Summary of Contents for JPK instruments nanowizard afm
- Page 1 NanoWizard ® Handbook Version 2.2a 05 / 2012 © 2004-2012 JPK Instruments AG all rights reserved...
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Page 3: Table Of Contents
Imaging hints - contact mode in liquid ......................34 Imaging hints – imaging in air ........................35 Simple DNA protocol for imaging tests ......................35 Artifacts ...............................37 Tip shape issues ............................37 Artifacts from damaged tips ........................... 38 Contamination ............................... 39 ® JPK Instruments NanoWizard Handbook Version 2.2a... - Page 4 Home made gel packs for cantilever storage ....................49 Suppliers of AFM accessories ........................49 References ............................51 10.1 General AFM Papers ............................. 51 10.2 Spring constant calibration references ......................53 10.3 Books ................................54 ® JPK Instruments NanoWizard Handbook Version 2.2...
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Page 5: Introduction
The pointed tip is brought into contact with the sample and moved across the surface. The instrument measures the deflection of the cantilever as it scans, and from this information about the tip movement a three-dimensional image of the sample is built up. ® JPK Instruments NanoWizard Handbook Version 2.2a... -
Page 6: Scanning Probe Microscopy
The list is not complete, as there are many different forms of scanning probe microscopy, and new techniques are still being developed. The information in this handbook is mainly concerned with Atomic Force Microscopy. ® JPK Instruments NanoWizard Handbook Version 2.2... -
Page 7: Atomic Force Microscopy
“top” and “bottom” halves of the detector. The lateral twisting of the cantilever can also be calculated by comparing the “left” and “right” halves of the detector. AFM is particularly suited for biological applications, because the samples can be ® JPK Instruments NanoWizard Handbook Version 2.2a... -
Page 8: Afm Cantilevers
0.005 N/m and 40 N/m are commercially available. You can deduce the properties of a cantilever from its outer shape. Thicker and shorter ones tend to be stiffer and have higher resonant frequencies. ® JPK Instruments NanoWizard Handbook Version 2.2... -
Page 9: Imaging Modes
In these dynamic modes, a setpoint amplitude is chosen, and the height adjusted to match this amplitude through the feedback system. In addition to the height and error signal information from this constant amplitude mode, the phase between the ® JPK Instruments NanoWizard Handbook Version 2.2a... -
Page 10: Another Way Of Thinking About Imaging Modes
(above the axis) is repulsive. As the tip and sample approach from a long distance, the attractive force increases to some minimum in the curve. Approaching beyond this minimum reaches a relatively sharp upwards part of the curve into the repulsive regime. ® JPK Instruments NanoWizard Handbook Version 2.2... - Page 11 The ranges for the operation of the different modes also vary a lot, so the force values can overlap for different modes, but this overview shows the general operating regimes for the different imaging modes. ® JPK Instruments NanoWizard Handbook Version 2.2a...
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Page 12: Operation
As there can be very low average deflection values during careful imaging, the stiffer cantilevers do not necessarily damage the surface. ® JPK Instruments NanoWizard Handbook Version 2.2... -
Page 13: Phase Imaging
This feature is marked with an arrow in the phase image. This is typical of the case where material property differences show up in the phase, independently of the height. ® JPK Instruments NanoWizard Handbook Version 2.2a... -
Page 14: Force Adjustment In Imaging Modes
Radmacher 1997 Nano-scribing ~ 5 nN, depending on the material Nano-manipulation < 1 nN in any case < 500 pN to move molecules in case of H-bonds ~ 100 pN to move molecules ® JPK Instruments NanoWizard Handbook Version 2.2... -
Page 15: Applications
The applications ® ® page for the NanoWizard AFM and the NanoWizard image gallery on the JPK www.jpk.com website contain more examples of the range of AFM applications and experiments that are possible. ® JPK Instruments NanoWizard Handbook Version 2.2a... -
Page 16: Force Spectroscopy
At some point, the attractive forces (usually Van der Waals and capillary forces) overcome the cantilever spring constant and the tip jumps into contact with the surface. ® JPK Instruments NanoWizard Handbook Version 2.2... -
Page 17: Data Processing For Analysis
(in Volts, as the signal from the photodiode) that is proportional to the cantilever deflection. In the example below, Approach (red) and retract (blue) curves are both plotted on the same axes. ® JPK Instruments NanoWizard Handbook Version 2.2a... - Page 18 (the flat part of the curve on the right hand side). This offset , which may be due to change in piezo height the initial settings of the equipment, or to thermal drift, should be subtracted from all the deflection data in order to calculate the true interaction force. ® JPK Instruments NanoWizard Handbook Version 2.2...
- Page 19 See Section 4.5 for more details. The example above has had the deflection multiplied by the spring constant to express the deflection as a force and would now be ready for analysis. ® JPK Instruments NanoWizard Handbook Version 2.2a...
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Page 20: Applications
Adhesion maps over the surface are also possible to investigate the distribution of receptors. The following table gives an overview of some interactions, and the part of the force curves that they are measured in. ® JPK Instruments NanoWizard Handbook Version 2.2... - Page 21 (1-5 microns) Adhesion strength can be measured between attached molecules and the surface when the attachments break. Overview of some of the interactions measured at different points during a force spectroscopy cycle. Receptor- ligand ® JPK Instruments NanoWizard Handbook Version 2.2a...
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Page 22: More About Cantilevers
Larger amounts of cantilevers are often delivered as a whole wafer in nitrogen-filled plastic containers. In this case no ® JPK Instruments NanoWizard Handbook Version 2.2... -
Page 23: Cantilever Types For Different Imaging Modes
Cantilevers with especially sharp tips such as EBD cantilevers (see below) and sharply edged cantilevers are so delicate that their very easily be damaged if used in contact mode. ® JPK Instruments NanoWizard Handbook Version 2.2a... -
Page 24: Tip Modification
120 – 130 Force Constant 42 N/m 21 – 78 Resonant Frequency 320 kHz 250 - 390 4.4 Tip modification For many applications, the tip is modified chemically or physically to give particular ® JPK Instruments NanoWizard Handbook Version 2.2... -
Page 25: Spring Constant
This means the data from different cantilevers can be combined well to give good statistics. Then it is just important to realize that there will be some systematic difference between the results from different methods. ® JPK Instruments NanoWizard Handbook Version 2.2a... - Page 26 Note that the force constant is independent of the mass of the cantilever, but the ρ resonance frequency is not. Typical values for silicon; E = 168.1 GPa) and <110> = 2.33 · 10 kg m ® JPK Instruments NanoWizard Handbook Version 2.2...
- Page 27 JPK SPM software, so more information on the method can be found in the NanoWizard User manual. The theory for the thermal noise calibration can be found in this Handbook in Section 8.2. ® JPK Instruments NanoWizard Handbook Version 2.2a...
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Page 28: Cell Imaging
Consequently, while AFM imaging of cells can generate novel information, the combination of AFM with other light microscopy techniques expands the scope of possible experiments from structural studies, to structure/function studies. ® JPK Instruments NanoWizard Handbook Version 2.2... -
Page 29: Sample Preparation
Care must be taken to wash cells thoroughly after week (or sometimes more) in PBS, fixation as residual fixant can make the surface sticky and cause problems during but cells should not be allowed to dry scanning. out at any stage. ® JPK Instruments NanoWizard Handbook Version 2.2a... - Page 30 If the fluorescent images are taken between AFM scans, then this is not such a problem, as the cantilever can be lifted off the surface while the fluorescence shutter is open. ® JPK Instruments NanoWizard Handbook Version 2.2...
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Page 31: Imaging Modes
A slightly stiffer cantilever may have general give better imaging, particularly for living a better resonant behavior and hence give more cells sensitive imaging. higher resonance frequency will also enable faster imaging. ® JPK Instruments NanoWizard Handbook Version 2.2a... -
Page 32: Critical Imaging Parameters
50 – 100 nm is a good starting range, though this may need to be optimized for different cell types, depending how sticky or soft they are. 5.5 Using the oscilloscope to optimize the imaging parameters ® JPK Instruments NanoWizard Handbook Version 2.2... - Page 33 The trace and retrace scan lines in the oscilloscope show these structures moving in the direction of the scan. ® JPK Instruments NanoWizard Handbook Version 2.2a...
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Page 34: Artifacts
If the cells to be investigated have large height differences and an image cannot be obtained with out this artifact present then special cantilevers with a long tip should be used, such as EBD tips (see Section 4.4). ® JPK Instruments NanoWizard Handbook Version 2.2... -
Page 35: Single Molecule Imaging
Almost all other surfaces have a higher roughness, and so are only suitable for larger molecules or complexes. Coverslips generally have the smoothest surface of glass samples, and can be conveniently cleaned with water and ethanol, although this may not be enough to remove all contamination. ® JPK Instruments NanoWizard Handbook Version 2.2a... - Page 36 Large mica steps are also visible, particularly where there are broken or damaged regions. Regions on the mica near large steps or broken regions should be avoided for high resolution imaging. ® JPK Instruments NanoWizard Handbook Version 2.2...
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Page 37: Imaging Hints - Intermittent Contact Mode In Liquid
Twisted protein fiber on mica measurement. Cantilevers like the ones recommended here have a resonant frequency around 10 - 13 kHz in liquid. The oscillation amplitude should generally be low for sensitively imaging small objects. ® JPK Instruments NanoWizard Handbook Version 2.2a... -
Page 38: Imaging Hints - Contact Mode In Liquid
Uncoated silicon cantilevers are much less sensitive to these changes than silicon nitride, because a metal coating is necessary for silicon nitride cantilevers and Bacteriorhodopsin the different surface materials make them very sensitive to environmental changes. ® JPK Instruments NanoWizard Handbook Version 2.2... -
Page 39: Imaging Hints - Imaging In Air
Sometimes it is good to start with a simple example to become familiar with the settings. This DNA protocol is fairly simple and reliable, so well suited to imaging testing. ® JPK Instruments NanoWizard Handbook Version 2.2a... - Page 40 Prepare and deposit the DNA as above, then the sample can be rinsed with the imaging buffer instead of water, and left wet for imaging without the drying step. ® JPK Instruments NanoWizard Handbook Version 2.2...
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Page 41: Artifacts
− − R >> For R = 10 nm and d = 5 nm, the observed width would be W = 20 nm ® JPK Instruments NanoWizard Handbook Version 2.2a... -
Page 42: Artifacts From Damaged Tips
All the features have a characteristic shape and the same orientation. Another example of repeatedly occurring features caused by a dull tip. Here the tip is quite blunt, and has a triangular shape. ® JPK Instruments NanoWizard Handbook Version 2.2... -
Page 43: Contamination
See also Section 5.5 and Section 5.6 for a discussion of imaging soft samples with a large height difference, for the specific example of cell imaging. ® JPK Instruments NanoWizard Handbook Version 2.2a... - Page 44 AFM imaging. It is best to locate an AFM in a relatively small room, where there are not many people passing through, in a stable environment. ® JPK Instruments NanoWizard Handbook Version 2.2...
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Page 45: Useful Physics For Spm
This method is based on measuring the free fluctuations of the cantilever, so the main advantages are because it is a passive measurement and can be made in liquid and actually in-situ during an experiment. ® JPK Instruments NanoWizard Handbook Version 2.2a... - Page 46 In fact, there are various correction factors that are needed to get a more accurate value from the fit. The online SPM software from JPK Instruments is equipped with automatic thermal noise analysis for cantilever calibration, several corrections are included and there is space for user input of specific correction factors, depending on the type of cantilever and resonance peak.
- Page 47 Butt and Jaschke paper for more information. Peak Correction factor Comments Example correction factors from Butt 0.817 Generally used and Jaschke, Nanotechnology (1995) 0.251 Used when first resonance frequency is too low 0.0863 Not generally used ® JPK Instruments NanoWizard Handbook Version 2.2a...
- Page 48 The online SPM software from JPK Instruments is equipped with automatic thermal noise analysis for cantilever calibration, fixed corrections for temperature, mounting angle etc.
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Page 49: Young's Modulus Of Materials
The Hertz model is the standard model used to analyze AFM force-distance curves to extract the elasticity. However, the Hertz model makes serious assumptions about the sample, for example that it is ® JPK Instruments NanoWizard Handbook Version 2.2a... - Page 50 The Hertz models for many common tip shapes are included in the JPK IP processing software; the equations can be found in the Image Processing Software Manual. ® JPK Instruments NanoWizard Handbook Version 2.2...
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Page 51: Useful Chemistry And Sample/Tip Preparations
McIntyre. “Use of biaxially oriented the tip. polypropylene film for evaluating and cleaning contaminated atomic force http://publish.uwo.ca/~hnie/spmman.html microscopy probe tips: An http://publish.uwo.ca/~hnie/pdf/rsi02.pdf application to blind tip reconstruction Rev. Sci. Instr. 73 (2002) 3831-3836 ® JPK Instruments NanoWizard Handbook Version 2.2a... -
Page 52: Silanization And Aptes Treatment
At the end, the droplet of APTES will have turned into a whitish silicate solid, this is not used, and the mica can be cleaved again to remove the reacted APTES. The other pieces of mica should now have reacted with the vapor. The surface is best ® JPK Instruments NanoWizard Handbook Version 2.2... -
Page 53: Home Made Gel Packs For Cantilever Storage
Suppliers of AFM accessories (small parts) TED PELLA Inc., USA Small parts for microscopy www.tedpella.com Agar Scientific Ltd., England Small parts for microscopy www.agarscientific.com PLANO W. Plannet GmbH Elektronenmikroskopie, Germany Small parts, esp. for electron ® JPK Instruments NanoWizard Handbook Version 2.2a... - Page 54 MatTek Corporation, USA Plastic petri dishes with integrated www.glass-bottom-dishes.com glass coverslip for enhanced optical microscopy ® Dow Corning Sylgard184 Silicone Elastomer for In Germany distributed by Sasco Semiconductor GmbH, Dreieich gel pack preparation ® JPK Instruments NanoWizard Handbook Version 2.2...
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Page 55: References
AFM and FCS study“, Langmuir 23(14):7659- 7665 (2007) M.H. Jensen, E.J. Morris, A.C. Simonsen, “Domain shapes, coarsening, and Lipid bilayer biochemistry random patterns in ternary membranes“, Langmuir 23: 8135-8141 (2007) ® JPK Instruments NanoWizard Handbook Version 2.2a... - Page 56 Soft Matter 4: 277–285 (2008) F. Balzer, L. Kankate, H. Niehus, H.-G. Rubahn, "Epitaxy vs. dipole assisted growth Fluorescent polymers for organic oligomer nanoaggregates" Proc. SPIE 5925:31-38 (2005) ® JPK Instruments NanoWizard Handbook Version 2.2...
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Page 57: Spring Constant Calibration References
Rev. Sci. Instrum. 64 (1993) 403-405. Thermal noise calibration J.L. Hutter, J. Bechhoefer Original paper where thermal noise "Calibration of atomic-force microscope tips" analysis is described Rev. Sci. Instrum. 64 (1993) 1868-1873. ® JPK Instruments NanoWizard Handbook Version 2.2a... -
Page 58: Books
Academic press, San Diego & Atomic force microscopy in cell biology. London 2002. ISBN: 0-12544171-1 (hb), ISBN: 0-12383851-7 (pb) Colton, Engel, Frommer, Gaub, Gewirth, Guckenberger, Heckl, Parkinson, Rabe Wiley, 1997. ISBN: 047195912X Procedures in Scanning Probe Microscopies. ® JPK Instruments NanoWizard Handbook Version 2.2... - Page 59 Note: All trademarked names mentioned in this manual remain the exclusive property of their respective owners. JPK Instruments AG Bouchéstrasse 12 12435 Berlin Germany Tel: +49 30 5331 12070 Fax: +49 30 5331 22555 support@jpk.com www.jpk.com JPK-DOC0025 All rights reserved.
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