Electromechanical imaging of tooth dentin and enamel has been performed with
sub-10 nm resolution using piezoresponse force microscopy. Characteristic
piezoelectric domain size and local protein fiber ordering in dentin have been
determined. The shape of a single collagen fibril in enamel is visualized in
real space and local hysteresis loops are measured. Because of the ubiquitous
presence of piezoelectricity in biological systems, this approach is expected
to find broad application in high-resolution studies of a wide range of
biomaterials.Comment: 12 pages, 4 figures, submitted for publication in Appl. Phys. Let

A. Gruverman

B. J. Rodriguez

Bazhenov V. A.

S. Jesse

Sergei V. Kalinin

T. Thundat

English

arXiv

Electromechanical imaging of tooth dentin and enamel has been performed with sub-10 nm resolution using piezoresponse force microscopy. Characteristic piezoelectric domain size and local protein fiber ordering in dentin have been determined. The shape of a single protein fibril in enamel is visualized in real space and local hysteresis loops are measured. Because of the ubiquitous presence of piezoelectricity in biological systems, this approach is expected to find broad application in high-resolution studies of a wide range of biomaterials

Kalinin, Sergei V.

Rodriguez, B. J.

Jesse, Stephen

Thundat, T.

Gruverman, Alexei

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Alexei Gruverman Publications Research Papers in Physics and Astronomy 7-28-2005 Electromechanical imaging of biological systems with sub-10 nm resolution Sergei V. Kalinin Condensed Matter Sciences Division, Oak Ridge National Laboratory, Tennessee, sergei2@ornl.gov B. J. Rodriguez North Carolina State University, Raleigh, brian.rodriguez@ucd.ie Stephen Jesse Oak Ridge National Laboratory, sjesse@ornl.gov T. Thundat Life Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee Alexei Gruverman University of Nebraska-Lincoln, agruverman2@unl.edu Follow this and additional works at: https://digitalcommons.unl.edu/physicsgruverman  Part of the Physics Commons Kalinin, Sergei V.; Rodriguez, B. J.; Jesse, Stephen; Thundat, T.; and Gruverman, Alexei, "Electromechanical imaging of biological systems with sub-10 nm resolution" (2005). Alexei Gruverman Publications. 18. https://digitalcommons.unl.edu/physicsgruverman/18 This Article is brought to you for free and open access by the Research Papers in Physics and Astronomy at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Alexei Gruverman Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Electromechanical imaging of biological systems with sub-10 nmresolutionSergei V. KalininaCondensed Matter Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831B. J. RodriguezDepartment of Physics, North Carolina State University, Raleigh, North Carolina 27695S. JesseCondensed Matter Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831T. ThundatLife Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831A. GruvermanaDepartment of Materials Science and Engineering, North Carolina State University, Raleigh,North Carolina 27695Received 11 March 2005; accepted 15 June 2005; published online 28 July 2005Electromechanical imaging of tooth dentin and enamel has been performed with sub-10 nmresolution using piezoresponse force microscopy. Characteristic piezoelectric domain size and localprotein fiber ordering in dentin have been determined. The shape of a single protein fibril in enamelis visualized in real space and local hysteresis loops are measured. Because of the ubiquitouspresence of piezoelectricity in biological systems, this approach is expected to find broad applicationin high-resolution studies of a wide range of biomaterials. © 2005 American Institute of Physics.DOI: 10.1063/1.2006984Electromechanical interactions at hierarchically differentstructural levels, from molecular to intra- and intercellular,play an important role in the functionality in biological sys-tems. One of the most common examples of electromechani-cal coupling in biological systems is piezoelectricity, discov-ered almost 50 years ago in such dissimilar biomaterials asbones, tendon, and wood.1–4 Since then, the direct and con-verse piezoelectric effect has been observed in most biologi-cal materials based on ordered molecular arrays of proteinsor polysaccharides. It has been argued that the presence ofpolar groups, such as -CO–NH- and -CO–O-, coupled withintrinsic chirality of biomolecules, determines the linearelectromechanical coupling in these systems. A number ofstudies have addressed the biological role of piezoelectricityas an intrinsic property of biological systems.5–7However, experimental measurements of piezoelectricityin biological systems have been hindered by the complexhierarchical structure of calcified and connective tissues andeffects related to conductivity and streaming potentials. Inthe former case, the assumption of sample uniformity, im-plicitly used in the interpretation of electromechanical mea-surements in single crystals, is no longer valid. In fact, eventhe symmetries of the piezoelectric tensors in previouslystudied biosystems are generally unknown. Furthermore, thepresence of ionic conductivity significantly complicates mac-roscopic electromechanical measurements. Thus, understand-ing of the fundamental mechanisms of electromechanicalcoupling in biological systems necessitates studies on thelevel of a single structural element, such as protein fibril inthe calcified and connective tissues, a task still not achieveddespite several attempts.8Here, we demonstrate electromechanical imaging andmicrostructural analysis of dentin and enamel in a humantooth with sub-10 nm resolution using vertical and lateralpiezoresponse force microscopy PFM. In PFM, a conduc-tive tip is brought into contact with the surface, and the pi-ezoelectric response is detected as the first harmonic compo-nent, A1, of the tip deflection, A=A0+A1 cost+,induced by the application of the periodic bias Vtip=Vdc+Vac cost to the tip. Principles and image formationmechanism of PFM are described in detail elsewhere.9–11PFM is implemented on a commercial scanning probe mi-croscopy SPM system Veeco MultiMode NS-IIIAequipped with additional function generators and lock-in am-plifiers DS 345 and SRS 830, Stanford Research Instru-ments, and Model 7280, Signal Recovery. A custom-builtsample holder was used to allow direct tip biasing and toavoid capacitive cross talk in the SPM electronics. Measure-ments were performed using Pt- and Au-coated tipsNCSC-12 C, Micromasch, l130 m, resonant frequency150 kHz, spring constant k4.5 N/m. Vertical PFMVPFM measurements were performed at frequencies50–100 kHz, which minimizes the longitudinal contributionto measured vertical signal.12 For lateral PFM LPFM, theoptimal conditions for contrast transfer were 10 kHz; forhigher frequencies, the onset of sliding friction minimizesin-plane oscillation transfer between the tip and the surface.Custom LABVIEW software was developed for simultaneousacquisition of VPFM and LPFM phase and amplitude data,emulating additional SPM data acquisitions channels.The surface layer of tooth enamel is comprised primarilyof hydroxyapatite HAP crystals and a small fraction3–5%  of organic fibers concentrated mostly in the vicin-ity of the dentin-enamel junction DEJ. The dentin layerbelow the enamel has significantly higher fraction of organicaAuthors to whom correspondence should be addressed; electronic mail:sergei2@ornl.gov and alexeigruverman@ncsu.eduAPPLIED PHYSICS LETTERS 87, 053901 20050003-6951/2005/875/053901/3/$22.50 © 2005 American Institute of Physics87, 053901-1material, up to 30–40%. The dominant organic constituent ofdentin and other calcified and connective tissues is collagen.Collagen, the most abundant protein in biological systems,exhibits piezoelectric properties, resulting in the nearly ubiq-uitous presence of piezoelectricity in connective and partiallycalcified tissues.13A deciduous human tooth was cross sectioned parallel togrowth direction and polished using diamond polishing padsdown to 0.5 m grit size. The enamel and dentin regions canbe readily identified using optical microscopy. Surface to-pography of dentin and enamel regions is shown in Figs. 1aand 1b.14 It can be seen that both regions are comprised ofelongated grains of about 100–300 nm in size, however, thedifference in microstructure between the two regions issmall. Shown in comparison are simultaneously acquiredamplitude and phase VPFM images Figs. 1c–1f. Inenamel, several isolated regions of 50–200 nm in size with ahigh piezoresponse signal appearing as bright spots in am-plitude image are observed, while the majority of material isnonpiezoelectric. At the same time, most of the dentin regionexhibits a strong response signal, with the typical size ofregions of constant phase and amplitude piezoelectric do-mains of the order of 200 nm. These observations are con-sistent with a high density of piezoelectrically active pro-teins, such as collagen, in dentin13 and a low fraction ofproteins in enamel.Simultaneous measurements of the vertical and lateralpiezoresponse map allow us to determine two components oflocal electromechanical response vector, which can be fur-ther correlated with topography or elasticity data from, e.g.,phase atomic force microscopy. Particularly, VPFM andLPFM data provide the basis for the statistical description ofthe electromechanical microstructure of the material. Shownin Fig. 2a is the double histogram of normalized VPFM andLPFM signals obtained in the dentin region, representing thecount number of points with the signal level in the intervalvpr+v , lpr+l, where vpr , lpr −1,1. Shown in Figs.2b and 2c are the amplitude, A2D, and angle, 2D, signaldistributions, where A2D=Absvpr+ I lpr, 2D=Argvpr+ I lpr calculated using commercial image analysissoftware.15 Note that the angle calculated from VPFM andLPFM data represents the orientation of the piezoresponsevector in the plane perpendicular to the cantilever axis andthus serves as the measure of local collagen fibril orientation.Data shown in Figs. 2a and 2c illustrate that there aretwo primary antiparallel orientations of the piezoresponsevector. Thus, the local dentin microstructure can be well rep-resented by axially ordered antiparallel collagen fibers, asshown in the inset of Fig. 2d. The characteristic fiber sizecan be determined using self-correlation function analysis asis illustrated in Fig. 2d. The normalized experimental func-tion can be well approximated using a simple phenomeno-logical form Cx=A exp−x /, where characteristic domainsize  is 160±2 nm, in agreement with data from Fig. 1d.To get further insight into the structure of the isolatedprotein presumably ameliogenin fibrils in enamel, we haveperformed high-resolution VPFM and LPFM imaging of theenamel region in the DEJ vicinity, as illustrated in Fig. 3.Both VPFM and LPFM images show a very strong electro-mechanical response that we attribute to a protein fibril em-bedded within a nonpiezoelectric matrix. Comparison of theVPFM and LPFM images shows different patterns of piezo-electric domains, suggesting a complicated structure of theprotein fibril, consisting of several protein molecules. Nota-bly, the spatial resolution of PFM, determined as a half-widthof the boundary between differently oriented piezoelectricregions, is 5 nm. This is comparable to the best resultsachieved to date for thin films of ferroelectric perovskitesand well above 30–100 nm resolution for single crystals.FIG. 1. Surface topography a,b, vertical piezoresponse amplitude c,d andphase e,f for enamel a,c,e and dentin b,d,f. Vertical scale is 50 nm. Theamplitude images illustrate regions with strong white and weak blackpiezoelectric coupling, the phase images provide information of polarizationorientation black—upward, white—downward.FIG. 2. a Double histogram of the VPFM and LPFM signals over 33 m2 region of dentin. b,c Amplitude and phase distribution of piezo-response vector. d Self-correlation function for the PFM signal. Dotted lineis fitting using Cx=A exp−x /, where the characteristic domain size  is160 nm. The inset shows the simplified nanostructural model for dentinformed by antiparallel collagen fibrils.FIG. 3. a Surface topography and b VPFM and c LPFM x signals of asingle-collagen fibril in enamel.053901-2 Kalinin et al. Appl. Phys. Lett. 87, 053901 2005Finally, to check the presence of the ferroelectric polar-ization in the material, PFM was used to acquire local hys-teresis loop in a manner similar to ferroelectric materials.16The piezoresponse, measured as a function of a dc bias offseton the tip, is shown in Fig. 4. No inversion of the strain signupon application of the dc bias was observed, indicating thatcollagen molecules possess strong piezoelectric properties,comparable to quartz, but are not ferroelectric. Notably, theslope of the amplitude response is small, indicative of mini-mal electrostatic contribution to measured signal. The effec-tive piezoelectric coefficient is dlocal=0.15–0.25 pm/V,nearly ten times larger than d=0.028 pm/V for a macro-scopic dentin sample and comparable to d=0.28 pm/V fordry bone.13 Interestingly, these observations are consistentwith the antiparallel orientation of collagen fibrils in dentin,since the electromechanical response will be cancelled out onthe macroscopic scale.To summarize, we have demonstrated the electrome-chanical imaging of dental tissues with sub-10 nanometerresolution. PFM provides an approach to distinguish betweenmaterials with different electromechanical properties, such asproteins and calcified tissues. PFM allows statistical descrip-tion of dental tissue structure, including the characteristicfiber size and type of local ordering. The collagen fibril hasbeen visualized in real space. The local electromechanicalproperties of dental tissues are measured as a function of tipbias and the material is shown to be piezoelectric, rather thanferroelectric. Due to the ubiquity of piezoelectricity in bio-logical systems, we expect this approach to find broad appli-cation in future high-resolution studies of biomaterials.Research performed in part as a Eugene P. Wigner Fel-low and staff member at the Oak Ridge National Laboratory,managed by UT-Battelle, LLC, for the U.S. Department ofEnergy under Contract DE-AC05-00OR22725 for S.V.K.A.G. acknowledges financial support from the National Sci-ence Foundation Grant No. DMR02-35632.1E. Fukada and I. Yasuda, J. Phys. Soc. Jpn. 12, 1158 1957.2E. Fukada, Jpn. J. Appl. Phys. 3, 117 1964.3E. Fukada, J. Phys. Jpn. 10, 149 1955.4V. A. Bazhenov, Piezoelectric Properties of Wood Consultants Bureau,New York, 1961.5E. Fukada, Biorheology 32, 593 1995.6C. A. L. Bassett, Calcif. Tissue Res. 1, 252 1968.7A. A. Marino and R. O. Becker, Nature London 228, 473 1970.8C. Halperin, S. Mutchnik, A. Agronin, M. Molotskii, P. Urenski, M. Salai,and G. Rosenman, Nano Lett. 4, 1253 2004.9A. Gruverman, O. Auciello, and H. Tokumoto, Annu. Rev. Mater. Sci. 28,101 1998.10L. M. Eng, S. Grafstrom, C. Loppacher, F. Schlaphof, S. Trogisch, A.Roelofs, and R. Waser, Adv. Solid State Phys. 41, 287 2001.11S. V. Kalinin, E. Karapetian, and M. Kachanov, Phys. Rev. B 70, 1841012004.12S. V. Kalinin, B. J. Rodriguez, S. Jesse, J. Shin, A. P. Baddorf, P. Gupta, H.Jain, D. B. Williams, and A. Gruverman unpublished.13A. A. Marino and B. D. Gross, Arch. Oral Biol. 34, 507 1989.14The enamel region has been imaged close to the DEJ where nonzeroconcentration of the protein fibers can be expected.15Mathematika 5.0, Wolfram Research, Champaign, IL.16M. Alexe, A. Gruverman, C. Harnagea, N. D. Zakharov, A. Pignolet, D.Hesse, and J. F. Scott, Appl. Phys. Lett. 75, 1158 1999.FIG. 4. Piezoelectric hysteresis loops of a single-collagen fibril in toothenamel.053901-3 Kalinin et al. Appl. Phys. Lett. 87, 053901 2005

Electromechanical imaging of biological systems with sub-10 nm resolution

Electromechanical imaging of tooth dentin and enamel has been performed with sub-10 nm resolution using piezoresponse force microscopy. Characteristic piezoelectric domain size and local protein fiber ordering in dentin have been determined. The shape of a single protein fibril in enamel is visualized in real space and local hysteresis loops are measured. Because of the ubiquitous presence of piezoelectricity in biological systems, this approach is expected to find broad application in high-resolution studies of a wide range of biomaterials. (c) 2005 American Institute of Physics.National Science FoundationAuthor has checked copyrightAD 09/01/201

Kalinin, S. V.

Rodriguez, Brian J.

Jesse, S.

et al.

Research Repository UCD

Title Electromechanical imaging of biological systems with sub-10 nm resolutionAuthors(s) Kalinin, S. V., Rodriguez, Brian J., Jesse, S., et al.Publication date 2005-08Publication information Kalinin, S. V., Brian J. Rodriguez, S. Jesse, and et al. “Electromechanical Imaging of Biological Systems with Sub-10 Nm Resolution” 87, no. 5 (August, 2005).Publisher American Institute of PhysicsItem record/more informationhttp://hdl.handle.net/10197/5334Publisher's version (DOI) 10.1063/1.2006984Downloaded 2023-11-01T04:02:15ZThe UCD community has made this article openly available. Please share how this accessbenefits you. Your story matters! (@ucd_oa)© Some rights reserved. For more informationElectromechanical imaging of biological systems with sub-10 nmresolutionSergei V. KalininaCondensed Matter Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831B. J. RodriguezDepartment of Physics, North Carolina State University, Raleigh, North Carolina 27695S. JesseCondensed Matter Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831T. ThundatLife Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831A. GruvermanaDepartment of Materials Science and Engineering, North Carolina State University, Raleigh,North Carolina 27695Received 11 March 2005; accepted 15 June 2005; published online 28 July 2005Electromechanical imaging of tooth dentin and enamel has been performed with sub-10 nmresolution using piezoresponse force microscopy. Characteristic piezoelectric domain size and localprotein fiber ordering in dentin have been determined. The shape of a single protein fibril in enamelis visualized in real space and local hysteresis loops are measured. Because of the ubiquitouspresence of piezoelectricity in biological systems, this approach is expected to find broad applicationin high-resolution studies of a wide range of biomaterials. © 2005 American Institute of Physics.DOI: 10.1063/1.2006984Electromechanical interactions at hierarchically differentstructural levels, from molecular to intra- and intercellular,play an important role in the functionality in biological sys-tems. One of the most common examples of electromechani-cal coupling in biological systems is piezoelectricity, discov-ered almost 50 years ago in such dissimilar biomaterials asbones, tendon, and wood.1–4 Since then, the direct and con-verse piezoelectric effect has been observed in most biologi-cal materials based on ordered molecular arrays of proteinsor polysaccharides. It has been argued that the presence ofpolar groups, such as -CO–NH- and -CO–O-, coupled withintrinsic chirality of biomolecules, determines the linearelectromechanical coupling in these systems. A number ofstudies have addressed the biological role of piezoelectricityas an intrinsic property of biological systems.5–7However, experimental measurements of piezoelectricityin biological systems have been hindered by the complexhierarchical structure of calcified and connective tissues andeffects related to conductivity and streaming potentials. Inthe former case, the assumption of sample uniformity, im-plicitly used in the interpretation of electromechanical mea-surements in single crystals, is no longer valid. In fact, eventhe symmetries of the piezoelectric tensors in previouslystudied biosystems are generally unknown. Furthermore, thepresence of ionic conductivity significantly complicates mac-roscopic electromechanical measurements. Thus, understand-ing of the fundamental mechanisms of electromechanicalcoupling in biological systems necessitates studies on thelevel of a single structural element, such as protein fibril inthe calcified and connective tissues, a task still not achieveddespite several attempts.8Here, we demonstrate electromechanical imaging andmicrostructural analysis of dentin and enamel in a humantooth with sub-10 nm resolution using vertical and lateralpiezoresponse force microscopy PFM. In PFM, a conduc-tive tip is brought into contact with the surface, and the pi-ezoelectric response is detected as the first harmonic compo-nent, A1, of the tip deflection, A=A0+A1 cost+,induced by the application of the periodic bias Vtip=Vdc+Vac cost to the tip. Principles and image formationmechanism of PFM are described in detail elsewhere.9–11PFM is implemented on a commercial scanning probe mi-croscopy SPM system Veeco MultiMode NS-IIIAequipped with additional function generators and lock-in am-plifiers DS 345 and SRS 830, Stanford Research Instru-ments, and Model 7280, Signal Recovery. A custom-builtsample holder was used to allow direct tip biasing and toavoid capacitive cross talk in the SPM electronics. Measure-ments were performed using Pt- and Au-coated tipsNCSC-12 C, Micromasch, l130 m, resonant frequency150 kHz, spring constant k4.5 N/m. Vertical PFMVPFM measurements were performed at frequencies50–100 kHz, which minimizes the longitudinal contributionto measured vertical signal.12 For lateral PFM LPFM, theoptimal conditions for contrast transfer were 10 kHz; forhigher frequencies, the onset of sliding friction minimizesin-plane oscillation transfer between the tip and the surface.Custom LABVIEW software was developed for simultaneousacquisition of VPFM and LPFM phase and amplitude data,emulating additional SPM data acquisitions channels.The surface layer of tooth enamel is comprised primarilyof hydroxyapatite HAP crystals and a small fraction3–5%  of organic fibers concentrated mostly in the vicin-ity of the dentin-enamel junction DEJ. The dentin layerbelow the enamel has significantly higher fraction of organicaAuthors to whom correspondence should be addressed; electronic mail:sergei2@ornl.gov and alexeigruverman@ncsu.eduAPPLIED PHYSICS LETTERS 87, 053901 20050003-6951/2005/875/053901/3/$22.50 © 2005 American Institute of Physics87, 053901-1 This article is copyrighted as indicated in the abstract. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP:137.43.140.249 On: Fri, 22 Nov 2013 17:09:11material, up to 30–40%. The dominant organic constituent ofdentin and other calcified and connective tissues is collagen.Collagen, the most abundant protein in biological systems,exhibits piezoelectric properties, resulting in the nearly ubiq-uitous presence of piezoelectricity in connective and partiallycalcified tissues.13A deciduous human tooth was cross sectioned parallel togrowth direction and polished using diamond polishing padsdown to 0.5 m grit size. The enamel and dentin regions canbe readily identified using optical microscopy. Surface to-pography of dentin and enamel regions is shown in Figs. 1aand 1b.14 It can be seen that both regions are comprised ofelongated grains of about 100–300 nm in size, however, thedifference in microstructure between the two regions issmall. Shown in comparison are simultaneously acquiredamplitude and phase VPFM images Figs. 1c–1f. Inenamel, several isolated regions of 50–200 nm in size with ahigh piezoresponse signal appearing as bright spots in am-plitude image are observed, while the majority of material isnonpiezoelectric. At the same time, most of the dentin regionexhibits a strong response signal, with the typical size ofregions of constant phase and amplitude piezoelectric do-mains of the order of 200 nm. These observations are con-sistent with a high density of piezoelectrically active pro-teins, such as collagen, in dentin13 and a low fraction ofproteins in enamel.Simultaneous measurements of the vertical and lateralpiezoresponse map allow us to determine two components oflocal electromechanical response vector, which can be fur-ther correlated with topography or elasticity data from, e.g.,phase atomic force microscopy. Particularly, VPFM andLPFM data provide the basis for the statistical description ofthe electromechanical microstructure of the material. Shownin Fig. 2a is the double histogram of normalized VPFM andLPFM signals obtained in the dentin region, representing thecount number of points with the signal level in the intervalvpr+v , lpr+l, where vpr , lpr −1,1. Shown in Figs.2b and 2c are the amplitude, A2D, and angle, 2D, signaldistributions, where A2D=Absvpr+ I lpr, 2D=Argvpr+ I lpr calculated using commercial image analysissoftware.15 Note that the angle calculated from VPFM andLPFM data represents the orientation of the piezoresponsevector in the plane perpendicular to the cantilever axis andthus serves as the measure of local collagen fibril orientation.Data shown in Figs. 2a and 2c illustrate that there aretwo primary antiparallel orientations of the piezoresponsevector. Thus, the local dentin microstructure can be well rep-resented by axially ordered antiparallel collagen fibers, asshown in the inset of Fig. 2d. The characteristic fiber sizecan be determined using self-correlation function analysis asis illustrated in Fig. 2d. The normalized experimental func-tion can be well approximated using a simple phenomeno-logical form Cx=A exp−x /, where characteristic domainsize  is 160±2 nm, in agreement with data from Fig. 1d.To get further insight into the structure of the isolatedprotein presumably ameliogenin fibrils in enamel, we haveperformed high-resolution VPFM and LPFM imaging of theenamel region in the DEJ vicinity, as illustrated in Fig. 3.Both VPFM and LPFM images show a very strong electro-mechanical response that we attribute to a protein fibril em-bedded within a nonpiezoelectric matrix. Comparison of theVPFM and LPFM images shows different patterns of piezo-electric domains, suggesting a complicated structure of theprotein fibril, consisting of several protein molecules. Nota-bly, the spatial resolution of PFM, determined as a half-widthof the boundary between differently oriented piezoelectricregions, is 5 nm. This is comparable to the best resultsachieved to date for thin films of ferroelectric perovskitesand well above 30–100 nm resolution for single crystals.FIG. 1. Surface topography a,b, vertical piezoresponse amplitude c,d andphase e,f for enamel a,c,e and dentin b,d,f. Vertical scale is 50 nm. Theamplitude images illustrate regions with strong white and weak blackpiezoelectric coupling, the phase images provide information of polarizationorientation black—upward, white—downward.FIG. 2. a Double histogram of the VPFM and LPFM signals over 33 m2 region of dentin. b,c Amplitude and phase distribution of piezo-response vector. d Self-correlation function for the PFM signal. Dotted lineis fitting using Cx=A exp−x /, where the characteristic domain size  is160 nm. The inset shows the simplified nanostructural model for dentinformed by antiparallel collagen fibrils.FIG. 3. a Surface topography and b VPFM and c LPFM x signals of asingle-collagen fibril in enamel.053901-2 Kalinin et al. Appl. Phys. Lett. 87, 053901 2005 This article is copyrighted as indicated in the abstract. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP:137.43.140.249 On: Fri, 22 Nov 2013 17:09:11Finally, to check the presence of the ferroelectric polar-ization in the material, PFM was used to acquire local hys-teresis loop in a manner similar to ferroelectric materials.16The piezoresponse, measured as a function of a dc bias offseton the tip, is shown in Fig. 4. No inversion of the strain signupon application of the dc bias was observed, indicating thatcollagen molecules possess strong piezoelectric properties,comparable to quartz, but are not ferroelectric. Notably, theslope of the amplitude response is small, indicative of mini-mal electrostatic contribution to measured signal. The effec-tive piezoelectric coefficient is dlocal=0.15–0.25 pm/V,nearly ten times larger than d=0.028 pm/V for a macro-scopic dentin sample and comparable to d=0.28 pm/V fordry bone.13 Interestingly, these observations are consistentwith the antiparallel orientation of collagen fibrils in dentin,since the electromechanical response will be cancelled out onthe macroscopic scale.To summarize, we have demonstrated the electrome-chanical imaging of dental tissues with sub-10 nanometerresolution. PFM provides an approach to distinguish betweenmaterials with different electromechanical properties, such asproteins and calcified tissues. PFM allows statistical descrip-tion of dental tissue structure, including the characteristicfiber size and type of local ordering. The collagen fibril hasbeen visualized in real space. The local electromechanicalproperties of dental tissues are measured as a function of tipbias and the material is shown to be piezoelectric, rather thanferroelectric. Due to the ubiquity of piezoelectricity in bio-logical systems, we expect this approach to find broad appli-cation in future high-resolution studies of biomaterials.Research performed in part as a Eugene P. Wigner Fel-low and staff member at the Oak Ridge National Laboratory,managed by UT-Battelle, LLC, for the U.S. Department ofEnergy under Contract DE-AC05-00OR22725 for S.V.K.A.G. acknowledges financial support from the National Sci-ence Foundation Grant No. DMR02-35632.1E. Fukada and I. Yasuda, J. Phys. Soc. Jpn. 12, 1158 1957.2E. Fukada, Jpn. J. Appl. Phys. 3, 117 1964.3E. Fukada, J. Phys. Jpn. 10, 149 1955.4V. A. Bazhenov, Piezoelectric Properties of Wood Consultants Bureau,New York, 1961.5E. Fukada, Biorheology 32, 593 1995.6C. A. L. Bassett, Calcif. Tissue Res. 1, 252 1968.7A. A. Marino and R. O. Becker, Nature London 228, 473 1970.8C. Halperin, S. Mutchnik, A. Agronin, M. Molotskii, P. Urenski, M. Salai,and G. Rosenman, Nano Lett. 4, 1253 2004.9A. Gruverman, O. Auciello, and H. Tokumoto, Annu. Rev. Mater. Sci. 28,101 1998.10L. M. Eng, S. Grafstrom, C. Loppacher, F. Schlaphof, S. Trogisch, A.Roelofs, and R. Waser, Adv. Solid State Phys. 41, 287 2001.11S. V. Kalinin, E. Karapetian, and M. Kachanov, Phys. Rev. B 70, 1841012004.12S. V. Kalinin, B. J. Rodriguez, S. Jesse, J. Shin, A. P. Baddorf, P. Gupta, H.Jain, D. B. Williams, and A. Gruverman unpublished.13A. A. Marino and B. D. Gross, Arch. Oral Biol. 34, 507 1989.14The enamel region has been imaged close to the DEJ where nonzeroconcentration of the protein fibers can be expected.15Mathematika 5.0, Wolfram Research, Champaign, IL.16M. Alexe, A. Gruverman, C. Harnagea, N. D. Zakharov, A. Pignolet, D.Hesse, and J. F. Scott, Appl. Phys. Lett. 75, 1158 1999.FIG. 4. Piezoelectric hysteresis loops of a single-collagen fibril in toothenamel.053901-3 Kalinin et al. Appl. Phys. Lett. 87, 053901 2005 This article is copyrighted as indicated in the abstract. Reuse of AIP content is subject to the terms at: http://scitation.aip.org/termsconditions. Downloaded to IP:137.43.140.249 On: Fri, 22 Nov 2013 17:09:11

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Electromechanical imaging of biological systems with sub-10nm resolution

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