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Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
Spm And Sicm Lecture
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Spm And Sicm Lecture

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  • 1. Scanning Probe Microscopy in general  No use of optics.  A probe senses a physical quantity which changes when the probe approaches the sample surface.  Sample or probe is moved by some kind of microactuator. Methods to obtain information:  Constant current mode: the probe is moved at a specified distance above the surface thus following the topology of the specimen. The height dependend signal (current) is kept constant this way. Slow scans, surfaces may be irregular.  Constant height mode: the height of the probe above the suface is fixed. The changes in the signal can be recorded. Fast scans, surfaces should be more even.
  • 2. Scanning Tunneling Microscopy - STM  The scanning probe consists of a  Signal: Tunneling current metallic tip biased with a voltage  Probe: Metallic tip against a conducting sample surface.  Resolution: Down to subÅ  The voltage induces a tunneling  Requisites: Conducting Surface, current between tip and surface. usually UHV  Can be used for microstructuring: by reversing the bias polarity single atoms can be picked up from the surface.
  • 3. Atomic Force Microscopy - AFM  The AFM operates by measuring  Signal: Deflection of cantilever attractive or repulsive forces  Probe: Diamond tip on cantilever between the tip and the sample.  Resolution: Down to 10pm  In ist repulsive contact mode a  Requisites: Regular surface, UHV for detection apparatus measures the high resolutions vertical deflection of the cantilever while it is draged over sthe surface.  In so called non-contact mode, the AFM derives topographic images from measurements of attractive forces. The lever is exited with a vibration at ist resonance frequency. When the tip is now attraced by near atoms (van der Waals forces) the vibration frequenca changes.
  • 4. Other techniques  Friction force microskopy (FFM)  Magnetic force microskopy (MFM)  Electrostatic force microskopy (EFM)  Scanning thermal microskopy (SThM)  Optical absorption microskopy  Scanning acoustic microskopy (SAM)  Molecular dip-stick microskopy  Shear force microskopy (ShFM)  Scanning near-field optical microskopy (SNOM)
  • 5. Patch clamp technique  Patch-clamping is an electro- physiological method used to monitor the ion current of single ion-channels in the membranes of living cells.  Currents are in the pA range – thus they are hard to distinguish from background noise.  Forming of a „Gigaseal“  Various configurations  „loose patch“ configuration is used in the SICM method  Publication: Neher and Sakmann. Die Erforschung von Zellsignalen mit der Patch-Clamp-Technik. Spektrum der Wissenschaft, pages 48–56, May 1992.
  • 6. Scanning ion conductance microscopy - SICM Working conditions:  Isolating samples  Probe: Micropipette  Environment: conductive liquid  Opening diameter of the pipette determines the resolution (500nm-  Atmospheric pressure 20nm)  Ideally suited for living cells.  Measurement of ion currents.  contact free Developed 1989 by Hansma Group, University of Santa Barbara.
  • 7. SICM - Principle  Ion current is flowing between bath electrode and electrode in the pipette.  Approach of the pipette towards the isolating sirface.  current drop  detection of the surface.  Backstepping.
  • 8. SICM – Model  Resistance: R =L/Aκ  Frustrum: RF =Lk/rpriπκ  Hollow cylinder: RH =ln( ro/ri)/2πhκ  Total resistance: RT = RK +RH =U/I  Resolved for the current: I =Uκπ/((Lk/rpri)+ln( ro/ri)/2h)  Saturation current (h  ∞): Isat = lim(Uκπ/((Lk/rpri)+ln( ro/ri)/2h))  =Uκπ/(Lk/rpri)  Normalized quantity: I/ Isat  It is possible to estimate the opening diameter from the measured resistance.
  • 9. SICM – Approaching curves
  • 10. SICM - Setup
  • 11. SICM – Setup description 1. (a) Optical microscope 1. Piezo controller (b) Object table 2. Patch-clamp amplifier (c) Condenser 3. Oscilloscope 2. Micro-manipulator 4. Function generator 3. Piezo-actuator 5. Vibration damping 4. Headstage 6. Connection to PC – data acquisition 5. Pipette holder  Farady cage (not shown) 6. Micropipette  Pipette puller (not shown)
  • 12. SICM – Signal diagram  Pipette movement: Lateral: via Piezo controller (commands over RS232). Vertical: per Modulationvoltage.  Output signal of the EPC7 unit: Proportional to the ion current, signal gets sampled. The vertical piezo position is controlled by a voltage delivered by the analog output of the NI-DAQ card. This method is much faster than the step-by-step method used in the approach function. The controlling voltage is dropped in a slope, thus the pipette is moved towards the surface. While the pipette moves the output of the patch-clamp amplifier (the actual ion-current) is sampled at 1 KHz and analyzed in realtime. An average of 20 samples is taken and compared with the last measurement by the data acquisition hardware. If the difference exceeds a defined ratio, the voltage slope is stopped and the position of the tip is determined by the function readheight.
  • 13. SICM – Manufacturing pipette tips  In principle the required small opening diameters are obtained by heating up a glass tube until it begins to melt. Then a longitudinal force is applied, pulling the tube apart until it is tearing. To get reproducible tips so called pullers are used.  In the puller the clamped glass tube is heated up by a platinum filament or by a laser beam. The force is applied by electromagnets or by gravity. Often the tubes are pulled with varying forces or in several pulling cycles.
  • 14. SICM – Pipette tip SEM
  • 15. SICM - Using the SICM  Fill and mount the tip  Enter liquid and measure saturation current  Find a sample object  Bring the tip into position  Approach the surface  Start scan
  • 16. SICM – Picture of red blood cells
  • 17. SICM – 3D picture
  • 18. SICM - A single cell
  • 19. SICM - Outlook Proposed improvements:  Reprogramming the software  A faster computer  Acquisition of a pipette puller  Use of the computer as function generator  Construction of a perfusion chamber Experiments:  Calibration  Frequency – and step-responses  Manufacturing and behavior of micropipettes  Localization of ion channels
  • 20. Bibliography 1  [Aea88] Alexander and Schneir et al. An atomic resolution afm implemented using an optical lever. Journal of Applied Physics, 65:164–167, 1988.  [AP03] Alexeev and Popkov. Magnetic Force Microscopy. NTMDT, State Institute for Physical Problems, Moscow, 2003. http://www.ntmdt.ru/applicationnotes/MFM/.  [Bea82] Binnig and Rohrer et al. Surface studies by scanning tunneling microscopy. Physical Review Letters, 49:57–61, 1982.  [BQG86] Binnig, Quate, and Gerber. Atomic force microscopy. Phys. Rev. Lett., 56:930–933, 1986.  [BR87] Binnig and Rohrer. Scanning tunneling microscopy – from birth to adolescence. Rev. Mod. Phys., 59:615–625, 1987.  [CGL92] A. Cavali´e, R. Grantyn, and H. D. Lux. Practical Electrophysiological Method, chapter Fabrication of patch clamp pipettes, pages 235–241. Wiley-Liss, New York, 1992.  [Dea89] Drake and Prater et al. Imaging crystals, polymers, and processes in water with the atomic force microscope. Science, 243:1586–1589, March 1989.  [Hea89] Hansma and Drake et al. The scanning ion-conductance microscope. Science, 24:641–643, February 1989.
  • 21. Bibliography 2  [Kam95] Jörg Kamp. Aufbau und Erprobung eines kombinierten Rasterionenleitungs- und Scherkraftmikroskops. Diploma thesis, Physikalisches Institut der Westfälischen Wilhelms-Universität, March 1995. in german language.  [KBM97] Korchev, Bashford, and Milovanovic. Scanning ion conductance microscopy of living cells. Biophysical Journal, 73:653–658, August 1997.  [Kea00] Korchev and Negulyaev et al. Functional localization of single active ion channels on the surface of a living cell. Nature Cell Biology, pages 616–619, September 2000.  [KMB97] Korchev, Milovanovic, and Bashford. Specialized ion-conductance microscope for imaging of living cells. Journal of Microscopy, 188(1):17– 23, October 1997.  [MDH87] Marti, Drake, and Hansma. Atomic force microscopy of liquid-covered surfaces: Atomic resolution images. Appl. phys. Lett., 51:484–486, 1987.  [Mea88] Marti and Elings et al. Scanning probe microscopy of biological samples and other surfaces. Journal of Microscopy, 152:803–809, 1988.  [ND96] Numberger and Draguhn. Patch-Clamp Technik. Spektrum Akademischer Verlag, 1996.  [NS92] Neher and Sakmann. Die Erforschung von Zellsignalen mit der Patch- Clamp-Technik. Spektrum der Wissenschaft, pages 48–56, May 1992. in german language.
  • 22. Bibliography 3  [OR95] O´Reilly and Richardson. A practical vibration isolation workstation for electrophysiology. journal of Neuroscientific Methods, 60:175–180, 1995.  [PH91] Prater and Hansma. Improved scanning ion-conductance microscope using microfabricated probes. Review of Scientific Instruments., 62(11):2634–2637, November 1991.  [PLH96] Proksch, Lal, and Hansma. Imaging the internal and external pore structure of membranes in fluid: Tappingmode scanning ion conductance microscopy. Biophysical Journal, 71:2155–2157, October 1996.  [Sch90] E. Schwab. Aufbau und Erprobung eines kombinierten Rasterionenleitungsmikroskops (RILM). Diploma thesis, Wiesbaden, 1990. in german language.  [Sch00] Stefan Schraml. Setup of a SICM. WE-Heraeus Ferienkurs Nanophysik, Sept. 2000. poster presentation.  [WW86] Williams and Wickramasinghe. Scanning thermal profiler. Appl. Phys. Lett., 49:1587–1589, 1986. Contact: DI Stefan Schraml sschraml@gmx.net ©2005

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