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Krystyn J. Van Vliet krystyn@mit 3.052 Spring 2003 March 4, 2003

HIGH-RESOLUTION IMAGING WITH FORCES (ATOMIC FORCE MICROSCOPY). Krystyn J. Van Vliet krystyn@mit.edu 3.052 Spring 2003 March 4, 2003. Review: Typical HRFS output on stiff substrate. Review: Typical HRFS output on stiff substrate. Review: Typical HRFS output on stiff substrate.

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Krystyn J. Van Vliet krystyn@mit 3.052 Spring 2003 March 4, 2003

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  1. HIGH-RESOLUTION IMAGING WITH FORCES (ATOMIC FORCE MICROSCOPY) Krystyn J. Van Vliet krystyn@mit.edu 3.052 Spring 2003 March 4, 2003

  2. Review: Typical HRFS output on stiff substrate

  3. Review: Typical HRFS output on stiff substrate

  4. Review: Typical HRFS output on stiff substrate

  5. Review: Experimental Aspects of Force Spectroscopy Conversion of raw data in a high-resolution force spectroscopy experiment : • sensor output, s  transducer displacement, d force, F • z-piezo deflection, z  tip-sample separation distance, D Typical force spectroscopy data for a weak cantilever on stiff substrate (ksample>> kcantilever) : APPROACH : (*sample and tip come together) • A: tip and sample out of contact, no interaction, cantilever undeflected, zero force (set F=0) • B/C: attractive interaction pulls tip down to surface and tip jumps to contact, cantilever exhibits mechanical instability • D: contact, constant compliance regime, no sample indentation, tip and sample move in unison (Ds/Dz=1) RETRACT :(*sample and tip move apart) • D: repulsive contact, constant compliance regime, tip deflected up • E: attractive force (adhesion) keep tip attached to surface, tip deflected down • F: tip pulls off from surface, cantilever instability • G: same as region A d = s/m F = k d D=zd D D D D A A B/C B/C G F G E E F *Note: For an adhesive interaction

  6. Atomic Force Microscopy Imaging • BASIC PRINCIPLES :  piezo rasters or scans in x/y direction across sample surface  cantilever deflects in response to a topographical feature  computer adjusts the z-piezo distance to keep the cantilever deflection constant and equal to the setpoint value “feedback loop” : system continuously changes in response to an experimental output (cantilever deflection) ERROR SIGNAL = actual signal- set point (*used to produce 2D topographical image in contact mode)

  7. Atomic Force Microscopy: General components and functions cantilever

  8. AFM : Normal Force Spectroscopy Modes of Operation *AC=dynamic(tip is driven to oscillate), DC=static(no external oscillation on tip) Contact (DC and AC) : Force Modulation Intermittent Contact : Tapping (AC) Non-Contact (AC)

  9. AFM : Contact Mode Output: “Isoforce” Height Feedback Error: Deflection http://www.physik3.gwdg.de/~radmacher/publications/osteoblasts.html

  10. AFM : Tapping Mode Feedback Error: Amplitude Additional Feedback: Phase Output: “Isoamplitude” Height Evaporated gold surface

  11. AFM : Normal Force Spectroscopy Modes of Operation

  12. AFM : First high resolution images LAYERED HARD CRYSTALLINE SOLID MATERIALS Highly Oriented Pyrolytic Graphite (HOPG) (http://www.energosystems.ru/fgallery.htm) http://stm2.nrl.navy.mil/how-afm/how-afm.html http://www.physics.sfasu.edu/afm/afm.htm

  13. TOP VIEW 100 nm SIDE VIEW 100 nm SIDE VIEW AFM: Tip Functionalization 1. Gold coating Purpose: Methods: TOP VIEW Si3N4 cantilever ONE-TIME Au COATING : heterogeneous, rougher larger polydomain microstructure INTERVAL Au COATING : homogeneous, smoother smaller polydomain microstructure Si chip

  14. microfabricated Si3N4 probe tip - - - - - - - - - - - - - - - - - + + + + + + AFM: Tip Functionalization 2. Chemical coating Purpose: Methods: Applications: http://www.di.com/AppNotes/LatChem/LatChemMain.html • Molecular Elasticity of Individual Polymer Chains • Protein Folding • DNA Interatomic Bonds • Receptor-Ligand Interactions • Covalent Bonds • Colloidal forces • Van der Waals forces • Hydration forces • Hydrophobic forces • Surface Adhesion • Nanoindentation • Electrostatic DLVO forces • Cell Adhesion • Steric Forces of Polymer Brushes proteins polyelectrolytes self-assembling monolayer ligands antibodies synthetic polymers

  15. AFM: Tip Functionalization (c) (c) colloidal particle (a)Single Cell Dictyostelium Discoideum (d) nanotube with individual ligand (b) E. Coli Bacteria (a) Benoit, M.; Gabriel, D.; Gerisch, G.; Gaub, H. E. Nature Cell. Bio2000, 2 (6), 313. (b) Ong, Y-L.; Razatos, A.; Georgiou, G.; Sharma, M. K. Langmuir1999, 15, 2719. (c) J . Seog, Ortiz/ Grodzinsky Labs 2001 (d) Wong S.S.; Joselevich E.; Woolley, A.T.; Cheung, C. L.; Lieber, C. M. Nature1998, 394 (6688), 52.

  16. AFM: Applications of modes Timeline: Contact DC and AC (Force Modulation Microscopy (FMM), Phase Imaging): Hansma, et al.,1991 Intermittant Contact/Tapping / Lift (AC): Hansma, et al.,1994 Noncontact (NC) 1995 I. Normal Force Microscopy II. Friction or Lateral Force Microscopy (FFM/ LFM) Frisbie, et al.,1994 III. Force / Volume Adhesion Microscopy Radmacher, et al.,1994 Surface Maps: Topography & Roughness, Electrostatic Interactions, Friction Chemical, Adhesion , Hardness, Elasticity /Viscoelasticity Dynamic Processes : Erosion, Degradation, Protein-DNA Interactions X X X X X X X X X http://www.di.com/AppNotes/ForceVol/FV.array.html X X X X X X X X X X X X=-OH,-CH3, -NH2 IV. Chemical Force Microscopy (CFM) Frisbie, et al., 1994

  17. AFM: Resolution factors/Artifact sources SPECIMEN DEFORMATION & THERMAL FLUCTUATIONS Hoh, et al. Biophys. J.1998, 75, 1076. PIEZO AMPLIFIER, SENSOR AND CONTROL ELECTRONICS, MECHANICAL PARAMETERS Physik Instruments, Nanopositioning1998 ADHESION FORCE Yang, et al. Ultramicroscopy1993, 50, 157 (*http://cnst.rice.edu/pics.html Lieber, et al., 2000) PROBE TIP SHARPNESS Sheng, et al. J. Microscopy1999, 196, 1. CANTILEVER THERMAL NOISE Lindsay Scanning Tunneling Microscopy and Spectroscopy1993, 335. Shao, et al. Ultramicroscopy1996, 66, 141.

  18. AFM: Advantages as tool to assess biological responses

  19. Biological Applications: AFM Images of Cells Contact mode image of human red blood cells - note cytoskeleton is visible. blood obtained from Johathan Ashmore, Professor of Physiology University College, London. A false color table has been used here, as professorial blood is in fact blue. 15µm scan courtesy M. Miles and J. Ashmore, University of Bristol, U.K. Red Blood Cells Shao, et al., : http://www.people.virginia.edu/~js6s/zsfig/random.html Height image of endothelial cells taking in fluid using Contact Mode AFM. 65 µm scan courtesy J. Struckmeier, S. Hohlbauch, P. Fowler, Digital Intruments/Veeco Metrology, Santa Barbara, USA. Rat Embryo Fibroblast(*M. Stolz,C. Schoenenberger, M.E. Müller Institute, Biozentrum, Basel Switzerland) Radmacher, et al., Cardiac Cells http://www.physik3.gwdg.de/~radmacher/

  20. Biological Applications: Manipulation of Living Cells • rest cantilever on top of cell and monitor cantilever deflection up and down = beating of cell • I. confluent layer of cells : beat regularly in terms of frequency and amplitude, enormous stability of pulsing, cell are synchronized and coupled together : diverse pulse shapes due to macroscopic moving centers of contraction and relaxation • II. individual cell : sequences of high mechanical activity alternate with times of quietness, irregular beating which often last for minutes, active sequences were irregular in frequency and amplitude • III. group of cells: “pulse mapping”

  21. Biological Applications: AFM Images of DNA Image of PtyrTlac supercoiled DNA. 750 nm scan courtesy C. Tolksdorf, Digital Instruments/Veeco, Santa Barbara, USA, and R. Schneider and G. Muskhelishvili, Istitut für Genetik und Mikrobiologie, Germany. TappingMode image of nucleosomal DNA was the highlight of the "Practical Course on Atomic Force Microscopy in Biology," held at the Biozentrum in Basel, Switzerland, July 1998. Image courtesy of Y. Lyubchenko. http://www.people.virginia.edu/~js6s/zsfig/DNA.html AFM image of short DNA fragment with RNA polymerase molecule bound to transcription recognition site. 238nm scan size. Courtesy of Bustamante Lab, Chemistry Department, University of Oregon, Eugene OR The high resolution of the SPM is able to discern very subtle features such as these two linear dsDNA molecules overlapping each other. 155nm scan. Image courtesy of W. Blaine Stine

  22. AFM: From Nano to MicroStructures Human hair (C. Ortiz) Eggshell

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