JPK instruments nanowizard afm, Handbook

Page 1: ...NanoWizard AFM Handbook Version 2 2a 05 2012 2004 2012 JPK Instruments AG all rights reserved...

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Page 3: ...ications 16 4 More about cantilevers 18 4 1 General points 18 4 2 Handling information 18 4 3 Cantilever types for different imaging modes 19 4 4 Tip modification 20 4 5 Spring constant 21 5 Cell imag...

Page 4: ...stant calibration 41 8 3 Young s Modulus of materials 45 9 Useful chemistry and sample tip preparations 47 9 1 Cleaning cantilevers and tips 47 9 2 Silanization and APTES treatment 48 9 3 Home made ge...

Page 5: ...und for more experienced users 1 2 What is an Atomic Force Microscope AFM is very different from optical microscopy There are no lenses there is no requirement for a light source to illuminate the sam...

Page 6: ...uch as the electron tunneling current used in STM the resolution can be good enough to see individual atoms Since the measured signal should be dominated by the small region of probe and sample that a...

Page 7: ...ill deflect towards or away from the surface This cantilever deflection must be detected in some way and converted into an electrical signal to produce the images The detection system that has become...

Page 8: ...ere are two or more cantilevers attached to the narrow edges of the chip What you are unable to see without a good optical microscope is the tip at the end of the cantilever Typically the tip is a few...

Page 9: ...in These two values control how quickly the feedback responds to a change in sample height The actual values need to be optimized for different imaging conditions depending on the sample topography an...

Page 10: ...the tip does not leave the surface at all during the oscillation cycle This is something like a dynamic form of contact mode and is usually called force modulation mode 2 3 Another way of thinking ab...

Page 11: ...etween the attractive and repulsive parts of the curve The maximum force perpendicular to the sample may be higher or lower than in contact mode but this is only applied for a short part of the cantil...

Page 12: ...de for the oscillation and also to provide phase information The phase of the cantilever oscillation can give information about the sample properties such as stiffness and mechanical information or ad...

Page 13: ...not damaged by the measurement and no extra equipment is required These methods are discussed further in Section 4 5 2 5 Phase imaging During an AFM experiment in intermittent contact mode the cantile...

Page 14: ...tact mode cantilever is known it is easy to get information about the force applied to a sample during imaging In the JPK NanoWizard software the setpoint can be displayed in units of force if the can...

Page 15: ...een studied The details of the cytoskeleton are usually visible in the images of live cells while fixed cells show the highest resolution features of the membrane surface Many possibilities open up if...

Page 16: ...e study of interaction forces with the AFM has led to deeper understanding of many biological and physical processes down to the single molecule level Simple force curves The data from an experiment i...

Page 17: ...tip may indent the sample enough to feel the supporting surface below The contact area will change as the tip indents a soft surface so the actual interactions involved in compression can be hard to...

Page 18: ...er tip and a bare hard substrate is used to determine the sensitivity of the experimental setup This is a measurement of the deflection of the tip in nanometers for a given movement of the detection l...

Page 19: ...to the surface The deflection can then be plotted against the tip sample separation rather than the piezo height Now that the deflection is in units of length it can be subtracted from the piezo heig...

Page 20: ...an be investigated Titin and bacteriorhodopsin are examples of proteins that have been intensively studied Membrane proteins can be pulled out of the membrane and the popping out of individual alpha h...

Page 21: ...ion Non specific including chemical affinity surface coatings Ligand receptor e g antibody antigen DNA hybridization e g matched or mismatched pairs Cell surface interactions Tip further away nanomete...

Page 22: ...d batch fabrication of probes gives reasonably consistent physical properties AFM cantilevers generally have a metallic coating on the back side to increase the reflectivity Aluminium is a common choi...

Page 23: ...ilever types for different imaging modes The geometry and the material of the cantilever both contribute to the properties that make a cantilever suitable for any particular imaging modes Most cantile...

Page 24: ...is reduced this can improve the situation however For intermittent contact mode in liquid softer cantilevers are often used because when the cantilever and surface are immersed in liquid there is not...

Page 25: ...orphous carbon surface may also be less adhesive than the bare silicon tips for imaging samples such as proteins Each tip must be individually modified however and this is not practical for most AFM u...

Page 26: ...esonant frequency f 12 2 8751 1 2 2 E l t f Note that the force constant is independent of the mass of the cantilever but the resonance frequency is not Typical values for silicon E 110 168 1 GPa and...

Page 27: ...lculation is 1 r s r c s s k k Ideally the two cantilevers are brought in contact with their very tips If there is a significant offset away from the end of the reference lever a correction needs to b...

Page 28: ...ion Obviously the fundamental requirement of any imaging technique is that contrast is somehow generated In terms of conventional optical microscopy this may be due to a difference in material density...

Page 29: ...Wash the surface with water and dry under nitrogen Incubate coated surface with sample of cells in suspension until cells settle and stick to surface 5 10 minutes gently rinse with relevant buffer me...

Page 30: ...siderations always exist but are more important when the force must be reduced as much as possible The use of unsharpened as opposed to sharpened cantilevers is recommended particularly for imaging li...

Page 31: ...ely to ensure that the imaging conditions remain optimal and the applied force does not increase too much due to deflection drift Responds less well to steep height changes such as sides of dendrites...

Page 32: ...cture of the cells to be imaged Imaging of flat well spread cells such as fibroblasts or endothelial cells will mean that scan speeds of up to 5 Hz can be used However with cells that have a high cell...

Page 33: ...race and retrace scan lines overlap at the bottom of any steep edge In this example trace is in bright red and retrace in dull red it can be seen that in the retrace line the tip has not followed the...

Page 34: ...dicates an unavoidable tip artifact where the edge of the tip has been imaged as it slides down a steep surface 5 6 Artifacts General artifacts arising from normal tip geometry damaged tips and contam...

Page 35: ...lose the stock solution immediately Make up buffers fresh whenever possible filter before use with a syringe filter If anything needs drying use a nitrogen cylinder with a suitable gas regulator to bl...

Page 36: ...his kind of mounting only to discover that the sample drifts too much to get good images Use all the advantages available from the AFM system The NanoWizard AFM system has several features to optimize...

Page 37: ...ty is low The resonant frequency of very soft cantilevers is also too low for good imaging for example a cantilever with a spring constant around 0 1 N m often has a resonance of only 1 5 kHz The pixe...

Page 38: ...ably stuck down since the area of such a membrane patch is so much larger than a single molecule and the proteins are extremely well supported laterally so much less likely to be swept aside by the AF...

Page 39: ...in air the choice of cantilever is quite wide Since the dry proteins are much harder than the hydrated proteins and they are usually very well stuck down then the range of spring constants goes from t...

Page 40: ...nteract with the DNA in solution to induce kinks in the DNA strands The DNA will still adsorb to the surface for imaging as normal but sharp bends can be seen in the DNA strands Magnesium ions are als...

Page 41: ...est narrowest tip produces the most accurate representation of the surface tip sample scan A practical example is shown on the right The AFM image shows a 3D view of a red blood cell with protrustions...

Page 42: ...l source of artifacts in AFM Even unused tips may have two points at their very end Double tips may also occur on worn tips that have been damaged during scanning Double tips Sometimes rather than hav...

Page 43: ...erts some force on the sample This can cause problems of distortion or damage to the sample which may move under this force particularly since many biological samples are soft and delicate and require...

Page 44: ...or mechanical drift from thermal or other effects The general approach is usually to construct AFMs from small light rigid components that will have high resonant frequencies The AFM is then located o...

Page 45: ...5 in water This is because of the much higher damping from the viscosity of the water compared with air In water the effective mass also increases since the cantilever carries some of the surrounding...

Page 46: ...the system kB T where kB is the Boltzmann constant not related to the spring constant The measured energy in the is given by the spring constant and the average value of the vertical deflection of th...

Page 47: ...tion factors are needed The correction factors given in the software Butt and Jaschke 0 817 for the first mode 0 251 for the second mode and 0 0863 for the third mode are only valid when the laser spo...

Page 48: ...best to combine results from force curves using the same type of cantilever where the shape differences are minimized The online SPM software from JPK Instruments is equipped with automatic thermal n...

Page 49: ...teins 0 5 GPa 0 5 10 9 Pa 5 000 bar wood 1 GPa 1 10 9 Pa 10 000 bar water 2 2 GPa 2 2 10 9 Pa 22 000 bar PMMA 3 GPa 3 10 9 Pa 30 000 bar silicon 110 170 GPa 170 10 9 Pa 1 700 000 bar steel 200 GPa 200...

Page 50: ...eneous and purely elastic so it is recommended to read about this analysis before designing elasticity experiments The Hertz models for many common tip shapes are included in the JPK IP processing sof...

Page 51: ...the tip is quickly contaminated but relatively easy to clean with detergent for example UV irradiation Irradiation of the tip with a UV lamp produces ozone and burns organic material from the tip Str...

Page 52: ...e NH2 groups that are positively charged in aqueous solutions The chemical needs to be high quality high purity or the surface contamination will be larger than the proteins or other sample to be imag...

Page 53: ...nt and sealing for electronic parts but it can also serve for gel pack preparation Together with a catalyst it is delivered in aluminum containers of 1 1 kg or more Distributed in Germany by Sasco Sem...

Page 54: ...roscopy MatTek Corporation USA www glass bottom dishes com Plastic petri dishes with integrated glass coverslip for enhanced optical microscopy Dow Corning In Germany distributed by Sasco Semiconducto...

Page 55: ...visualized by atomic force microscopy FEBS Letters 565 53 58 2004 Cell imaging J M Burns A Cuschieri P A Campbell Optimisation of fixation period on biological cells via time lapse elasticity mapping...

Page 56: ...nd inhibitor interactions of a single antiporter J Mol Biol 362 925 932 2006 Protein unfolding A Kedrov M Appel H Baumann C Ziegler D J M ller Examining the dynamic energy landscape of an antiporter u...

Page 57: ...Zimmermann M Textor J V r s E Reimhult From particle self assembly to functionalized sub micron protein patterns Nanotechnology 19 075301 2008 Surface patterning 10 2Spring constant calibration refere...

Page 58: ...y 13 2002 33 37 Comparison of thermal noise with other dynamic methods for triangular cantilevers A Maali C Hurth R Boisgard C Jai T Cohen Bouhacina J P Aim Hydrodynamics of oscillating atomic force m...

Page 59: ...trademarked names mentioned in this manual remain the exclusive property of their respective owners JPK Instruments AG Bouch strasse 12 12435 Berlin Germany Tel 49 30 5331 12070 Fax 49 30 5331 22555...