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

2. 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,   force, F z-piezo deflection, z  tip-sample separation distance, D Typical force spectroscopy data for a weak cantilever on stiff substrate (k sample >> k cantilever ) : 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 (  s/  z=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  s/m D=z  A B/C D D E F G A DD E F G *Note: For an adhesive interaction F = k 

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 Non-Contact (AC) Intermittent Contact : Tapping (AC)

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

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

11. AFM : Normal Force Spectroscopy Modes of Operation

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

13. INTERVAL Au COATING : homogeneous, smoother smaller polydomain microstructure Si chip Si 3 N 4 cantilever TOP VIEW AFM: Tip Functionalization ONE-TIME Au COATING : heterogeneous, rougher larger polydomain microstructure 1. Gold coating Purpose: Methods: 100 nm SIDE VIEW 100 nm SIDE VIEW TOP VIEW

14. microfabricated Si 3 N 4 probe tip - - - - - - - - - - - - - - - - - + + + + + + synthetic polymers polyelectrolytes self-assembling monolayer ligands proteins 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 AFM: Tip Functionalization 2. Chemical coating Purpose: Methods: Applications: antibodies http://www.di.com/AppNotes/LatChem/LatChemMain.html

15. (c) (d) nanotube with individual ligand (a) Benoit, M.; Gabriel, D.; Gerisch, G.; Gaub, H. E. Nature Cell. Bio 2000, 2 (6), 313. (b) Ong, Y-L.; Razatos, A.; Georgiou, G.; Sharma, M. K. Langmuir 1999, 15, 2719. (c) J. Seog, Ortiz/ Grodzinsky Labs 2001 (d) Wong S.S.; Joselevich E.; Woolley, A.T.; Cheung, C. L.; Lieber, C. M. Nature 1998, 394 (6688), 52. (a) Single Cell Dictyostelium Discoideum (b) E. Coli Bacteria (c) colloidal particle AFM: Tip Functionalization

16. IV. Chemical Force Microscopy (CFM) Frisbie, et al., 1994 Noncontact (NC) 1995 II. Friction or Lateral Force Microscopy (FFM/ LFM) Frisbie, et al., 1994 I. Normal Force Microscopy III. Force / Volume Adhesion Microscopy Radmacher, et al., 1994 Contact DC and AC (Force Modulation Microscopy (FMM), Phase Imaging): Hansma, et al., 1991 Intermittant Contact/Tapping / Lift (AC): Hansma, et al., 1994 X=-OH,-CH 3, -NH 2 X X X X X X X X X X X X X X X X X X X X Surface Maps: Topography & Roughness, Electrostatic Interactions, Friction Chemical, Adhesion, Hardness, Elasticity /Viscoelasticity Dynamic Processes : Erosion, Degradation, Protein-DNA Interactions AFM: Applications of modes Timeline: http://www.di.com/AppNotes/ForceVol/FV.array.html

17. PROBE TIP SHARPNESS Sheng, et al. J. Microscopy 1999, 196, 1. CANTILEVER THERMAL NOISE Lindsay Scanning Tunneling Microscopy and Spectroscopy 1993, 335. Shao, et al. Ultramicroscopy 1996, 66, 141. PIEZO AMPLIFIER, SENSOR AND CONTROL ELECTRONICS, MECHANICAL PARAMETERS Physik Instruments, Nanopositioning 1998 SPECIMEN DEFORMATION & THERMAL FLUCTUATIONS Hoh, et al. Biophys. J. 1998, 75, 1076. ADHESION FORCE Yang, et al. Ultramicroscopy 1993, 50, 157 (*http://cnst.rice.edu/pics.html Lieber, et al., 2000) AFM: Resolution factors/Artifact sources

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. Rat Embryo Fibroblast(*M. Stolz,C. Schoenenberger, M.E. Müller Institute, Biozentrum, Basel Switzerland) 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. Red Blood Cells Shao, et al., : http://www.people.virginia.edu/~js6s/zsfig/random.html Radmacher, et al., Cardiac Cells http://www.physik3.gwdg.de/~radmacher/

20. 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” Biological Applications: Manipulation of Living Cells

21. 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 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. 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 Biological Applications: AFM Images of DNA

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