proteins 10 nm Bacteria 1μm Red Blood Cell 5μm Human Hair 75μm Si Atom Spacing 0.4nm DNA 2nm Virus 50nm Cell 30μm Ant 5mm Angstrom Nanometer TEM AFM Micron Millimeter Meter Near Field Optical Optical Microscope SEM

3 A C B D AFM Basic Configuration Z Y X

4 Liquid Cell and Perfusion (flow through) Cell

5 Intermolecular/Atomic Interactions Important in AFM Ionic Interactions (charge-charge) Si AFM probes (SiO - ) SiN AFM probes (SiO - and NH 3+ ) 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 away Hydrophobic (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)

6 VDW Interactions: AFM Probe - Sample Cantilever Deflection in Force (N) or Voltage (V) VDW Repulsion contact non-contact Distance (tip position) WDW radius Dipole-Dipole Attractions Adhesion greatly exaggerated

7 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 Surface Thermal Fluctuations (< noise) Oscillatory Forces (F liquid ) 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 Additional Forces in Liquid

8 Oscillatory Forces & Viscosity Force curve showing increase in viscosity of confined liquid between tip and sample. As tip approaches surface, viscosity increases. O Shea Jpn. J. Appl. Phys. 40 (2001) 4309 AFM tip surface roughness can disrupt tip-liquidsurface interface. Roughness introduces disorder, reduces packing and helps to minimize oscillatory forces. Smaller solvent molecules also increase averaging. AFM tip immersed in liquid. In high res imaging, force between tip-sample (F image ) should be small. Solvent forces (F liquid ) also interact between tip and sample.

9 Immobilization Chemistry is Critical for Samples in Liquid (e.g., Biological Samples) OH OH OH OEt Si O Si O Si + EtO Si NH 2 OEt APTES mica EtOH Electrostatic Attachment positively charged protein OEt EtO Si NH 2 OH O OH Si O Si O Si Negatively charged mica O O H H Glutaraldehyde EtO Si N H OH Si OEt O OH O Si O Si OH Glut-Mica O H 2 N H 2 N H 2 N NH 2 H 2 N NH 2 Proteins (lysine groups) Covalent Protein Immobilization on Mica

10 Contact Mode Imaging y X Dynamic in x and y Tip in contact or near contact with surface Small vertical force Probe exerts lateral forces on sample Damage to soft samples Weakly bound samples move which lowers lateral resolution X 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

11 Deflection Images of Live BCE Cell LOW FORCE HIGH FORCE 200 nm 100 um 25 um

12 AC Mode Imaging z y X X Piezoelectric transducer shakes cantilever holder at or near resonant frequency of cantilever Dynamic in x, y, & z Intermittent contact Interaction with sample reduces oscillation amplitude. Reduction in amplitude used as feedback signal Soft surfaces stiffened by viscoelastic response Impact forces predominately vertical Large vertical force, small lateral force Higher lateral resolution than contact mode ~ V

13 Short Range Interactions (Attraction/Repulsion) Effect Resonance Frequency Attractive gradient is equivalent to additional spring tension on tip, which reduces resonance frequency of cantilever ΔF Attractive Repulsive Attractive ΔF Repulsive Repulsive gradient is equivalent to additional spring compression on tip, which increases resonance frequency of cantilever

14 Phase Imaging Drive Signal Detected Signal Before contact 500nm Contact with hard material dampens amplitude but has little effect on phase Topography image of block copolymer Contact with soft material effects phase and amplitude Phase lag related to material stiffness 500nm Phase image of block copolymer

15 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 Topography Amplitude Phase Image courtesy of Nanotechnology Center of Tsinghua University AC Mode images of inner surface of blood vessel in buffer

16 AC AFM in Liquid AAC 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 MAC Mode Cantilever is coated with magnetic film Magnetic field drives cantilever ONLY Cleaner Oscillation in Liquids

17 Human Rhino Virus 2 (HRV2) 300 nm 70 nm 20 nm Protein Loops 10 nm nm HRV2 Absorbed on Mica/NiCl Kienberger et al., Single Mol. 2 (2001) 99

18 In situ Experiment: RNA Release from HRV2 ph= hours 300 nm 300 nm 900 nm Kienberger et al., J. Virol. 78 (2004) 3203

19 Thank You Questions?