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Atomic Force Microscopy – More Than Microscopy

报告题目： Atomic force microscopy more than microscopy ( 原子力显微镜 – 不仅仅是显微镜) 报 告 人：陈新镛 教授 University of Nottingham. Atomic Force Microscopy – More Than Microscopy. Xinyong Chen Laboratory of Biophysics and Surface Analysis School of Pharmacy The University of Nottingham.

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Atomic Force Microscopy – More Than Microscopy

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  1. 报告题目：Atomic force microscopy more than microscopy (原子力显微镜 – 不仅仅是显微镜) 报 告 人：陈新镛 教授University of Nottingham

  2. Atomic Force Microscopy– More Than Microscopy Xinyong Chen Laboratory of Biophysics and Surface Analysis School of Pharmacy The University of Nottingham

  3. Outline • Force measurement • Force/distance (f/d) • Two-dimensional Force measurement • AC mode “force” measurement • Amplitude/distance (a/d) • Phase/distance (p/d) • Tapping mode imaging and a-p/d • Other force-related afm techniques • Force-modulation imaging • Lateral-force (friction) imaging • Lateral resonance (AC mode lateral force)

  4. Force measurement

  5. Force/distance curves Point of maximum load c Contact region Deflection/Force b a Maximum adhesion force d Scanner Displacement (nm)

  6. Retract Experimental f/d curves Approach

  7. AFM probe Lactose Salbutamol Measurement of particle-particle interaction Force (nN) Increase in strength of interaction with salbutamol in air Salbutamol Lactose 1µm 60000 Lactose 50000 PTFE Salbutamol 40000 PTFE Average Adhesion Force/nN 30000 0 250 500 20000 Distance (nm) 10000 0 Tip 3 Tip 1 Tip 2 International Journal of Pharmaceutics238 (2002) 17-27 Single particle interaction between salbutamol, lactose and PTFE

  8. Adhesion between a solid sphere and a solid plane (Hertz Model) • JKR (Johnson, Kendall, Roberts – 1964-1971) model: • DMI (Derjagin, Muller, Toropov – 1975) model: • Maugis(1992): R F Stiff materials (DMT) l  0 Compliant materials (JKR) l   With complex and implicit parameter equations

  9. AFM Cantilever and Probe Ferritin Anti - Ferritin Antibody Substrate Specific interaction

  10. 1.6 1.2 0.8 Approach Force (nN) Retract 0.4 0 -25 25 75 125 175 -0.4 Distance moved by the Cantilever (nm) Antigen-antibody interaction 1.4 1 Approach 0.6 Force (nN) Retract 0.2 -0.2 -0.6 -25 25 75 125 175 Distance moved by the Cantilever (nm) Biochemistry, 36, 7457 – 7463(1997)

  11. Multiple link in tip/surface modification Force versus probe-sample separation curves for a BBSA coated probe and (a) a streptavidin coated silicon surface, (b) a streptavidin functionalised surface after incubation in a solution of the bis-biotinylated peptide, and (c) the streptavidin/bis-biotinylated peptide/streptavidin multilayer. The Analyst, 2000, 125(2), 245 - 250

  12. Force curve of chain-like molecules

  13. Force measurement of RNA molecules Examples of the types of force curves observed between RNA functionalized AFM surfaces and tips; each curve has been corrected to show force against tip-sample displacement. In each example the approach trace is shown in gray and the retract trace in black. (A) No interaction has occurred between the tip and substrate. (B) A nonspecific interaction between the two surfaces, with no observable change in the gradient of the retraction slope. (C) A specific interaction between the two RNA functionalized surfaces, showing a stretch of ~10 nm before the rupture event. Biophysical Journal 86:3811-3821 (2004)

  14. Representative force-extension traces for DNA molecules of three different lengths Biochem. Soc. Trans. (2003) 31, (1052–1057)

  15. AFM Cantilever and Probe Ferritin Anti - Ferritin Antibody Substrate Antigen-antibody interaction - Single antigen-antibody bond?

  16. Antigen-antibody interaction- Single antigen-antibody bond? Analysis of the distribution of adhesive forces (n = 140) obtained with three functionalized AFM probes. Each data point represents an individual force measurement performed at any position on the sample surface. The force distribution data is plotted, using data bin sizes from 15 to 35 pN, as a stacked area graph (smallest bin size plotted lowermost). In this way, common features of the distributions are reinforced by the stacking process. A quantization in the forces, with a period of 49 ± 10 pN, is suggested by this analysis. Biochemistry, 36, 7457 – 7463(1997)

  17. PEG attached Antibody Mono-functional Spacer PEG molecules Covalently attached hCG Silicon Substrate “Spacers” – a way to measure single molecular interaction

  18. 2.5 2.5 (a) (b) 2 2 1.5 1.5 Approach Approach Force (nN) 1 1 Force (nN) Retract Retract 0.5 0.5 0 0 0 50 100 150 0 50 100 150 -0.5 -0.5 -1 -1 Distance Moved by the Cantilever (nm) Distance moved by the Cantilever (nm) (c) (d) 1.4 8 1.2 1 6 Force (nN) Approach 0.8 Approach Force (nN) 4 Retract Retract 0.6 0.4 2 0.2 0 0 0 50 100 150 200 0 100 200 300 -0.2 -2 Distance Moved by the Cantilever (nm) -0.4 Distance Moved by the Cantilever (nm) Force measurements between PEG-antibody coated probes and bhCG coated surfaces Low probability in detecting single molecular pair interaction!

  19. Antibody-antigen interaction using polymeric linkers AFM probe Polymeric linker e.g. agarose, PEG Antigen Biotinylated antibody Streptavidin PLA-PEG-Biotin

  20. Exploring surface hardness Hertz Model: Deflection on hard surfaces (no indentation) F Z crystalline budesonide Lactose DZ: Indentation on soft surfaces

  21. Functionalized probe Array of Potential Interaction Sites Specific Molecular Interaction Searching for specificinteraction sites: Two dimensional force measurement

  22. Force-Volume (FV) mode

  23. AFM probe A A Salbutamol Scan probe whilst recording adhesion events B B Lactose 1µm FV measurement of salbutamol particles on lactose-coated surfaces Force distance curves extracted from the marked locations on the force volume image. The Salbutamol particle has greater adhesion to the lactose carrier at position B.

  24. 500 nm BSA Buffer PS Si Topography Adhesion FV measurement on BSA partially coated surfaces with a BSA-coated tip Langmuir 13, 4106 (1997)

  25. FV measurement on BSA partially coated surfaces with a BSA-coated tip:Time evolution Dynamic observation of BSA adsorption onto a polystyrene surface. Retract f-d curves were measured on a polystyrene spin-cast surface with a BSA-coated probe after the surface was exposed to a BSA solution (0.01 mg/mL, pH 7, 100 mM phosphate buffer) for 0 min (I), 4 min (II), 5 min (III), 7 min (IV), 10 min (V), and 60 min (VI) without movement of the sampling location. Langmuir 13, 4106 (1997)

  26. Maximum force (Topography) Stiffness Adhesion Sample: a silicon surface AFM tip: silicon, 4 N/m PF frequency: 905Hz Pulsed-Force AFM Topography Tip oscillation Adhesion Drive

  27. Height Adhesion Stiffness 1 mm PF-AFM images of PLMA/PmMl6 blend in water PLMA: poly (lauryl methacrylate) PmMl6: 2-methacryloyloxyethyl phosphorylcholine-co-lauryl methacrylate (1:6)

  28. Polystyrene Albumin PF-AFM images of a polystyrene surface partially coated with albumin Buffer Albumin 5 mm Height Adhesion

  29. PS Si PF-AFM images of a polystyrene surface partially coated with fibrinogen Fibrinogen Fibrinogen Polystyrene pH7 Phosphate Buffer 1 mm Height Adhesion

  30. Outline • Force measurement • Force/distance (f/d) • Two-dimensional Force measurement • AC mode “force” measurement • Amplitude/distance (a/d) • Phase/distance (p/d) • Tapping mode imaging and a-p/d • Other force-related afm techniques • Force-modulation imaging • Lateral-force (friction) imaging • Lateral resonance (AC mode lateral force)

  31. Force measurement in AC mode

  32. gel mica PS mica gel PS A-p/d curves measured on different material surfaces

  33. Phase imaging

  34. 250 nm Dark phase Tapping mode images of gelatine on a mica surface Height Phase Ultramicroscopy 75, 171(1998)

  35. 500 nm Light phase Tapping mode images of gelatine on a polystyrene surface Height Phase Ultramicroscopy 75, 171(1998)

  36. Phase imaging and a-p/d curves Ultramicroscopy 75, 171(1998)

  37. Energy dissipation model (J. Cleveland et al., 1998)     =    +     = – J.P. Cleveland et al., Appl. Phys. Lett. 72 (1998) 2613. Physical meaning of phase contrast Free of tip/sample interactions: f = 90

  38. Height Phase Height Phase TM-AFM imaging of an EDMPC partially-coated mica surface

  39. Height Phase Physical meaning of phase contrast Bistable status of tapping tips

  40. Outline • Force measurement • Force/distance (f/d) • Two-dimensional Force measurement • AC mode “force” measurement • Amplitude/distance (a/d) • Phase/distance (p/d) • Tapping mode imaging and a-p/d • Other force-related afm techniques • Force-modulation imaging • Lateral-force (friction) imaging • Lateral resonance (AC mode lateral force)

  41. Force-modulation imaging

  42. FM-AFM images of a two-phase block copolymer Height Force Modulation 900nm scans Veeco Instruments www.veeco.com

  43. Lateral-Force AFM

  44. Lateral-Force AFM Height Lateral Force

  45. HOPG surface imaged with interleaved TappingMode height (left) and TRmode phase (right). 3µm scan. (Supplied by Veeco.) Lateral resonance (LR) AFM

  46. Acknowledgements • Dr Ardeshir Danesh, • Dr Giles Sanders, Dr Jianxin Zhang, Dr Wenke Zhang • Prof Martyn Davies, Prof Saul Tendler, Prof Clive Roberts, Dr Phil Williams, Dr Steph Allen • NT-MDT • http://www.ntmdt.ru/ 王宽诚教育基金会 Funding: BBSRC, EPSRC, RSPGB, GSK, Pfizer Global R&D, Nektar, Molecular Profiles.

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