probing nano-scale forces with the atomic force...
TRANSCRIPT
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Probing Nano-Scale Forceswith the Atomic Force Microscope
TheWorldLeader InScanningProbeMicroscopy
ties are similarly important,often affecting the structural anddynamic behavior of systemsfrom composite materials toblood cells. AFM offers a newtool to study these importantparameters on the micron tonanometer scale using a tech-nique that measures forces onthe AFM probe tip as it ap-proaches and retracts from asurface. This application notedescribes measurement andconstruction of “force curves”and some of the importantapplications for this newtechnology.
The ability of the atomic forcemicroscope (AFM) to createthree-dimensional micrographswith resolution down to thenanometer and Angstrom scaleshas made it an essential tool forimaging surfaces in applicationsranging from semiconductorprocessing to cell biology. Inaddition to this topographicalimaging, however, the AFM canalso probe nanomechanical andother fundamental properties ofsample surfaces, including theirlocal adhesive or elastic (compli-ance) properties.
Microscopic adhesionaffects a huge varietyof events, from thebehavior of paintsand glues, ceramicsand compositematerials, to DNAreplication andthe action ofdrugs in thehuman body.Elastic proper-
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Position SensitiveDetector
CantileverWithProbe Tip
Laser
Basic AFM Operation
A key element of the AFM is itsmicroscopic force sensor, orcantilever (Figure 1). Thecantilever is usually formed byone or more beams of silicon orsilicon nitride that is 100 to500µm long and about 0.5 to5µm thick. Mounted on the endof the cantilever is a sharp tipthat is used to sense a forcebetween the sample and tip. Fornormal topographic imaging, theprobe tip is brought intocontinuous or intermittentcontact with the sample and
Figure 1: Schematic of an atomic force microscope (AFM) showing theforce sensing cantilever.
Figure 2: Typical 3-D surface topographic imageproduced by the AFM. Sample shown is aTetrahymena Protozoan. 50µm scan courtesy E.Henderson, BioForce Labs and Iowa State University.
By C.B. Prater,P.G. Maivald,K.J. Kjoller,M.G. Heaton
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69. Weisenhorn, A.L., et al. Deformationobserved on soft surfaces with an AFM. inProc. SPIE - Int. Soc. Opt. Eng. (USA).1993. Los Angeles, CA, USA.
70. Schoenenberger, C.A. and J.H. Hoh,Slow cellular dynamics in MDCK and R5cells monitored by time-lapse atomic forcemicroscopy. Biophys. J. (USA), 1994.67,(2): p. 929-36.
71. Radmacher, M., et al., Measuring theviscoelastic properties of human plateletswith the atomic force microscope.submitted, 1995.
72. Radmacher, M., M. Fritz, and P.K.Hansma, Imaging soft samples with theatomic force microscope—gelatin in waterand propanol. Biophysical journal, 1995. inpress.
73. Maivald, P., et al., Using forcemodulation to image surface elasticities withthe atomic force microscope.Nanotechnology (UK), 1991. 2,(2):p. 103-6.
74. Radmacher, M., R.W. Tillmann, andH.E. Gaub, Imaging viscoelasticity by forcemodulation with the atomic forcemicroscope. Biophys. J. (USA), 1993.64,(3): p. 735-42.
75. Yamada, H., et al. Atomic forcemicroscopy studies of photosyntheticprotein membrane Langmuir-Blodgettfilms. in Thin Solid Films (Switzerland).1994. Trois-Riveries, Que., Canada.
76. Florin, E.L., et al., Atomic forcemicroscope with magnetic force modula-tion. Rev. Sci. Instrum. (USA), 1994.65,(3): p. 639-43.
77. Fritz, M., et al. Visualization andidentification of intracellular structures byforce modulation microscopy and druginduced degradation. in J. Vac. Sci. Technol.B, Microelectron. Nanometer Struct.(USA). 1994. Beijing, China.
78. Baselt, D.R. and J.D. Baldeschwieler,Imaging spectroscopy with the atomic forcemicroscope. J. Appl. Phys. (USA), 1994.76,(1): p. 33-8.
79. van der Werf, K.O., et al., Adhesionforce imaging in air and liquid by adhesionmode atomic force microscopy. Appl. Phys.Lett. (USA), 1994. 65,(9): p. 1195-7.
80. Radmacher, M., et al., Mappinginteraction forces with the atomic forcemicroscope. Biophys. J., 1994. 66: p. 2159-65.
81. Hues, S.M., et al., Scanning probemicroscopy of thin films. MRS Bull. (USA),1993. 18,(1): p. 41-9.
82. Mizes, H.A., et al., Submicron probe ofpolymer adhesion with atomic forcemicroscopy: dependence on topography andmaterial inhomogeneities. Appl. Phys. Lett.(USA), 1991. 59,(22): p. 2901-3.
83. Aime, J.P., et al., Comments on the useof the force mode in atomic forcemicroscopy for polymer films. J. Appl. Phys.(USA), 1994. 76,(2): p. 754-62.
84. Overney, R.M., et al. Elasticity, wear,and friction properties of thin organic filmsobserved with atomic force microscopy. in J.Vac. Sci. Technol. B, Microelectron.Nanometer Struct. (USA). 1994. Beijing,China.
85. Juai, R. and B. Bhushan, Nanoindenta-tion studies of sublimed fullerene filmsusing atomic force microscopy. J. Mater.Res. (USA), 1993. 8,(12): p. 3019-22.
86. Torii, A., et al., An elasticity evaluationmethod for micro-mechanical structures byusing load-deflection curve of forcemicroscope. Trans. Inst. Electr. Eng. Jpn. A(Japan), 1992. 112-A,(12): p. 979-86.
87. Hues, S.M., C.F. Draper, and R.J.Colton. Measurement of nanomechanicalproperties of metals using the atomic forcemicroscope. in J. Vac. Sci. Technol. B,Microelectron. Nanometer Struct. (USA).1994. Beijing, China.
88. Sumomogi, T., et al. Micromachiningof metal surfaces by scanning probemicroscope. in J. Vac. Sci. Technol. B,Microelectron. Nanometer Struct. (USA).1994. Beijing, China.
89. Bhushan, B. and V.N. Koinkar,Nanoindentation hardness measurements
using atomic force microscopy. Appl. Phys.Lett. (USA), 1994. 64,(13): p. 1653-5.
90. Sader, J.E., et al., Method for thecalibration of atomic force microscopecantilevers. submitted, 1995.
91. Cleveland, J.P., et al., A nondestructivemethod for determining the spring constantof cantilevers for scanning force microscopy.Rev. Sci. Instrum. (USA), 1993. 64,(2): p.403-5.
92. Hutter, J.L. and J. Bechhoefer,Calibration of atomic-force microscope tips.Rev. Sci. Instrum. (USA), 1993. 64,(7): p.1868-73.
93. Butt, H.J., et al., Scan speed limit inatomic force microscopy. J. Microsc. (UK),1993. 169, pt.1: p. 75-84.
94. Senden, T.J., et al., Experimentaldetermination of spring constants in atomicforce microscopy. Langmuir, 1994. 10(4):p. 1003-1004.
95. Smith, S.T. and L.P. Howard, Aprecision, low-force balance and itsapplication to atomic force microscopeprobe calibration. Rev. Sci. Instrum. (USA),1994. 65,(4, pt.1): p. 903-9.
96. Sader, J.E. and L. White, Theoreticalanalysis of the static deflection of plates foratomic force microscope applications. J.Appl. Phys. (USA), 1993. 74,(1): p. 1-9.
97. Neumeister, J.M. and W.A. Ducker,Lateral, normal, and longitudinal springconstants of atomic force microscopycantilevers. Rev. Sci. Instrum. (USA), 1994.65,(8): p. 2527-31.
98. Drummond, C.J. and T.J. Senden,Characterisation of the mechanicalproperties of thin film cantilevers with theatomic force microscope. Materials ScienceForum Trans Tech Publications, to bepublished.
99. Chan, D., “AFM analysis”, : Dept. ofMathematics, University of Melbourne,Parkville 3052 Australia. Fax +61-3-344-4599, email [email protected].
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raster-scanned over the surface.Fine-motion piezoelectricscanners are used to generate theprecision motion needed togenerate topographic images andforce measurements. A piezo-electric scanner is a device thatmoves by a microscopic amountwhen a voltage is applied acrossits electrodes. Depending on theAFM design, scanners are usedto translate either the sampleunder the cantilever or thecantilever over the sample.Piezoelectric scanners for AFMsusually can translate in threedirections (x, y, and z axes) andcome in different sizes to allowmaximum scan ranges of 0.5 to125µm in the x and y axes andseveral microns in the vertical (z)axis. A well-built scanner cangenerate stable motion on a scalebelow 1 Angstrom.
By scanning the AFM cantileverover a sample surface (or scan-ning a sample under the cantile-ver) and recording the deflectionof the cantilever, the local heightof the sample is measured.Three-dimensional topographi-cal maps of the surface are thenconstructed by plotting the localsample height versus horizontalprobe tip position (a summaryarticle on the basics of AFM isavailable on request from DigitalInstruments). Other imagingtechniques are also used includ-ing measuring the change inamplitude or phase of anoscillating cantilever, usingTappingMode™, for example.
Force CurveMeasurements
In addition to these topographicmeasurements, the AFM canalso provide much more infor-mation. The AFM can alsorecord the amount of force feltby the cantilever as the probe tipis brought close to — and evenindented into — a samplesurface and then pulled away.This technique can be used tomeasure the long range attractiveor repulsive forces between theprobe tip and the samplesurface, elucidating local chemi-cal and mechanical propertieslike adhesion and elasticity, andeven thickness of adsorbedmolecular layers or bond rupturelengths.
To help examine the basics ofAFM force measurements,Figure 4-1 shows a typical“force-versus-distance” curve orforce curve, for short, as gener-ated by a NanoScope III AFMsystem (generation and analysisof these curves is discussed inmore detail in the sidebar,“Details of Force Measure-ments”). Force curves typicallyshow the deflection of the freeend of the AFM cantilever as thefixed end of the cantilever isbrought vertically towards andthen away from the samplesurface. Experimentally, this isdone by applying a triangle-wavevoltage pattern to the electrodesfor the z-axis scanner. Thiscauses the scanner to expand andthen contract in the verticaldirection, generating relativemotion between the cantileverand sample. The deflection of
Figure 4-1: AFM Force Measurement (“force curve”)as displayed on Digital Instruments NanoScope®SPM system. The force curve plots the deflection ofthe force-sensing cantilever as the tip is extendedtoward the sample (yellow) and retracted from thesample (white). The labeled points on the curve (A-E)are illustrated in Fig. 4-2 (opposite).
Figure 3: Digital Instruments MultiMode™ AFM,optimized for high resolution force measurements andtopographic imaging.
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the free end of the cantilever ismeasured and plotted at manypoints as the z-axis scannerextends the cantilever towardsthe surface and then retracts itagain. By controlling theamplitude and frequency of thetriangle-wave voltage pattern,the researcher can vary thedistance and speed that the AFMcantilever tip travels during theforce measurement.
Similar measurements can bemade with oscillating probesystems like TappingMode andnon-contact AFM. This sort ofwork is just beginning foroscillating probe systems, butmeasurements of cantileveramplitude and/or phase versusseparation can provide moreinformation about the details ofmagnetic and electric fields oversurfaces and also provideinformation about viscoelasticproperties of sample surfaces.
Anatomy of aForce Curve
Several points along a typicalforce curve are shown schemati-cally in Figure 4-2. The cantile-ver starts (point A) not touchingthe surface. In this region, if thecantilever feels a long-rangeattractive (or repulsive) force itwill deflect downwards (orupwards) before making contactwith the surface. In the caseshown, there is minimal long-range force, so this “non-contact” part of the force curveshows no deflection. As theprobe tip is brought very close tothe surface, it may jump into
contact (B) if it feels sufficientattractive force from the sample.Once the tip is in contact withthe surface, cantilever deflectionwill increase (C) as the fixed endof the cantilever is broughtcloser to the sample. If thecantilever is sufficiently stiff, theprobe tip may indent into thesurface at this point. In thiscase, the slope or shape of thecontact part of the force curve(C) can provide informationabout the elasticity of the samplesurface.
After loading the cantilever to adesired force value, the process isreversed. As the cantilever iswithdrawn, adhesion or bondsformed during contact with thesurface may cause the cantileverto adhere to the sample (sectionD) some distance past the initialcontact point on the approachcurve (B). A key measurementof the AFM force curve is thepoint (E) at which the adhesionis broken and the cantilevercomes free from the surface.This can be used to mesure therupture force required to breakthe bond or adhesion.
Beginnings — Control-ling Surface Adhesion
One of the first uses of forcemeasurements was to improvethe quality of AFM images bymonitoring and minimizing theattractive forces between the tipand sample. Large adhesiveforces can reduce imagingresolution, damage the sampleand probe, and/or create un-wanted artifacts.
Figure 4-2: AFM ForceMeasurement (“force curve”) ofcantilever-sample interactionshown schematically at severalpoints along the force curve shownin Fig. 4-1 (opposite), including:A) the approach (“non-contact”region), B) jump to contact,C) contact, D) adhesion, andE) pulloff.
A
B
C
D
E
2
raster-scanned over the surface.Fine-motion piezoelectricscanners are used to generate theprecision motion needed togenerate topographic images andforce measurements. A piezo-electric scanner is a device thatmoves by a microscopic amountwhen a voltage is applied acrossits electrodes. Depending on theAFM design, scanners are usedto translate either the sampleunder the cantilever or thecantilever over the sample.Piezoelectric scanners for AFMsusually can translate in threedirections (x, y, and z axes) andcome in different sizes to allowmaximum scan ranges of 0.5 to125µm in the x and y axes andseveral microns in the vertical (z)axis. A well-built scanner cangenerate stable motion on a scalebelow 1 Angstrom.
By scanning the AFM cantileverover a sample surface (or scan-ning a sample under the cantile-ver) and recording the deflectionof the cantilever, the local heightof the sample is measured.Three-dimensional topographi-cal maps of the surface are thenconstructed by plotting the localsample height versus horizontalprobe tip position (a summaryarticle on the basics of AFM isavailable on request from DigitalInstruments). Other imagingtechniques are also used includ-ing measuring the change inamplitude or phase of anoscillating cantilever, usingTappingMode™, for example.
Force CurveMeasurements
In addition to these topographicmeasurements, the AFM canalso provide much more infor-mation. The AFM can alsorecord the amount of force feltby the cantilever as the probe tipis brought close to — and evenindented into — a samplesurface and then pulled away.This technique can be used tomeasure the long range attractiveor repulsive forces between theprobe tip and the samplesurface, elucidating local chemi-cal and mechanical propertieslike adhesion and elasticity, andeven thickness of adsorbedmolecular layers or bond rupturelengths.
To help examine the basics ofAFM force measurements,Figure 4-1 shows a typical“force-versus-distance” curve orforce curve, for short, as gener-ated by a NanoScope III AFMsystem (generation and analysisof these curves is discussed inmore detail in the sidebar,“Details of Force Measure-ments”). Force curves typicallyshow the deflection of the freeend of the AFM cantilever as thefixed end of the cantilever isbrought vertically towards andthen away from the samplesurface. Experimentally, this isdone by applying a triangle-wavevoltage pattern to the electrodesfor the z-axis scanner. Thiscauses the scanner to expand andthen contract in the verticaldirection, generating relativemotion between the cantileverand sample. The deflection of
Figure 4-1: AFM Force Measurement (“force curve”)as displayed on Digital Instruments NanoScope®SPM system. The force curve plots the deflection ofthe force-sensing cantilever as the tip is extendedtoward the sample (yellow) and retracted from thesample (white). The labeled points on the curve (A-E)are illustrated in Fig. 4-2 (opposite).
Figure 3: Digital Instruments MultiMode™ AFM,optimized for high resolution force measurements andtopographic imaging.
3
the free end of the cantilever ismeasured and plotted at manypoints as the z-axis scannerextends the cantilever towardsthe surface and then retracts itagain. By controlling theamplitude and frequency of thetriangle-wave voltage pattern,the researcher can vary thedistance and speed that the AFMcantilever tip travels during theforce measurement.
Similar measurements can bemade with oscillating probesystems like TappingMode andnon-contact AFM. This sort ofwork is just beginning foroscillating probe systems, butmeasurements of cantileveramplitude and/or phase versusseparation can provide moreinformation about the details ofmagnetic and electric fields oversurfaces and also provideinformation about viscoelasticproperties of sample surfaces.
Anatomy of aForce Curve
Several points along a typicalforce curve are shown schemati-cally in Figure 4-2. The cantile-ver starts (point A) not touchingthe surface. In this region, if thecantilever feels a long-rangeattractive (or repulsive) force itwill deflect downwards (orupwards) before making contactwith the surface. In the caseshown, there is minimal long-range force, so this “non-contact” part of the force curveshows no deflection. As theprobe tip is brought very close tothe surface, it may jump into
contact (B) if it feels sufficientattractive force from the sample.Once the tip is in contact withthe surface, cantilever deflectionwill increase (C) as the fixed endof the cantilever is broughtcloser to the sample. If thecantilever is sufficiently stiff, theprobe tip may indent into thesurface at this point. In thiscase, the slope or shape of thecontact part of the force curve(C) can provide informationabout the elasticity of the samplesurface.
After loading the cantilever to adesired force value, the process isreversed. As the cantilever iswithdrawn, adhesion or bondsformed during contact with thesurface may cause the cantileverto adhere to the sample (sectionD) some distance past the initialcontact point on the approachcurve (B). A key measurementof the AFM force curve is thepoint (E) at which the adhesionis broken and the cantilevercomes free from the surface.This can be used to mesure therupture force required to breakthe bond or adhesion.
Beginnings — Control-ling Surface Adhesion
One of the first uses of forcemeasurements was to improvethe quality of AFM images bymonitoring and minimizing theattractive forces between the tipand sample. Large adhesiveforces can reduce imagingresolution, damage the sampleand probe, and/or create un-wanted artifacts.
Figure 4-2: AFM ForceMeasurement (“force curve”) ofcantilever-sample interactionshown schematically at severalpoints along the force curve shownin Fig. 4-1 (opposite), including:A) the approach (“non-contact”region), B) jump to contact,C) contact, D) adhesion, andE) pulloff.
A
B
C
D
E
5
Air
Water
In 1989, Weisenhorn andHansma of the University ofCalifornia at Santa Barbara (1)noted that the typical adhesiveforce between a standard siliconnitride AFM tip and a micasurface imaged in ambient airwas about 100 nanoNewtons(nN = 10-9 Newtons); however,they found that this force couldbe reduced by a factor of 100 (toabout 1nN) by simply immers-ing the sample in water (Fig 5).The reason for the large adhe-sion in ambient air is that mostsamples in air are covered by athin layer of water and othercondensed contaminants. Thesecontaminants often form acapillary bridge between the tipand sample, generating largeadhesive forces. When the entirecantilever is completely sub-merged in liquid, these capillaryforces largely disappear.
Mate and others used the onsetof the capillary force to theiradvantage. By recording thedistance between the onset ofcapillary force and the pointwhen the AFM tip contacts thesurface, they could measure thethickness of the adsorbedmolecular layers, down to athickness of 2nm. (2)
Since these discoveries, manyrefinements have been made.Weihs and others (3-5) noticedthat the adhesive force betweenthe AFM tip and sample de-creased with tip radius. Sharpertips yield lower adhesive forcesand, therefore, lower imagingforces. Force measurementswere also used to demonstratesimilarly reduced capillary forcesfor samples in vacuum (6) and
Figure 5: Measurement of theforces between an AFM tip and amica surface in air and water. Themeasurement in air shows a largeadhesive force due to capillaryforces from the liquid contaminantlayer on the surface. Once thewhole cantilever probe isimmersed in water, capillary forcesare mostly eliminatedand adhesion is substantiallydiminished. From A.L.Weisenhorn, reference (1).
in reduced humidity environ-ments. (5, 7)
In 1990, Hartmann (8) calcu-lated that attractive Van derWaals forces could be substan-tially reduced by operating inappropriate solvents, especiallyethanol. Weisenhorn and others(9-11) confirmed with AFMforce measurements that tip-sample adhesion was dramati-cally reduced in ethanol as wellas other solvents. Even smalladditions of ethanol to waterreduced adhesive forces up to afactor of 100 over pure water;the effect was probably enhancedby ethanol’s ability to removesome organic contaminants fromthe sample surface. Thundat(12) also noticed reducedadhesion between the AFM tipand sample by first treating thetip with ozone or ultravioletlight to remove organic contami-nation. The combination ofthese and other improvementscan reduce tip-sample forcesduring AFM imaging to lessthan 0.1nN. AFM users nowroutinely use force curve mea-surements to check tip-surfaceadhesion to ensure that the tipand sample are free of contami-nation that could hinder AFMimaging.
However, controlling surfacecontaminants may only be halfthe story. Alley (13) recentlyobserved improved image qualityon silicon surfaces by treatingthe AFM tip with a silanemonolayer usingoctadecyltrichlorosilane (OTS).Even in ambient air, Alley notedadhesive forces of less than 1nN,
100nm
60nm
150nm
60nm
Water
Air
4
Details of Force MeasurementsThis section discusses in more detail the process used tomake AFM force measurements. As described above, forcemeasurements are made by recording the deflection of thefree end of the AFM cantilever as the fixed end of thecantilever is extended towards and retracted from thesample. AFMs typically measure cantilever deflections bybouncing a laser beam off the free end of the cantilever(Figure 1). Deflections of the cantilever cause the reflectedlaser beam to change its angle. The motion of the reflectedlaser beam is detected by a multiple segment photodiodeknown as a Position Sensitive Detector (PSD). The verticalsensitivity of an AFM measurement depends on the lengthof the cantilever— for the same vertical deflection shortercantilevers produce a larger angular change than longercantilevers.
The basic “force curve” as measured by an AFM is actually acurve showing “PSD voltage versus scanner position.” ThePSD voltage can be converted into a cantilever deflectiondistance by a simple calibration procedure: The AFMcantilever tip is brought in contact with a hard surface andthen the cantilever or sample is moved by a known amountusing the z-axis scanner. The AFM system records howmuch PSD voltage is generated for a known deflection, andthis conversion factor is used to convert future PSD mea-surements into cantilever deflections.
Calculating ForcesThe force sensed by the AFM probe is calculated bymultiplying the deflection of the cantilever by thecantilever’s spring constant. For example, if a cantileverwith a spring constant of 1 Newton per meter is deflected by1nm, the spring force exerted by the cantilever is 1nN. Thespring constant of AFM cantilevers are determined both bythe geometric properties (lengths, widths, and thickness),and by the elastic modulus of the cantilever material.Cantilevers are available with a variety of spring constantsfrom roughly 0.01 to 100 Newtons/meter (ornanoNewtons/nanometer), providing a range of measure-ment capabilities for various surfaces. Because cantileverspring constants can vary from cantilever to cantilever,several methods have been developed to measure (90-95) orcalculate (96-98) the cantilever spring constant.
Setpoint and Tracking ForcesThe majority of this article discusses using force curves tomeasure forces between surfaces under study or probing themechanical properties of sample surfaces. Another commonuse of force curves is to set the tracking force applied to thesample by the AFM tip during imaging. AFMs usuallywork by maintaining a constant deflection of the cantileveror, for oscillating cantilever techniques, a constant ampli-tude. The tracking force is adjusted by changing the“setpoint” deflection or amplitude. Force curves provide agraphic way of seeing how much force is exerted by a givendeflection or amplitude setpoint (see Figure). For contact
mode AFM, the total amount of force exerted on a samplecomes from two sources—the cantilever spring force plusany adhesion between the tip and sample. These twosources are indicated in the figure. The spring force resultsfrom bending the cantilever from its equilibrium (zero-force) position. The “setpoint” shown in the figure isadjusted by the user depending on the amount of cantileverspring force desired. Any adhesion forces between the tipand sample will add to the selected spring force, resulting ina “total tracking force,” as shown. Force curves are usefulfor adjusting the tracking force because they allow the userto visualize the amounts of cantilever spring force andadhesion, and then to select an appropriate setpoint. Inpractice, the setpoint is usually selected to minimize thecantilever spring force. Sometimes, however, it is possibleto choose a setpoint that results in a “negative” spring force.In this case, the cantilever bends downwards toward thesample and its spring force opposes the adhesive force.“Negative” spring forces can produce the lowest totaltracking forces, but imaging can also be unstable as thecantilever may unexpectedly spring off the sample surface.
Force vs. SeparationFor some measurements it is also desirable to replace the“force-versus-scanner-position” curve with a “force-versus-separation” curve. In the presence of long-range forces, thefree end of the cantilever will bend towards or away fromthe sample surface during the measurement. In this case,the motion of the end of the cantilever will no longer matchthe motion of the z-axis scanner. But “force-versus scannerposition” curves can easily be converted to “force-versus-separation” plots by simply correcting the recorded scannerposition by the measured deflection of the cantilever. Forexample, if the scanner moves 10nm toward a surface, andthe cantilever tip is attracted to the surface and also deflects2nm towards the surface, the actual tip-sample separation isreduced by 12 nm. Force-versus-separation curves are oftenmore useful for comparing measurements with theorybecause they show directly the dependence of the force ondistance between the tip and sample. In fact, Dr. DerekChan of the University of Melbourne has written analysissoftware for AFM force measurements. (99) The softwaregraphically compares force-versus-separation curves topredictions for Van der Waals, electrostatic, and other forces,and it can extract such properties as sample surface charge.
Force
Total Tracking Force
SetpointCantilerverSpring Force
Adhesion Force
Scanner Z-position
}}
5
Air
Water
In 1989, Weisenhorn andHansma of the University ofCalifornia at Santa Barbara (1)noted that the typical adhesiveforce between a standard siliconnitride AFM tip and a micasurface imaged in ambient airwas about 100 nanoNewtons(nN = 10-9 Newtons); however,they found that this force couldbe reduced by a factor of 100 (toabout 1nN) by simply immers-ing the sample in water (Fig 5).The reason for the large adhe-sion in ambient air is that mostsamples in air are covered by athin layer of water and othercondensed contaminants. Thesecontaminants often form acapillary bridge between the tipand sample, generating largeadhesive forces. When the entirecantilever is completely sub-merged in liquid, these capillaryforces largely disappear.
Mate and others used the onsetof the capillary force to theiradvantage. By recording thedistance between the onset ofcapillary force and the pointwhen the AFM tip contacts thesurface, they could measure thethickness of the adsorbedmolecular layers, down to athickness of 2nm. (2)
Since these discoveries, manyrefinements have been made.Weihs and others (3-5) noticedthat the adhesive force betweenthe AFM tip and sample de-creased with tip radius. Sharpertips yield lower adhesive forcesand, therefore, lower imagingforces. Force measurementswere also used to demonstratesimilarly reduced capillary forcesfor samples in vacuum (6) and
Figure 5: Measurement of theforces between an AFM tip and amica surface in air and water. Themeasurement in air shows a largeadhesive force due to capillaryforces from the liquid contaminantlayer on the surface. Once thewhole cantilever probe isimmersed in water, capillary forcesare mostly eliminatedand adhesion is substantiallydiminished. From A.L.Weisenhorn, reference (1).
in reduced humidity environ-ments. (5, 7)
In 1990, Hartmann (8) calcu-lated that attractive Van derWaals forces could be substan-tially reduced by operating inappropriate solvents, especiallyethanol. Weisenhorn and others(9-11) confirmed with AFMforce measurements that tip-sample adhesion was dramati-cally reduced in ethanol as wellas other solvents. Even smalladditions of ethanol to waterreduced adhesive forces up to afactor of 100 over pure water;the effect was probably enhancedby ethanol’s ability to removesome organic contaminants fromthe sample surface. Thundat(12) also noticed reducedadhesion between the AFM tipand sample by first treating thetip with ozone or ultravioletlight to remove organic contami-nation. The combination ofthese and other improvementscan reduce tip-sample forcesduring AFM imaging to lessthan 0.1nN. AFM users nowroutinely use force curve mea-surements to check tip-surfaceadhesion to ensure that the tipand sample are free of contami-nation that could hinder AFMimaging.
However, controlling surfacecontaminants may only be halfthe story. Alley (13) recentlyobserved improved image qualityon silicon surfaces by treatingthe AFM tip with a silanemonolayer usingoctadecyltrichlorosilane (OTS).Even in ambient air, Alley notedadhesive forces of less than 1nN,
100nm
60nm
150nm
60nm
Water
Air
4
Details of Force MeasurementsThis section discusses in more detail the process used tomake AFM force measurements. As described above, forcemeasurements are made by recording the deflection of thefree end of the AFM cantilever as the fixed end of thecantilever is extended towards and retracted from thesample. AFMs typically measure cantilever deflections bybouncing a laser beam off the free end of the cantilever(Figure 1). Deflections of the cantilever cause the reflectedlaser beam to change its angle. The motion of the reflectedlaser beam is detected by a multiple segment photodiodeknown as a Position Sensitive Detector (PSD). The verticalsensitivity of an AFM measurement depends on the lengthof the cantilever— for the same vertical deflection shortercantilevers produce a larger angular change than longercantilevers.
The basic “force curve” as measured by an AFM is actually acurve showing “PSD voltage versus scanner position.” ThePSD voltage can be converted into a cantilever deflectiondistance by a simple calibration procedure: The AFMcantilever tip is brought in contact with a hard surface andthen the cantilever or sample is moved by a known amountusing the z-axis scanner. The AFM system records howmuch PSD voltage is generated for a known deflection, andthis conversion factor is used to convert future PSD mea-surements into cantilever deflections.
Calculating ForcesThe force sensed by the AFM probe is calculated bymultiplying the deflection of the cantilever by thecantilever’s spring constant. For example, if a cantileverwith a spring constant of 1 Newton per meter is deflected by1nm, the spring force exerted by the cantilever is 1nN. Thespring constant of AFM cantilevers are determined both bythe geometric properties (lengths, widths, and thickness),and by the elastic modulus of the cantilever material.Cantilevers are available with a variety of spring constantsfrom roughly 0.01 to 100 Newtons/meter (ornanoNewtons/nanometer), providing a range of measure-ment capabilities for various surfaces. Because cantileverspring constants can vary from cantilever to cantilever,several methods have been developed to measure (90-95) orcalculate (96-98) the cantilever spring constant.
Setpoint and Tracking ForcesThe majority of this article discusses using force curves tomeasure forces between surfaces under study or probing themechanical properties of sample surfaces. Another commonuse of force curves is to set the tracking force applied to thesample by the AFM tip during imaging. AFMs usuallywork by maintaining a constant deflection of the cantileveror, for oscillating cantilever techniques, a constant ampli-tude. The tracking force is adjusted by changing the“setpoint” deflection or amplitude. Force curves provide agraphic way of seeing how much force is exerted by a givendeflection or amplitude setpoint (see Figure). For contact
mode AFM, the total amount of force exerted on a samplecomes from two sources—the cantilever spring force plusany adhesion between the tip and sample. These twosources are indicated in the figure. The spring force resultsfrom bending the cantilever from its equilibrium (zero-force) position. The “setpoint” shown in the figure isadjusted by the user depending on the amount of cantileverspring force desired. Any adhesion forces between the tipand sample will add to the selected spring force, resulting ina “total tracking force,” as shown. Force curves are usefulfor adjusting the tracking force because they allow the userto visualize the amounts of cantilever spring force andadhesion, and then to select an appropriate setpoint. Inpractice, the setpoint is usually selected to minimize thecantilever spring force. Sometimes, however, it is possibleto choose a setpoint that results in a “negative” spring force.In this case, the cantilever bends downwards toward thesample and its spring force opposes the adhesive force.“Negative” spring forces can produce the lowest totaltracking forces, but imaging can also be unstable as thecantilever may unexpectedly spring off the sample surface.
Force vs. SeparationFor some measurements it is also desirable to replace the“force-versus-scanner-position” curve with a “force-versus-separation” curve. In the presence of long-range forces, thefree end of the cantilever will bend towards or away fromthe sample surface during the measurement. In this case,the motion of the end of the cantilever will no longer matchthe motion of the z-axis scanner. But “force-versus scannerposition” curves can easily be converted to “force-versus-separation” plots by simply correcting the recorded scannerposition by the measured deflection of the cantilever. Forexample, if the scanner moves 10nm toward a surface, andthe cantilever tip is attracted to the surface and also deflects2nm towards the surface, the actual tip-sample separation isreduced by 12 nm. Force-versus-separation curves are oftenmore useful for comparing measurements with theorybecause they show directly the dependence of the force ondistance between the tip and sample. In fact, Dr. DerekChan of the University of Melbourne has written analysissoftware for AFM force measurements. (99) The softwaregraphically compares force-versus-separation curves topredictions for Van der Waals, electrostatic, and other forces,and it can extract such properties as sample surface charge.
Force
Total Tracking Force
SetpointCantilerverSpring Force
Adhesion Force
Scanner Z-position
}}
6
With a force sensitivity on theorder of a few piconewtons (pN= 10-12 N), AFMs are excellenttools for probing these funda-mental force interactions. (14-18) For example, AFM has beenused to probe the nature ofattractive Van der Waals andattractive/repulsive electrostaticforces between such systems asmetal probes and insulating micasurfaces (19, 20), and insulatingprobes on insulating andconducting samples with suchmaterials as silicon nitride,diamond, alumina, mica, glassand graphite. (4, 9, 21, 22)
With an eye towards studies ofadhesion, friction and wear,Binggeli and Mate (23) haveused AFM to study the forma-tion or suppression of capillarycondensation on hydrophilicsilicon, amorphous carbon, andlubricated SiO
2 surfaces. Torii
has also studied variations incapillary adhesion on glass andrubber samples caused bymaterial differences and varia-tions in surface topography. (24)Radmacher et al. have evenmeasured the forces between anAFM tip and individual proteinmolecules. (25)
Measurementsin Liquids
As discussed above, forcemeasurements in water revealedthe benefits of AFM imaging inthis environment due to thelower tip-sample forces. Someof the most interesting forcemeasurements have also beenperformed with samples under
liquids where the environmentcan be quickly changed to adjustthe concentration of variouschemical components.
In liquids, electrostatic forcesbetween dissolved ions and othercharged groups play an impor-tant role in determining theforces sensed by an AFMcantilever. Butt, Jaschke andDucker (26) have recentlywritten an excellent review ofAFM force measurements inliquid environments. The liquidenvironment has become animportant stage for fundamentalforce measurement becauseresearchers can control many ofthe details of the probe surfaceforce interaction by adjustingproperties of the liquid. Experi-mentally, Butt and others (21,27, 28) have shown, for ex-ample, that electrostatic tip-sample forces depend stronglyon pH and salt concentration(Figure 6). In fact, Butt showedthat it is often possible to adjustthe pH or salt concentrationsuch that the attractive Van derWaals forces are effectivelynegated by repulsive electrostaticforces. This has been an impor-tant discovery because it canallow tuning of the liquidenvironment to minimizeadhesive tip-sample forces thatcan damage the sample duringimaging.
Lin et al. (29) have also mea-sured force profiles in liquid atvarious pH values to determinethe surface charge of an Si
3N
4
AFM tip and, as a result, theisoelectric point, the pH (6.0 intheir experiment) at which the
CantileverForce
pH 10.4
pH 8.1
pH 6.1
pH 3.1
Sample Position
Figure 6: Force measurements from a NanoScopeAFM system of the tip-sample forces between asilicon nitride cantilever and a mica surface undervarying pH. Notice that at low pH there is a verystrong attraction between the tip and sample. At highpH, the situation is reversed where long-rangerepulsion dominates. At an intermediate pH, theoverall tip-sample forces can be minimized. Reprintedby permission, reference (27).
again about two orders ofmagnitude less than for anuntreated tip. By reducing theforce that the AFM applies tothe sample surface, all of thesediscoveries have helped improveAFM image quality.
MeasuringFundamental Forces
Concern with the fundamentalinteractions between surfacesextends across physics, chemis-try, materials science and avariety of other disciplines.
0.02nN
20nm
7
AFM tip was electrically neutral.Further details of surfacechemistry and tip-sampleinteractions in fluid for Si
3N
4
AFM tips have been determinedby Senden and Drummond.(30) Drummond and Sendenhave also recently published amethod for determining theradius of a Si
3N
4 tip by measur-
ing electric double layer forces inliquid. (31) The liquid environ-ment can also be used to preparesurfaces or solutions that containmore complex molecules withmore detailed force interactions.For example, Torii and cowork-ers have compared adhesiveforces between the AFM tip anda silica surface in a variety oforganic and inorganic solutions.(32, 33)
Quantized Adhesion
Butt’s balancing of attractive andrepulsive forces has also beenused to quantify the adhesionbetween a silicon nitride tip anda glass surface. (34, 35) Hohnoticed that when he adjustedthe overall tip-sample adhesionto around 1nN, repeatedmeasurements of the adhesiveforce varied in discrete units ofaround 0.01nN. More recentwork by Cleveland et al.(36)suggests that these discreteinteractions may be caused bythe ordered layers of watermolecules that form on thesample surface, creating peri-odic force “steps” for the probetip. By analyzing the quantiza-tion of forces, the strength ofsingle binding events can bedetermined.
Colloidal Forces(Top Work fromDown Under)
Australia has been an importantsource of recent work on AFMforce measurements involvingcolloids. William Ducker andco-workers have pioneered theattachment of colloidal spheres(particles on the micron scale) toAFM cantilevers and thenmeasuring forces between thesphere and a sample of interest(Figure 7). This is an extremelyinteresting technique because, asDucker writes, “The forcesbetween colloidal particlesdominate the behavior of a greatvariety of materials, includingpaints, paper, soils, clays and, insome circumstances, biologicalcells.” (37) Colloid forces alsoplay a critical role in the forma-tion of ceramics and compositematerials.
Colloidal spheres of glass, silica,polystyrene, various metals andother materials are readilyavailable, allowing use of probesof known size and shape suchthat normalized force curves canbe calculated and quantified.These quantitative force mea-surements have allowed detailedstudies of such phenomena assurface charging in liquids,polymer-solid interactions, andhydrophobic interactions, toname a few.
Using colloidal probes, studieshave been made of interactionforces between silica, (37) gold,(38) titanium dioxide, (39, 40)zirconia, (41) and polystyreneand p-type silicon (42). Biggs
Figure 7: The AFM can measure forces on colloidalparticles. In this case, a silica bead was glued to anAFM cantilever to probe the force between the beadand water droplets or air bubbles. Reprinted bypermission, reference (44).
and Healy (41, 43) have mea-sured steric and bridging forcesbetween colloidal zirconia andpolymer chains of polyacrylicacid. Steric forces are forcesassociated with the entropicarrangement or ordering ofatoms and molecules; theseforces can dominate the struc-ture of slurries used in makingceramics. Biggs and Healy usedthe AFM to move the zirconiaprobe in and out of the polymerlayer at a rate similar to thoseexpected for Brownian collisionsin a ceramic slurry and tomeasure the effect of pH on this
6
With a force sensitivity on theorder of a few piconewtons (pN= 10-12 N), AFMs are excellenttools for probing these funda-mental force interactions. (14-18) For example, AFM has beenused to probe the nature ofattractive Van der Waals andattractive/repulsive electrostaticforces between such systems asmetal probes and insulating micasurfaces (19, 20), and insulatingprobes on insulating andconducting samples with suchmaterials as silicon nitride,diamond, alumina, mica, glassand graphite. (4, 9, 21, 22)
With an eye towards studies ofadhesion, friction and wear,Binggeli and Mate (23) haveused AFM to study the forma-tion or suppression of capillarycondensation on hydrophilicsilicon, amorphous carbon, andlubricated SiO
2 surfaces. Torii
has also studied variations incapillary adhesion on glass andrubber samples caused bymaterial differences and varia-tions in surface topography. (24)Radmacher et al. have evenmeasured the forces between anAFM tip and individual proteinmolecules. (25)
Measurementsin Liquids
As discussed above, forcemeasurements in water revealedthe benefits of AFM imaging inthis environment due to thelower tip-sample forces. Someof the most interesting forcemeasurements have also beenperformed with samples under
liquids where the environmentcan be quickly changed to adjustthe concentration of variouschemical components.
In liquids, electrostatic forcesbetween dissolved ions and othercharged groups play an impor-tant role in determining theforces sensed by an AFMcantilever. Butt, Jaschke andDucker (26) have recentlywritten an excellent review ofAFM force measurements inliquid environments. The liquidenvironment has become animportant stage for fundamentalforce measurement becauseresearchers can control many ofthe details of the probe surfaceforce interaction by adjustingproperties of the liquid. Experi-mentally, Butt and others (21,27, 28) have shown, for ex-ample, that electrostatic tip-sample forces depend stronglyon pH and salt concentration(Figure 6). In fact, Butt showedthat it is often possible to adjustthe pH or salt concentrationsuch that the attractive Van derWaals forces are effectivelynegated by repulsive electrostaticforces. This has been an impor-tant discovery because it canallow tuning of the liquidenvironment to minimizeadhesive tip-sample forces thatcan damage the sample duringimaging.
Lin et al. (29) have also mea-sured force profiles in liquid atvarious pH values to determinethe surface charge of an Si
3N
4
AFM tip and, as a result, theisoelectric point, the pH (6.0 intheir experiment) at which the
CantileverForce
pH 10.4
pH 8.1
pH 6.1
pH 3.1
Sample Position
Figure 6: Force measurements from a NanoScopeAFM system of the tip-sample forces between asilicon nitride cantilever and a mica surface undervarying pH. Notice that at low pH there is a verystrong attraction between the tip and sample. At highpH, the situation is reversed where long-rangerepulsion dominates. At an intermediate pH, theoverall tip-sample forces can be minimized. Reprintedby permission, reference (27).
again about two orders ofmagnitude less than for anuntreated tip. By reducing theforce that the AFM applies tothe sample surface, all of thesediscoveries have helped improveAFM image quality.
MeasuringFundamental Forces
Concern with the fundamentalinteractions between surfacesextends across physics, chemis-try, materials science and avariety of other disciplines.
0.02nN
20nm
7
AFM tip was electrically neutral.Further details of surfacechemistry and tip-sampleinteractions in fluid for Si
3N
4
AFM tips have been determinedby Senden and Drummond.(30) Drummond and Sendenhave also recently published amethod for determining theradius of a Si
3N
4 tip by measur-
ing electric double layer forces inliquid. (31) The liquid environ-ment can also be used to preparesurfaces or solutions that containmore complex molecules withmore detailed force interactions.For example, Torii and cowork-ers have compared adhesiveforces between the AFM tip anda silica surface in a variety oforganic and inorganic solutions.(32, 33)
Quantized Adhesion
Butt’s balancing of attractive andrepulsive forces has also beenused to quantify the adhesionbetween a silicon nitride tip anda glass surface. (34, 35) Hohnoticed that when he adjustedthe overall tip-sample adhesionto around 1nN, repeatedmeasurements of the adhesiveforce varied in discrete units ofaround 0.01nN. More recentwork by Cleveland et al.(36)suggests that these discreteinteractions may be caused bythe ordered layers of watermolecules that form on thesample surface, creating peri-odic force “steps” for the probetip. By analyzing the quantiza-tion of forces, the strength ofsingle binding events can bedetermined.
Colloidal Forces(Top Work fromDown Under)
Australia has been an importantsource of recent work on AFMforce measurements involvingcolloids. William Ducker andco-workers have pioneered theattachment of colloidal spheres(particles on the micron scale) toAFM cantilevers and thenmeasuring forces between thesphere and a sample of interest(Figure 7). This is an extremelyinteresting technique because, asDucker writes, “The forcesbetween colloidal particlesdominate the behavior of a greatvariety of materials, includingpaints, paper, soils, clays and, insome circumstances, biologicalcells.” (37) Colloid forces alsoplay a critical role in the forma-tion of ceramics and compositematerials.
Colloidal spheres of glass, silica,polystyrene, various metals andother materials are readilyavailable, allowing use of probesof known size and shape suchthat normalized force curves canbe calculated and quantified.These quantitative force mea-surements have allowed detailedstudies of such phenomena assurface charging in liquids,polymer-solid interactions, andhydrophobic interactions, toname a few.
Using colloidal probes, studieshave been made of interactionforces between silica, (37) gold,(38) titanium dioxide, (39, 40)zirconia, (41) and polystyreneand p-type silicon (42). Biggs
Figure 7: The AFM can measure forces on colloidalparticles. In this case, a silica bead was glued to anAFM cantilever to probe the force between the beadand water droplets or air bubbles. Reprinted bypermission, reference (44).
and Healy (41, 43) have mea-sured steric and bridging forcesbetween colloidal zirconia andpolymer chains of polyacrylicacid. Steric forces are forcesassociated with the entropicarrangement or ordering ofatoms and molecules; theseforces can dominate the struc-ture of slurries used in makingceramics. Biggs and Healy usedthe AFM to move the zirconiaprobe in and out of the polymerlayer at a rate similar to thoseexpected for Brownian collisionsin a ceramic slurry and tomeasure the effect of pH on this
9
Binding Forces
As with the chemically-modifiedprobes described above, biologi-cal researchers are findinggrowing applications for AFMprobes that have been tailored tosense a specific biologicalreaction. For example, Florin,Moy and Gaub recently mea-sured the binding forces ofindividual ligand-receptor pairs(58-60) (Ligands are moleculesthat bind to a specific site onanother molecule, usually aprotein. A receptor is a proteinthat receives and binds a ligandmolecule and then producessome response). These measure-ments were accomplished bymodifying an AFM probe tip sothat its outer surface was coatedwith avidin receptor molecules(Figure 10). Gaub’s team usedagarose beads coated withvarious forms of biotin, a ligandthat binds to avidin. By block-ing most of the adhesive forcewith free avidin molecules insolution, the group was able toreduce the total adhesion to apoint at which they were able tosee the rupture of a finitenumber of bonds. By analyzingthe variation in the adhesiveforces, the researchers deter-mined individual receptor-ligandbinding forces to be 160pN forbiotin-avidin and 85pN foriminobiotin-avidin. They werealso able to calculate the effectiverupture length of the biotin-avidin bond.
Lee and co-workers (61) haveused a technique similar to thatof Florin to directly measureinteraction forces between
terminating the monolayers onthe tip and sample, they wereable to study the adhesion andfriction between combinationsof acid (—COOH) and methyl(—CH
3) functional groups (55,
56) (Figure 8).
Biological Applications
The life sciences are a naturalarena for the study of nano-scaleforce interactions. Countlessbiological processes — DNAreplication, protein synthesis,drug interaction, and manyothers — are largely governed byintermolecular forces. And withits sensitivity down to tens ofpiconewtons, the AFM is anexcellent tool for probing theseinteractions. In addition, theAFM can make nanomechanicalmeasurements (like elasticity) onbiological specimens, offeringinsight into subjects like cellularand even protein dynamics.Recently, for example, Dammerand co-workers reported inScience that they had used theAFM to measure the bindingstrength of cell adhesionproteoglycans (moleculescontaining a mixture of proteinsand sugars) from a marinesponge (Figure 9). (57) Theymeasured the force between asingle pair of proteoglycanadhesion molecules to be as highas 400pN, strong enough tohold the weight of 1600 cells.They reasoned that this highbinding force per molecule andthe 1000 proteoglycan bonds percell are responsible for thesubstantial structural integrity ofthe marine sponge.
Figure 9: Typical AFM force curves for adhesionproteoglycan (AP) molecules on both the AFM tip andon a mica substrate. The retracting part of the forcecurves shows discrete jumps as AP-AP bonds arebroken. Reprinted by permission, reference (57).Note that the NanoScope MultiMode AFM can resolvepicoNewton-scale forces.
D
C
B
A
40 nm
100 pN
40 nm
100 pN
40 nm
100 pN
30 nm
100 pN
8
Similar measurements of electro-static charge density on biologi-cal membranes have beenperformed by Butt (see Biologi-cal Applications, below). Elec-tric double layer forces have alsobeen studied on self-assembledmonolayers(hexadecyltrimethylammoniumhalides), deposited on colloidalsilica spheres. (50, 51)
Chemical sensing has been takenone step further by bindingsensing molecules to the AFMtip or to colloidal spheresattached to the AFM cantilever.Surface chemistry techniquesalready used — for example inliquid chromatography — offermany model preparations forcreating AFM probes withchemically modified surfaces.Molecules bound to the AFMprobe can then be used aschemical sensors to detect forcesbetween molecules on the tipand target molecules on thesurface. Ducker and others haveused this technique to createhydrophobic AFM colloidalprobes to study the nature of thehydrophobic force. (52, 53)When octadecyltrichlorosilane(OTS) was bound to AFM tips,Nakagawa et al. (54) found thatthe AFM could discriminatebetween monolayers of 4different alkyltrichlorosilanes(CH
3(CH)
nSiCl
2 with n = 1, 8,
13, 17) deposited on silicon.Charles Lieber and his team atHarvard bound functionalizedthiol monolayers to gold-coatedAFM cantilever tips and also togold-coated target samples. Byvarying the functional group
steric force. Butt has evenmeasured the forces on acolloidal glass particle as it entersan air bubble or water drop. (44)
Chemical Sensitivity
The above discussion illustratesthat AFM force measurementsare extremely sensitive to tip andsurface properties. Extendingthis work to the chemical realm,Burnham and co-workers (45)used AFM force measurementsto distinguish two similarsamples, each covered with asingle chemical monolayer —the only difference being thatone surface was terminated withCH
3 groups, the other with CF
3.
O’Shea (46) also observedchanges in frictional forcesbetween the AFM tip andsurface that were dependent onthe presence or absence of asingle molecular layer. Olsson(47) studied methylated siliconsamples with a tungsten tip andfound he could identify 100nm-sized methylated patches on thesilicon by observing variations inthe capillary forces between thetip and sample.
Additionally, Ishino and co-workers have recently used theAFM to detect the chargepolarity of various functionalmonolayers deposited on gold.(48, 49) For this work, theyheld the gold sample surfaceunder electrochemical controland measured the AFM tip-sample force while adjusting thepotential on the gold surface.
Figure 8: (Top) AFM friction (chemical force)measurement of patterned organic monolayers ongold as scanned with tip coated with similarmonolayer. The difference in friction is due to differentfunctional groups terminating the patterned regions ofthe sample. 50µm scan.
(Bottom) Force curves from a NanoScope MultiModeAFM on patterned regions for different tip and samplefunctional group terminations. Reprinted bypermission, references (55, 56).
COOH – CH3
CH3 – CH3
COOH – COOH
1 nN
Z displacement (nm)0
Adhe
sion
Forc
e
Forc
e
5 10 15 20 25
9
Binding Forces
As with the chemically-modifiedprobes described above, biologi-cal researchers are findinggrowing applications for AFMprobes that have been tailored tosense a specific biologicalreaction. For example, Florin,Moy and Gaub recently mea-sured the binding forces ofindividual ligand-receptor pairs(58-60) (Ligands are moleculesthat bind to a specific site onanother molecule, usually aprotein. A receptor is a proteinthat receives and binds a ligandmolecule and then producessome response). These measure-ments were accomplished bymodifying an AFM probe tip sothat its outer surface was coatedwith avidin receptor molecules(Figure 10). Gaub’s team usedagarose beads coated withvarious forms of biotin, a ligandthat binds to avidin. By block-ing most of the adhesive forcewith free avidin molecules insolution, the group was able toreduce the total adhesion to apoint at which they were able tosee the rupture of a finitenumber of bonds. By analyzingthe variation in the adhesiveforces, the researchers deter-mined individual receptor-ligandbinding forces to be 160pN forbiotin-avidin and 85pN foriminobiotin-avidin. They werealso able to calculate the effectiverupture length of the biotin-avidin bond.
Lee and co-workers (61) haveused a technique similar to thatof Florin to directly measureinteraction forces between
terminating the monolayers onthe tip and sample, they wereable to study the adhesion andfriction between combinationsof acid (—COOH) and methyl(—CH
3) functional groups (55,
56) (Figure 8).
Biological Applications
The life sciences are a naturalarena for the study of nano-scaleforce interactions. Countlessbiological processes — DNAreplication, protein synthesis,drug interaction, and manyothers — are largely governed byintermolecular forces. And withits sensitivity down to tens ofpiconewtons, the AFM is anexcellent tool for probing theseinteractions. In addition, theAFM can make nanomechanicalmeasurements (like elasticity) onbiological specimens, offeringinsight into subjects like cellularand even protein dynamics.Recently, for example, Dammerand co-workers reported inScience that they had used theAFM to measure the bindingstrength of cell adhesionproteoglycans (moleculescontaining a mixture of proteinsand sugars) from a marinesponge (Figure 9). (57) Theymeasured the force between asingle pair of proteoglycanadhesion molecules to be as highas 400pN, strong enough tohold the weight of 1600 cells.They reasoned that this highbinding force per molecule andthe 1000 proteoglycan bonds percell are responsible for thesubstantial structural integrity ofthe marine sponge.
Figure 9: Typical AFM force curves for adhesionproteoglycan (AP) molecules on both the AFM tip andon a mica substrate. The retracting part of the forcecurves shows discrete jumps as AP-AP bonds arebroken. Reprinted by permission, reference (57).Note that the NanoScope MultiMode AFM can resolvepicoNewton-scale forces.
D
C
B
A
40 nm
100 pN
40 nm
100 pN
40 nm
100 pN
30 nm
100 pN
8
Similar measurements of electro-static charge density on biologi-cal membranes have beenperformed by Butt (see Biologi-cal Applications, below). Elec-tric double layer forces have alsobeen studied on self-assembledmonolayers(hexadecyltrimethylammoniumhalides), deposited on colloidalsilica spheres. (50, 51)
Chemical sensing has been takenone step further by bindingsensing molecules to the AFMtip or to colloidal spheresattached to the AFM cantilever.Surface chemistry techniquesalready used — for example inliquid chromatography — offermany model preparations forcreating AFM probes withchemically modified surfaces.Molecules bound to the AFMprobe can then be used aschemical sensors to detect forcesbetween molecules on the tipand target molecules on thesurface. Ducker and others haveused this technique to createhydrophobic AFM colloidalprobes to study the nature of thehydrophobic force. (52, 53)When octadecyltrichlorosilane(OTS) was bound to AFM tips,Nakagawa et al. (54) found thatthe AFM could discriminatebetween monolayers of 4different alkyltrichlorosilanes(CH
3(CH)
nSiCl
2 with n = 1, 8,
13, 17) deposited on silicon.Charles Lieber and his team atHarvard bound functionalizedthiol monolayers to gold-coatedAFM cantilever tips and also togold-coated target samples. Byvarying the functional group
steric force. Butt has evenmeasured the forces on acolloidal glass particle as it entersan air bubble or water drop. (44)
Chemical Sensitivity
The above discussion illustratesthat AFM force measurementsare extremely sensitive to tip andsurface properties. Extendingthis work to the chemical realm,Burnham and co-workers (45)used AFM force measurementsto distinguish two similarsamples, each covered with asingle chemical monolayer —the only difference being thatone surface was terminated withCH
3 groups, the other with CF
3.
O’Shea (46) also observedchanges in frictional forcesbetween the AFM tip andsurface that were dependent onthe presence or absence of asingle molecular layer. Olsson(47) studied methylated siliconsamples with a tungsten tip andfound he could identify 100nm-sized methylated patches on thesilicon by observing variations inthe capillary forces between thetip and sample.
Additionally, Ishino and co-workers have recently used theAFM to detect the chargepolarity of various functionalmonolayers deposited on gold.(48, 49) For this work, theyheld the gold sample surfaceunder electrochemical controland measured the AFM tip-sample force while adjusting thepotential on the gold surface.
Figure 8: (Top) AFM friction (chemical force)measurement of patterned organic monolayers ongold as scanned with tip coated with similarmonolayer. The difference in friction is due to differentfunctional groups terminating the patterned regions ofthe sample. 50µm scan.
(Bottom) Force curves from a NanoScope MultiModeAFM on patterned regions for different tip and samplefunctional group terminations. Reprinted bypermission, references (55, 56).
COOH – CH3
CH3 – CH3
COOH – COOH
1 nN
Z displacement (nm)0
Adhe
sion
Forc
e
Forc
e
5 10 15 20 25
11
Because a single molecule can bebound to the end of the tether,Schindler’s group is optimisticthat this technique will besuccessful in creating entireimages of binding sites byscanning the sensor moleculeacross the surface to detectbinding events. The highsensitivity of the AFM’s forcedetection and the growingexperience in measuring biologi-cal binding forces holds greatpromise for the study of suchtopics as DNA replication,DNA/protein interactions, cellimmune response and drugdevelopment and testing.
Charge Density on Surfaces
Another application of AFMforce measurements is to imageor quantify electrical surfacecharge. The dynamics of manybiological systems depends onthe electrical properties of thesample surface. Butt, forexample, used the force on theAFM probe to estimate thesurface charge of the purplemembrane of Halobacteriaholobium. (65) Leng andWilliams have made similarmeasurements in air on testsamples including red bloodcells. (66)
Biomechanical Measurements
In addition to measuringbinding forces and electrostaticforces, the AFM can also probethe micromechanical propertiesof biological samples. Specifi-cally, the AFM can observe the
elasticity and, in fact, theviscosity of samples rangingfrom live cells and membranes tobone and cartilage.
In 1992, Tao and co-workersfirst studied the microelasticproperties of hydrated cow tibiain comparison to standards likerubber and stainless steel. (67)Weisenhorn continued in thisvein, using force curves tomeasure the elasticity of cartilageand living cells. (68, 69) Inaddition to measuring the cells’mechanical properties,Weisenhorn et al. were also ableto predict the maximum probeforces necessary for high-resolution AFM imaging of cells.
Schoenenberger and Hoh haveused AFM force measurementsto determine the stiffness of amonolayer of living Madin-Darby canine kidney (MDCK)cells. (70) The authors were alsoable to record the increasingstiffness of these cells over timeduring gluteraldehyde fixation(Figure 12). Radmacher andcoworkers have recently madequantitative measurements ofviscoelasticity of human plateletsand other soft samples usingsimilar AFM techniques. (71,72) By making stiffness oradhesion measurements at manypoints on a sample, the AFMcan also create entire images ofsurface elasticity or surfaceadhesion using force modulation(73-77) or related techniques.(78-80) (An application noteon the force modulation tech-nique is available from DigitalInstruments.)
PEG20 PEG20
AbHSA
HSA
C=O
O=CS
HNMicaS
S
S
AFM Tip
NH
Chemistry: Spacer and Linkage
Figure 11: (Top) Schematic of“sensor force microscopy.” Abiological sensor molecule, in thiscase AbHSA, is attached to an AFMtip with a molecular tether, thePEG20 molecule. The sensormolecule is scanned across thesurface while force measurementsdetect whether a binding eventoccurs when a target molecule isencountered. (Bottom) Details ofchemical link of sensor and targetmolecules. Reprinted bypermission, reference (63, 64).
Force
Principle of Sensor Force Microscopy
yx
Scan
10
complementary chains of single-stranded DNA. Single-strandedDNA, twenty bases long, wasbound to a sample surface andalso to a spherical probe attachedto the AFM cantilever. TheDNA strands on the sphericalprobe contained a sequence thatwas complementary to that ofstrands on the sample. Leeobserved a distribution ofbinding forces with peaks atthree distinct values: 1.52, 1.11,and 0.52nN. The researchersattributed the distribution ofbinding forces to the formationand then rupture of 20, 16, or12 base pairs.
Boland and Ratner (62) at theUniversity of Washingtonrecently measured the bindingforces between various bareDNA nucleotide bases. Theycreated self-assembled monolay-ers of DNA bases on a goldsample substrate and also on agold-coated AFM tip. Bindingforces due to hydrogen bondingwere only observed when the tipand sample were coated withcomplementary bases, forexample adenine (A) on the tipand thymine (T) on the surface.
Tethered Molecular Sensors
Schindler and colleagues at theUniversity of Linz, Austria havedeveloped another novel tech-nique for measuring bindingforces and foresee a techniquefor imaging binding sites withthe AFM. Schindler’s team hascreated a technique they callsensor force microscopy (63, 64)in which a single biologicalmolecule (the sensor) is attachedto the AFM tip on the end of amolecular tether (Figure 11).The molecular tether is an elasticlinker molecule a few nanom-eters long based on polyethyleneglycol (PEG) molecules. ThePEG tether gives the sensormolecule the freedom to orientitself properly to bind to itstarget on the surface. When abinding event takes place, theAFM detects the additional forcerequired to break the molecularadhesion. Schindler’s group hasused this technique to recordbinding forces between humanserum albumin (HSA) and itsassociated antibody (Ab
HSA),
with measurements averaging470pN.
5 nN
Retraction
5 nN
Displacement
Forc
e
100 nm
ApproachA
Force Curves ForReceptor-Ligand Adhesion
B
C
100 nm
500pN
Fad
50 nm
5 nN
Retraction
5 nN
Displacement
Forc
e
100 nm
ApproachA
Force Curves ForReceptor-Ligand Adhesion
B
C
100 nm
500pN
Fad
50 nm
Figure 10: (Top) Schematic of the avidin-functionalizedAFM tip and the biotinylated agarose bead, shownhere partially blocked with avidin.
(Bottom) Cantilever deflection curves on approachand retraction of an avidin tip on a biotinylatedagarose bead (A) before and (B) after blockage withan excess of free avidin (200µg/ml). (C) Magnificationof a force scan on a biotinylated agarose beadapproximately 95% blocked with free avidin. Fad is themeasured unbinding force plotted. Reprinted bypermission, reference (58).
11
Because a single molecule can bebound to the end of the tether,Schindler’s group is optimisticthat this technique will besuccessful in creating entireimages of binding sites byscanning the sensor moleculeacross the surface to detectbinding events. The highsensitivity of the AFM’s forcedetection and the growingexperience in measuring biologi-cal binding forces holds greatpromise for the study of suchtopics as DNA replication,DNA/protein interactions, cellimmune response and drugdevelopment and testing.
Charge Density on Surfaces
Another application of AFMforce measurements is to imageor quantify electrical surfacecharge. The dynamics of manybiological systems depends onthe electrical properties of thesample surface. Butt, forexample, used the force on theAFM probe to estimate thesurface charge of the purplemembrane of Halobacteriaholobium. (65) Leng andWilliams have made similarmeasurements in air on testsamples including red bloodcells. (66)
Biomechanical Measurements
In addition to measuringbinding forces and electrostaticforces, the AFM can also probethe micromechanical propertiesof biological samples. Specifi-cally, the AFM can observe the
elasticity and, in fact, theviscosity of samples rangingfrom live cells and membranes tobone and cartilage.
In 1992, Tao and co-workersfirst studied the microelasticproperties of hydrated cow tibiain comparison to standards likerubber and stainless steel. (67)Weisenhorn continued in thisvein, using force curves tomeasure the elasticity of cartilageand living cells. (68, 69) Inaddition to measuring the cells’mechanical properties,Weisenhorn et al. were also ableto predict the maximum probeforces necessary for high-resolution AFM imaging of cells.
Schoenenberger and Hoh haveused AFM force measurementsto determine the stiffness of amonolayer of living Madin-Darby canine kidney (MDCK)cells. (70) The authors were alsoable to record the increasingstiffness of these cells over timeduring gluteraldehyde fixation(Figure 12). Radmacher andcoworkers have recently madequantitative measurements ofviscoelasticity of human plateletsand other soft samples usingsimilar AFM techniques. (71,72) By making stiffness oradhesion measurements at manypoints on a sample, the AFMcan also create entire images ofsurface elasticity or surfaceadhesion using force modulation(73-77) or related techniques.(78-80) (An application noteon the force modulation tech-nique is available from DigitalInstruments.)
PEG20 PEG20
AbHSA
HSA
C=O
O=CS
HNMicaS
S
S
AFM Tip
NH
Chemistry: Spacer and Linkage
Figure 11: (Top) Schematic of“sensor force microscopy.” Abiological sensor molecule, in thiscase AbHSA, is attached to an AFMtip with a molecular tether, thePEG20 molecule. The sensormolecule is scanned across thesurface while force measurementsdetect whether a binding eventoccurs when a target molecule isencountered. (Bottom) Details ofchemical link of sensor and targetmolecules. Reprinted bypermission, reference (63, 64).
Force
Principle of Sensor Force Microscopy
yx
Scan
10
complementary chains of single-stranded DNA. Single-strandedDNA, twenty bases long, wasbound to a sample surface andalso to a spherical probe attachedto the AFM cantilever. TheDNA strands on the sphericalprobe contained a sequence thatwas complementary to that ofstrands on the sample. Leeobserved a distribution ofbinding forces with peaks atthree distinct values: 1.52, 1.11,and 0.52nN. The researchersattributed the distribution ofbinding forces to the formationand then rupture of 20, 16, or12 base pairs.
Boland and Ratner (62) at theUniversity of Washingtonrecently measured the bindingforces between various bareDNA nucleotide bases. Theycreated self-assembled monolay-ers of DNA bases on a goldsample substrate and also on agold-coated AFM tip. Bindingforces due to hydrogen bondingwere only observed when the tipand sample were coated withcomplementary bases, forexample adenine (A) on the tipand thymine (T) on the surface.
Tethered Molecular Sensors
Schindler and colleagues at theUniversity of Linz, Austria havedeveloped another novel tech-nique for measuring bindingforces and foresee a techniquefor imaging binding sites withthe AFM. Schindler’s team hascreated a technique they callsensor force microscopy (63, 64)in which a single biologicalmolecule (the sensor) is attachedto the AFM tip on the end of amolecular tether (Figure 11).The molecular tether is an elasticlinker molecule a few nanom-eters long based on polyethyleneglycol (PEG) molecules. ThePEG tether gives the sensormolecule the freedom to orientitself properly to bind to itstarget on the surface. When abinding event takes place, theAFM detects the additional forcerequired to break the molecularadhesion. Schindler’s group hasused this technique to recordbinding forces between humanserum albumin (HSA) and itsassociated antibody (Ab
HSA),
with measurements averaging470pN.
5 nN
Retraction
5 nN
Displacement
Forc
e
100 nm
ApproachA
Force Curves ForReceptor-Ligand Adhesion
B
C
100 nm
500pN
Fad
50 nm
5 nN
Retraction
5 nN
Displacement
Forc
e
100 nm
ApproachA
Force Curves ForReceptor-Ligand Adhesion
B
C
100 nm
500pN
Fad
50 nm
Figure 10: (Top) Schematic of the avidin-functionalizedAFM tip and the biotinylated agarose bead, shownhere partially blocked with avidin.
(Bottom) Cantilever deflection curves on approachand retraction of an avidin tip on a biotinylatedagarose bead (A) before and (B) after blockage withan excess of free avidin (200µg/ml). (C) Magnificationof a force scan on a biotinylated agarose beadapproximately 95% blocked with free avidin. Fad is themeasured unbinding force plotted. Reprinted bypermission, reference (58).
12
Materials ScienceApplications
So far we have focused onmechanical and adhesive mea-surements on biological samples.It should be no surprise that thechemistry, physics and materialsscience communities are alsovery interested in the nano-scalemechanical properties of avariety of polymers, metals,semiconductors and insulators.Measurement of magnetic andelectric forces over industrialsamples like magnetic hard disksor doped semiconductors isalready a rapidly maturing field.In addition, measurements ofadhesion, elasticity and evenplastic deformation are becom-ing more common.
Nano-scale Mechanics
One of the main applicationareas of AFM force measure-ments in materials science is thestudy of mechanical propertiesof nanometer scale thin filmsand clusters. As microstructuressuch as integrated circuitscontinue to shrink, extrapolationof the mechanical behavior ofthin films from known proper-ties of the bulk materials be-comes increasingly inaccurate.Continuing demand for fastercomputers and larger capacitymemory and storage devicesplaces increasing importance onunderstanding nano-scalemechanics of metals and othercommonly used materials. Withits fine scale resolution and forcemeasurement capabilities, AFMis becoming an essential tool for
studying these nano-scalemechanics. An extensivediscussion of quantitativemeasurements of thin filmmechanical properties using theAFM is presented in the recentreview by Hues, Colton, Meyer,and Güntherodt. (81)
Polymers and Organic Films
Polymers and organic films areoften ideal samples for AFMforce studies since they areusually soft enough to be easilyindented by the AFM tip forces.In one study, Mizes and co-workers used AFM force mea-surements to perform nanoin-dentation and adhesion studieson doped and undoped polycar-bonate surfaces. (82) Usingforce curves to map localadhesion, they identified areaswhere adhesion varied indepen-dent of the surface topographyof the samples. Aimé and othershave recently described amacroscopic theory to allowinterpretation of AFM forcemeasurements on polymersurfaces. (83)
Organic films have also beenstudied by several groups. Forexample, Radmacher and othersused the force modulationtechnique to study elasticity andviscosity of Langmuir-Blodgett(LB) films. (74) Overney et al.used the AFM to study elasticity,wear and frictional properties oforganic thin films including LBfilms and mixtures offluorohydrocarbons. (84) Elasticproperties of fullerene(buckyball) films have also beenstudied with the AFM by Juai et
8
6
4
2
0 1 2
-10s10s20s80s
240s
Relative Sample Postion (µm)
Rela
tive
Forc
e (n
N)
ForceCurvesDuringCellFixation
Figure 12: (A) and (B) show the surface of an MDCKmonolayer before (A) and after (B) glutaraldehydefixation. The surface (B) is markedly rougher. Thegraph shows sequential AFM force measurements onthe cell monolayer during fixation. The force curvesbecome increasingly steep over time, illustrating themechanical stiffening of the monolayer during fixation.This work was done on a NanoScope MultiMode AFM.Reprinted by permission, reference (70).
13
AFM tip for molecular transpor-tation by moving fullerencemolecules from the originaldeposited film to anothersubstrate.
Thin Films, Clustersand Single Crystals
One way to study mechanicalproperties of metal thin films isto prepare unsupported metalfilms that are thin enough to bebent by the forces of the AFMcantilever. Torii and co-workersused this novel technique tomeasure elasticity of very thinaluminum samples. (86) Theshape of the contact region ofthe AFM force curve was used tocalculate sample elasticity. Toriicalculated the elastic modulus ofthe aluminum in the thin filmby first calibrating the springconstant of the AFM cantileverand then measuring the changein slope of the force curve forthe aluminum test sample.
Elastic measurements of thinmetal films on solid substratescan also be made. Chromium,molybdenum, and tungsten thinfilms were studied by Hues et al.(87), whereas Schaefer andcolleagues measured the elasticmodulus of nanometer-scalegold clusters.
Indentation and PlasticDeformation
Plastic deformation results whena material’s elastic limit isexceeded and a permanentdeformation is caused. Plastic
properties are usually studiedthrough a technique calledindentation, which refers toapplying a known force to asample with the AFM probe tip(or other probe) and thenobserving the size of the plasticindentation left by the probe. Aforce curve showing the processof plastic indentation on amagnetic recording tape isshown in Figure 13.
Indentation and, in fact,micromachining of copper andnickel surfaces has been per-formed by Sumomogi et al. (88)Bhushan and coworkers haveeven used the AFM with aspecially prepared diamond tipto perform indentation studieson single crystal silicon. (89)They used loading forces from60 to 100µN, creating indenta-tions as small as 1nm deep(Figure 14).
Summary
Atomic Force Microscopy hasmade its mark on a wide varietyof applications as a topographicmeasurement and mapping tool.Now AFM force measurementsare providing information onatomic- and molecular-scaleinteractions as well as nano-scaleadhesive and elastic response.These measurements are begin-ning to revolutionize the way wequantitatively observe and,indeed, think about our chemi-cal, biological and physicalworld. The vast majority ofAFM force measurements —including nearly all of those
Figure 13: NanoScope force curve showing plasticindentation on a magnetic tape sample. The graphshows two curves, one as the tip advances towardsand then indents into the surface (yellow), and another(white) as the tip withdraws from the surface. Theseparation between these two curves shows thatmaterial has been plastically displaced by the tip.Courtesy J. Elings, Digital Instruments.
described here — have beenperformed with NanoScopesystems and we are activelyadding functionality to makethese measurements increasinglyaccurate, repeatable and easier toperform. As described in thisarticle, much has already beenaccomplished in a variety ofapplications. With this technol-ogy still in its relative infancy,there is much more excitingwork yet to come.
al. (85) Juai’s team also used
12
Materials ScienceApplications
So far we have focused onmechanical and adhesive mea-surements on biological samples.It should be no surprise that thechemistry, physics and materialsscience communities are alsovery interested in the nano-scalemechanical properties of avariety of polymers, metals,semiconductors and insulators.Measurement of magnetic andelectric forces over industrialsamples like magnetic hard disksor doped semiconductors isalready a rapidly maturing field.In addition, measurements ofadhesion, elasticity and evenplastic deformation are becom-ing more common.
Nano-scale Mechanics
One of the main applicationareas of AFM force measure-ments in materials science is thestudy of mechanical propertiesof nanometer scale thin filmsand clusters. As microstructuressuch as integrated circuitscontinue to shrink, extrapolationof the mechanical behavior ofthin films from known proper-ties of the bulk materials be-comes increasingly inaccurate.Continuing demand for fastercomputers and larger capacitymemory and storage devicesplaces increasing importance onunderstanding nano-scalemechanics of metals and othercommonly used materials. Withits fine scale resolution and forcemeasurement capabilities, AFMis becoming an essential tool for
studying these nano-scalemechanics. An extensivediscussion of quantitativemeasurements of thin filmmechanical properties using theAFM is presented in the recentreview by Hues, Colton, Meyer,and Güntherodt. (81)
Polymers and Organic Films
Polymers and organic films areoften ideal samples for AFMforce studies since they areusually soft enough to be easilyindented by the AFM tip forces.In one study, Mizes and co-workers used AFM force mea-surements to perform nanoin-dentation and adhesion studieson doped and undoped polycar-bonate surfaces. (82) Usingforce curves to map localadhesion, they identified areaswhere adhesion varied indepen-dent of the surface topographyof the samples. Aimé and othershave recently described amacroscopic theory to allowinterpretation of AFM forcemeasurements on polymersurfaces. (83)
Organic films have also beenstudied by several groups. Forexample, Radmacher and othersused the force modulationtechnique to study elasticity andviscosity of Langmuir-Blodgett(LB) films. (74) Overney et al.used the AFM to study elasticity,wear and frictional properties oforganic thin films including LBfilms and mixtures offluorohydrocarbons. (84) Elasticproperties of fullerene(buckyball) films have also beenstudied with the AFM by Juai et
8
6
4
2
0 1 2
-10s10s20s80s
240s
Relative Sample Postion (µm)
Rela
tive
Forc
e (n
N)
ForceCurvesDuringCellFixation
Figure 12: (A) and (B) show the surface of an MDCKmonolayer before (A) and after (B) glutaraldehydefixation. The surface (B) is markedly rougher. Thegraph shows sequential AFM force measurements onthe cell monolayer during fixation. The force curvesbecome increasingly steep over time, illustrating themechanical stiffening of the monolayer during fixation.This work was done on a NanoScope MultiMode AFM.Reprinted by permission, reference (70).
13
AFM tip for molecular transpor-tation by moving fullerencemolecules from the originaldeposited film to anothersubstrate.
Thin Films, Clustersand Single Crystals
One way to study mechanicalproperties of metal thin films isto prepare unsupported metalfilms that are thin enough to bebent by the forces of the AFMcantilever. Torii and co-workersused this novel technique tomeasure elasticity of very thinaluminum samples. (86) Theshape of the contact region ofthe AFM force curve was used tocalculate sample elasticity. Toriicalculated the elastic modulus ofthe aluminum in the thin filmby first calibrating the springconstant of the AFM cantileverand then measuring the changein slope of the force curve forthe aluminum test sample.
Elastic measurements of thinmetal films on solid substratescan also be made. Chromium,molybdenum, and tungsten thinfilms were studied by Hues et al.(87), whereas Schaefer andcolleagues measured the elasticmodulus of nanometer-scalegold clusters.
Indentation and PlasticDeformation
Plastic deformation results whena material’s elastic limit isexceeded and a permanentdeformation is caused. Plastic
properties are usually studiedthrough a technique calledindentation, which refers toapplying a known force to asample with the AFM probe tip(or other probe) and thenobserving the size of the plasticindentation left by the probe. Aforce curve showing the processof plastic indentation on amagnetic recording tape isshown in Figure 13.
Indentation and, in fact,micromachining of copper andnickel surfaces has been per-formed by Sumomogi et al. (88)Bhushan and coworkers haveeven used the AFM with aspecially prepared diamond tipto perform indentation studieson single crystal silicon. (89)They used loading forces from60 to 100µN, creating indenta-tions as small as 1nm deep(Figure 14).
Summary
Atomic Force Microscopy hasmade its mark on a wide varietyof applications as a topographicmeasurement and mapping tool.Now AFM force measurementsare providing information onatomic- and molecular-scaleinteractions as well as nano-scaleadhesive and elastic response.These measurements are begin-ning to revolutionize the way wequantitatively observe and,indeed, think about our chemi-cal, biological and physicalworld. The vast majority ofAFM force measurements —including nearly all of those
Figure 13: NanoScope force curve showing plasticindentation on a magnetic tape sample. The graphshows two curves, one as the tip advances towardsand then indents into the surface (yellow), and another(white) as the tip withdraws from the surface. Theseparation between these two curves shows thatmaterial has been plastically displaced by the tip.Courtesy J. Elings, Digital Instruments.
described here — have beenperformed with NanoScopesystems and we are activelyadding functionality to makethese measurements increasinglyaccurate, repeatable and easier toperform. As described in thisarticle, much has already beenaccomplished in a variety ofapplications. With this technol-ogy still in its relative infancy,there is much more excitingwork yet to come.
al. (85) Juai’s team also used
15
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25. Radmacher, M., et al., Imagingadhesion forces and elasticity of lysozymeadsorbed to mica with the atomic forcemicroscope. Langmuir, 1994. 10(10): p.3809-3814.
26. Butt, H.-J., M. Jaschke, and W.Ducker, Measuring surface forces in aqueouselectrolyte solution with the atomic forcemicroscope. Bioelectrochemistry andBioenergetics, 1995. (38): p. 191-201in press.
27. Butt, H.J., Measuring electrostatic, vander Waals, and hydration forces inelectrolyte solutions with an atomic forcemicroscope. Biophys. J. (USA), 1991.60,(6): p. 1438-44.
65µN, 2.5nm, 16.6 GPa
100µN, 7.0nm, 11.7 GPa
60µN, 1.0nm
70µN, 3.0nm, 15.8 GPa
Figure 14: NanoScope AFMimages of silicon 111 surfacesafter indentation with a diamondAFM tip with loads of 60, 65, 70,and 100µN. The smallest loadproduced an indentation only 1nmdeep, the largest load created anindentation about 7nm deep.Reprinted by permission,reference (89).
15
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48. Ishino, T., et al., Electrical double-layerforces measured with an atomic forcemicroscope while electrochemicallycontrolling surface potential of thecantilever. Jpn. J. Appl. Phys. 2, Lett.(Japan), 1994. 33,(11A): p. L1552-4.
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50. Johnson, S.B., et al., Electrical doublelayer properties of hexadecyltrimethyl-ammonium chloride surfaces in aqueoussolution. Colloids and Surfaces A, 1995. inpress.
51. Johnson, S.B., et al., Comparison oftechniques for measuring the electricaldouble layer properties of surfaces inaqueous solution: hexadecyltrimethyl-ammonium bromide self-assemblystructures as a model system. Langmuir,1995. in press.
52. Ducker, W.A., Measurement of forcesin liquids using a force microscope.Langmuir, 1992. 8(7): p. 1831-36.
53. Rabinovich, Y.I. and R.-H. Yoon, Useof atomic force microscope for themeasurement of hydrophobic forcesbetween silanized silica plate and glassspheres. Langmuir, 1994. 10(6): p. 1903-1909.
54. Nakagawa, T., K. Ogawa, and T.Kurumizawa. Atomic force microscope forchemical sensing. in J. Vac. Sci. Technol. B,
Microelectron. Nanometer Struct. (USA).1994. Beijing, China.
55. Frisbie, C.D., et al., Functional groupimaging by chemical force microscopy.Science, 1994. 265(30 Sept. 1994): p. 2071-2073.
56. Noy, A., et al., Chemical fForcemicroscopy: Exploiting chemically-modifiedtips to quantify adhesion, friction, andfunctional group distributions in molecularassemblies. submitted, 1995.
57. Dammer, U., et al., Binding strengthbetween cell adhesion proteoglycansmeasured by atomic force microscopy.Science, 1995. 267(24 February 1995): p.1173-1175.
58. Florin, E.L., V.T. Moy, and H.E. Gaub,Adhesion forces between individual ligand-receptor pairs. Science (USA), 1994.264,(5157): p. 415-17.
59. Moy, V.T., E.-L. Florin, and H.E. Gaub,Inermolecular forces and energies betweenligands and receptors. Science, 1994. 265.
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61. Lee, G.U., L.A. Chrisey, and R.J.Colton, Direct measurement of the forcesbetween complementary strands of DNA.Science (USA), 1994. 266,(5186): p. 771-3.
62. Boland, T. and B.D. Ratner, Directmeasurement by atomic force microscopy ofhydrogen bonding in DNA NucleotideBases. Proc. Natl. Acad. Sci. USA, 1995. inpress.
63. Haselgrüber, T., et al., Synthesis andApplications of a new poly(ethylene glycol)derivative for the crosslinking of amines withthiols. Bioconjugate Chem., 1995. in press,.
64. Hinterdorfer, P., et al. Antibody-antigenunbinding forces measured by forcemicroscopy using antibodies bound to AFMtips via a specially designed flexiblecrosslinker. in presented at the BiophysicalSociety Meeting. 1994. San Francisco, CA.
65. Butt, H.J., Measuring local surfacecharge densities in electrolyte solutions witha scanning force microscope. Biophys. J.(USA), 1992. 63,(2): p. 578-82.
66. Leng, Y. and C.C. Williams. Molecularcharge mapping with the electrostatic forcemicroscope. in Proc. SPIE - Int. Soc. Opt.Eng. (USA). 1993. Los Angeles, CA, USA.
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14
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24. Torii, A., et al., Adhesive forcedistribution on microstructures investigatedby an atomic force microscope. Sens.Actuators A, Phys. (Switzerland), 1994.A44,(2): p. 153-8.
25. Radmacher, M., et al., Imagingadhesion forces and elasticity of lysozymeadsorbed to mica with the atomic forcemicroscope. Langmuir, 1994. 10(10): p.3809-3814.
26. Butt, H.-J., M. Jaschke, and W.Ducker, Measuring surface forces in aqueouselectrolyte solution with the atomic forcemicroscope. Bioelectrochemistry andBioenergetics, 1995. (38): p. 191-201in press.
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65µN, 2.5nm, 16.6 GPa
100µN, 7.0nm, 11.7 GPa
60µN, 1.0nm
70µN, 3.0nm, 15.8 GPa
Figure 14: NanoScope AFMimages of silicon 111 surfacesafter indentation with a diamondAFM tip with loads of 60, 65, 70,and 100µN. The smallest loadproduced an indentation only 1nmdeep, the largest load created anindentation about 7nm deep.Reprinted by permission,reference (89).
1
Probing Nano-Scale Forceswith the Atomic Force Microscope
TheWorldLeader InScanningProbeMicroscopy
ties are similarly important,often affecting the structural anddynamic behavior of systemsfrom composite materials toblood cells. AFM offers a newtool to study these importantparameters on the micron tonanometer scale using a tech-nique that measures forces onthe AFM probe tip as it ap-proaches and retracts from asurface. This application notedescribes measurement andconstruction of “force curves”and some of the importantapplications for this newtechnology.
The ability of the atomic forcemicroscope (AFM) to createthree-dimensional micrographswith resolution down to thenanometer and Angstrom scaleshas made it an essential tool forimaging surfaces in applicationsranging from semiconductorprocessing to cell biology. Inaddition to this topographicalimaging, however, the AFM canalso probe nanomechanical andother fundamental properties ofsample surfaces, including theirlocal adhesive or elastic (compli-ance) properties.
Microscopic adhesionaffects a huge varietyof events, from thebehavior of paintsand glues, ceramicsand compositematerials, to DNAreplication andthe action ofdrugs in thehuman body.Elastic proper-
���������
Position SensitiveDetector
CantileverWithProbe Tip
Laser
Basic AFM Operation
A key element of the AFM is itsmicroscopic force sensor, orcantilever (Figure 1). Thecantilever is usually formed byone or more beams of silicon orsilicon nitride that is 100 to500µm long and about 0.5 to5µm thick. Mounted on the endof the cantilever is a sharp tipthat is used to sense a forcebetween the sample and tip. Fornormal topographic imaging, theprobe tip is brought intocontinuous or intermittentcontact with the sample and
Figure 1: Schematic of an atomic force microscope (AFM) showing theforce sensing cantilever.
Figure 2: Typical 3-D surface topographic imageproduced by the AFM. Sample shown is aTetrahymena Protozoan. 50µm scan courtesy E.Henderson, BioForce Labs and Iowa State University.
By C.B. Prater,P.G. Maivald,K.J. Kjoller,M.G. Heaton
16
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99. Chan, D., “AFM analysis”, : Dept. ofMathematics, University of Melbourne,Parkville 3052 Australia. Fax +61-3-344-4599, email [email protected].
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