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Nanoscale elasticity mappings of micro-constituents of abalone shell by band excitation-contact resonance force microscopy† Tao Li and Kaiyang Zeng* The macroscopic mechanical properties of the abalone shell have been studied extensively in the literature, but the in situ nanoscale elasticity of various micro-constituents in the shell have not been characterized and reported yet. In this study, the nanoscale elasticity mappings including different micro-constituents in abalone shell were observed by using the Contact Resonance Force Microscopy (CR-FM) technique. CR-FM is one of the advanced scanning probe microscopy techniques that is able to quantify the local elastic moduli of various materials in a non-destructive manner. Instead of an average value, an elasticity mapping that reveals the nanoscale variations of elastic moduli with location can be extracted and
Received 4th October 2013 Accepted 14th November 2013
correlated with the topography of the structure. Therefore in this study, by adopting the CR-FM technique that is incorporated with the band excitation technique, the elasticity variations of the abalone
DOI: 10.1039/c3nr05292c
shell caused by different micro-constituents and crystal orientations are reported, and the elasticity
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values of the aragonite and calcite nanograins are quantified.
Introduction Mollusk shell is a typical natural nanocomposite and also an armour which has protected the host mollusk from predators over billions of years of evolution. The shell is generally composed by more than 95 wt% of calcium carbonate (CaCO3) and less than 5 wt% of biopolymers (mainly chitin and various proteins). The two micro-constituents together form multiple levels of hierarchical structures via a biomineralization process under ambient conditions.1,2 The mollusk shell is well-known for its outstanding mechanical properties (in terms of toughness and strength) compared to those of their individual microconstituents. Amongst various shell structures, the mechanical properties of nacre have been most extensively studied due to its better toughness behavior compared to others.3 The nacre structure is found in many species of mollusk shells, including abalone shell. The nacre structure is composed of CaCO3 mineral layers (around 500 nm in thickness) joined by thin layers of biopolymers, which are usually called interlamellar biopolymers (thickness range within 5 to 20 nm).4 Thus, when viewed on the cross-section, it shows a “bricks-and-mortar” type of structure. In addition, each mineral layer is constructed by closelypacked polygonal CaCO3 (aragonite) platelets (size range within 5 to 10 mm) that are separated by intertabular biopolymers.1 Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576. E-mail: [email protected] † Electronic supplementary 10.1039/c3nr05292c
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Furthermore, each platelet is composed of a large number of nanograins (38 21 nm of diameter) that are embedded in an intracrystalline biopolymer matrix (4 nm thick).5 There are many reports presenting the average value of the elastic moduli of the entire nacre structure and the micro-constituents, which were usually measured by traditional mechanical testing methods, such as micro-/nano-indentation with numerical modelling.5–9 Table 1 summarizes some of the reported elastic modulus values of the nacre and its constituents. Depending on the testing methods, large deviations of the elasticity values are reported, especially for the biopolymers. This is due to the difficulties of measuring the value of elastic modulus of the biopolymers in situ, especially for the intracrystalline biopolymers. Thus, many of the reported values are obtained by using nite element (FE) modelling. Furthermore, in abalone shell, there is an outermost shell section which works as the rst shield when mollusk is under attack, and this section consists mainly of CaCO3 in the calcite polymorph. In this shell section, large calcite prisms of tens of micrometers are enveloped in the interprismatic biopolymers.10 Each of these prisms is reported as a single crystal. The reported mechanical properties of the calcite section in abalone shell is scarce. The elastic modulus of pure calcite crystals was reported to be about 72 and 88 GPa, depending on the crystal orientations.11 Generally-speaking, the traditional mechanical testing methods are usually destructive, and by these only the average values can be obtained. These methods are not very suitable to depict the elasticity of a nanocomposite, especially the complex hierarchical structures, such as those in the abalone shell. In this case, contact resonance force microscopy (CR-FM) shows
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Elastic moduli of different component in abalone shell that reported in the literatures
E/GPa
Method
Shell species
Overall nacre section 72.6 5.8 69 7 79 15 62.5 11.2
Nanoindentation Nanoindentation Nanoindentation Nanoindentation
Haliotis rufescens Haliotis rufescens Haliotis rufescens Pinctada maxima
Biopolymers 40 11 3 15 2.84 0.27 6.3 0.56 3.81 0.41 49 3
CR-FM & FE analysis Inverse FE analysis 3D FE modelling Nanoindentation Modelling Mori–Tanaka's model Nanoindentation
Perna canaliculus Haliotis rufescens Haliotis rufescens Haliotis rufescens Pinctada maxima Pinctada maxima Haliotis rufescens
Interlamellar, b-chitin Interlamellar polymer strand Interlamellar Interlamellar Interlamellar Intracrystalline Interlamellar
14 12 15 13 16 5 12
Aragonite Nanograin 82.7 96.75 5.67
FE modelling Sharp nanoindentation
Haliotis rufescens Pinctada maxima
Single-crystal Nanograins
13 5
advantages to study the local elasticity of the nanocomposite structure. Moshe-Drezner et al. have demonstrated the usage of CR-FM in mollusk shell.14 In their study, the elastic modulus of b-chitin in nacre was reported to be 40 GPa based on experimental observation and nite element analysis. However, it did not provide any high spacial resolution mapping of the nanograins and biopolymers. CR-FM is a general name of the dynamic-approach atomic force microscopy (AFM) method, which is the most promising method for quantitative measurements of the elasticity of relatively stiff materials. Instead of an averaged value over the entire sample surface, CR-FM shows a high-resolution image (usually at the nanoscale) of elasticity mapping of the scanned surface. For highly nonhomogeneous materials, such as mollusk shell, CR-FM can provide more meaningful moduli mappings rather than a single averaged modulus value. During the CR-FM measurement, a cantilever is vibrated at its contact resonant frequency.17 Its working principle is based on the accurate measurements of the frequencies and quality factors at which the free and contact resonances occur. During the postmeasurement analysis, the mechanical properties of samples can be deduced based on two classical models: the dynamic motion of the cantilever beam and the tip–sample contact mechanics.17 In the CR-FM technique, a tested sample is welladhered on top of a resonator in order to initiate the ultrasonic oscillation of sample. Such oscillations, in terms of contact resonance frequency ( fc) and quality factor (Qc), are detected by an AFM probe in contact with the sample surface with a normal static loading force (Fn). Experimentally, the CR-FM measurement involves many precautions and procedures. First, the cantilever has to be calibrated to determine the free resonance frequency ( f0) and quality factor (Q0), as well as the sensitivity and spring constant. Therefore, the exact value of Fn can be determined (in Newtons). Second, a reference sample with a known elastic modulus, which needs to be similar to that of the tested sample, is required in order to quantify the elastic
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Note
Ref.
6 12 13 5
modulus of the tested sample. Finally, due to the relatively large contact force, the AFM tip may be blunted during the scanning processes, in which case the contact radius between the tip and sample may vary with the scanning process.18 Thus, the reference material needs to be examined again, i.e., during the measurement, the reference material was examined at least twice, before and aer the sample was tested. CR-FM can be conducted at a single point or over a pre-dened area. Because of the analysis relies on the accuracy of the measured values of fc and Qc at each data point, the tracking of contact resonance frequency is necessary. Thus, the band excitation (BE) technique is used for frequency tracking. BE is one of the newlydeveloped multifrequency SPM methods to trace shis of fc caused by surface roughness and property difference.19–22 The BE method allows a decoupling between conservative and dissipative tip–sample interactions and identifying the non-linear responses. It can also eliminate topographic crosstalk.22 This technique excites and detects responses at all frequencies simultaneously within a specied frequency band, within which the contact resonance frequency is roughly located in the center.21 During the operation of BE, cantilever oscillations are recorded at hundreds of frequency intervals within the frequency band, and the responsive amplitude and phase data can be tted within the whole frequency spectrum. Thus, the fc can be identied unambiguously for each scanned data point. Any change of the fc can be detected within this band. Most importantly, because of the usage of multiple frequencies, more data can be recorded and used for tting with the high-quality damped harmonic oscillation (DHO) model, i.e., tting the cantilever amplitude response to a Lorentzian curve,20 so that the real sample deformation can be extracted. Aer tting the raw data obtained by the Band Excitation CR-FM (BE-CR-FM) measurements, the fc and Qc images can be generated.21 For elasticity data analysis, the value of the storage modulus depends on the value of fc, while the loss modulus is more dependent on both the fc and Qc of the resonance
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spectrum.19 More detailed working principles of the BE technique can be found in some recent publications.21,23
Experimental Sample preparation Fresh abalones were purchased from a seafood market. The shucked shell was cut into small fragments using a water-cooled rotating diamond blade. The shell fragments were ground with SiC papers, in a sequence of #1200, #2400 and #4000, and then polished with 0.3 mm and 0.05 mm Al2O3 powder solution. Finally, the samples were cleaned by sonication in deionized water and blow-dried in air. Two types of samples were prepared for CR-FM studies. One was the cross-sectional abalone shell where both calcite and nacre sections were exposed. The crosssectional nacre can be clearly identied by its “bricks-andmortar” structure. The other type of sample was polished from the outer surface. Due to the uneven surface features, both calcite and nacre sections were able to be exposed in this surface. Nacre sections can be identied easily by their closely packed polygons or platelets. This type of sample is referred to as the nacre platelet surface or calcite outer surface in the following text. The polished samples have a surface roughness smaller than 6 nm (RMS value as measured by AFM), except for the cross-sectional nacre, whose RMS value can reach to about 35 nm due to the presence of bulging layers (this will be explained in the Results section). All of the polished samples were stored under ambient conditions for further examination. BE-CR-FM method In this study, CR-FM was conducted by using a commercial AFM (MFP-3D, Asylum Research, USA) with a contact resonance module. Each sample was adhered rmly onto the piezoelectric actuator of the CR-FM module by using a fast-setting epoxy adhesive. The elasticity mapping of these samples were examined by two individual rectangular AFM probes (AC200TS, Olympus, Japan), whose tip was located right at the end of the cantilever, and the nominal tip radius was 9 2 nm. The 1st eigenmode free resonance properties of both probes were summarized in Table 2. The cantilever spring constant was calibrated by the thermal noise method.24 To quantify the elastic modulus of the abalone shell, a reference sample with a similar stiffness to that of the abalone samples needed to be examined as well. In this work, SiO2, whose elastic modulus is around 72 GPa, was chosen as the reference sample. The exact elastic moduli of SiO2 associated with different ranges of loading force were characterized by nanoindentation tests with
Table 2 Calibrated cantilever properties by force curve and thermal methods, including free resonance frequency f0, free resonance quality factor Q0, and cantilever spring constant kc
Cantilever
f0/kHz
Q0
kc/N m1
#1 #2
126.96 131.01
237.30 216.40
5.84 6.56
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continuous stiffness measurement (Nanoindenter XP, MTS, USA). The fc and Qc of the reference sample were examined before and aer the CR-FM scanning of the abalone shell samples, with the identical cantilever, laser positioning, and loading force. Samples were scanned under two different loading forces, 36 and 72 nN, which were ensured to be larger than the tip–sample adhesion force (about 10 to 20 nN), and small enough to preserve the tip radius and also to avoid plastic deformation of the shell sample. As mentioned above, the BE method is used to track closely the frequency shi during the scanning process. Instead of dragging from one point to another point, the BE method allows the movement of tip in a way that lis up rst and then moves to the next data location. Thus, it greatly slows down tip wear and minimizes topographic cross-talk due to the surface roughness. In this study, a dynamic frequency band with a band width of 40 kHz was used for fc tracking under the chirp drive signal.20 For proper working of the BE method, small amplitude of cantilever tip oscillation is ensured (much smaller than 1 nm in the experiment).20 The area associated with each pixel in the mapping is less than 8 8 nm2. The method to obtain the elasticity mappings in this study followed strictly that reported by Killgore et al.25 In addition, the loss tangent [tan(d)] mappings are also calculated using the method proposed by Campbell, Ferguson and Hurley.26 tan(d) is a value which measures the sample damping and is equivalent to the ratio between the loss modulus and storage modulus. The analysis methods are briey described in the following. Input data to calculate the elasticity and dissipation mappings are: (i) total length of cantilever (L); (ii) tip location with respect to the free end of cantilever (L0 ); (iii) f0 and Q0 as illustrated in Table 2; and (iv) fc and Qc which are generated by tting the raw BE-CRFM experimental data with the DHO model. For the Kelvin– Voigt contact model, the cantilever dynamics of the 1st exural mode can be described with a complex normalized wave number l1L ¼ a1 + ib1. The values of a1, b1, and l1L can be determined from the measurements of the values of f0, Q0, fc, and Qc. In this study, small damping is ensured by satisfying the condition of a1 [ b1. Aerwards, the normalized tip–sample contact stiffness (a) and damping coefficient (b) can be calculated from l1L. This method applies to both the reference and tested samples. With the known elastic modulus of the reference sample, Eref, and by assuming a Hertzian contact mode, the reduced elastic modulus of the tested sample (ER) can be extracted from the equation of ER ¼ Eref(a/aref)3/2. Thus, by characterizing the reference sample under the same loading force, the determination of the tip–sample contact radius can be omitted. Furthermore, the true sample modulus can be extracted from the reduced modulus by reasonably assuming the Si cantilever properties and sample Poisson's ratio with the formula 1/ER ¼ (1 y2)/E + (1 ycant2)/Ecant, where y and E are the Poisson's ratio and elastic modulus of the sample and ycant and Ecant are the corresponding values for the cantilever. In this paper, all of the data points in the mappings presented in the results have been put through these calculations to determine the corresponding values of ER. The calculations for each data point in elasticity mappings were accomplished by a
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home-made computational routine using commercial soware (Matlab). In addition, the loss tangent can be expressed as tan(d) ¼ (bfcl2)/(af0), where l ¼ 1.8751 for the 1st exural mode. Based on this formula, tan(d) does not rely on a referencing or calibration technique, but only on the dissipative and conservative interactions of the tip–sample surface. Thus, the value of tan(d) for both mineral and biopolymer phases can be quantied unambiguously. However, BE-CR-FM imposes certain intrinsic limitations on quantication of the elasticity of different microconstituents in the nanocomposite if the elasticity values of the microconstituents differ by several orders of magnitude. As a result, the contact resonance frequencies for these components, for example mineral and biopolymer phases in abalone shell, can be far-separated, in such cases the 40 kHz frequency band may not be wide enough to capture the large frequency shis caused by these microconstituents. In addition, the selection of cantilever is also based on the approximate range of the elastic modulus of the tested sample. For so biopolymers, a soer cantilever should be used, while stiff mineral nanograins need to be examined by a stiffer cantilever. The dilemma restricts the quantication of elasticity in abalone shell on either nanograins or biopolymers, but not on both of them at the same time. Thus, in this study, the quantication of elastic moduli of various mineral nanograins is the major concern. The reference sample, (SiO2), is also selected based on the elasticity of the mineral phase. Under such circumstances, the observed values may not describe the true elastic moduli for pure biopolymers, thus they will not be reported explicitly. Although only one type of component can be quantied, the relative relations revealed by the BE-CR-FM mappings still provide valuable insights into the in situ elasticity variations and arrangement of different microconstituents in the abalone shell.
Results and discussions Nanoscale elasticity and loss tangent mappings of nacre The BE-CR-FM was rstly conducted on the surface of a random platelet in nacre. The mappings of the reduced elastic modulus (ER) and loss tangent [tan(d)] of this platelet surface are shown in Fig. 1, with approximately a 4 4 nm2 area associated with each pixel. The topography of the same platelet is also scanned by AFM tapping mode before and aer the BE-CR-FM imaging. Due to the long acquisition time (usually 1–2 hours), thermal dri cannot be avoided, especially for such small scan size. Thus, there is possible mis-alignment between the topography image and the BE-CR-FM mappings, but surface features can still be clearly identied and correlated. The values of ER in this particular region are found within the range of 85 to 110 GPa [Fig. 1(c)]. This implies a highly non-uniform nature of the nacre platelet at the nanoscale. Thus, an average value obtained over the sample surface (such as those by nanoindentation) cannot describe accurately the elasticity variations of the nacre platelets. Furthermore, the image contrast is similar among the mappings of contact resonance frequency, reduced modulus and sample modulus. In this scanned region, aragonite nanograins and intracrystalline biopolymers are the main
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Fig. 1 Topography and corresponding BE-CR-FM images of nacre platelet surface (500 500 nm2, 128 128 pixels, Fn ¼ 36 nN). (a) AFM topography obtained before the BE-CR-FM scanning; (b) AFM topography image obtained after the BE-CR-FM scanning; (c) mapping of reduced elastic modulus; (d) mapping of loss tangent, tan d.
components. Thus, the locations exhibiting the lowest stiffness are expected to be associated with high concentrations of intracrystalline biopolymers, while the highest stiffness should be contributed by the nanograins. By examining the elasticity mapping together with the topographic image, the average ER and tan(d) for pure aragonite nanograins (red-colour regions) can be quantied as about 103.10 GPa and 0.0113 respectively. The blue- and purple-color regions should have a higher concentration of the intracrystalline biopolymers due to the greatly reduced elasticity. These biopolymers are not evenly distributed over the sample surface and do not surround every nanograin. They are usually trapped at the relatively large void spaces. Furthermore, a bunch of nanograins usually form an aggregate and exhibit a similar elastic modulus, but not all of the nanograins have the same values. The subsurface biopolymers, if they are reached by the stress eld created due to the contact with the AFM tip, may slightly reduce the values of the observed elastic moduli of nanograins. It was reported that the pure geological aragonite crystal had an elastic modulus ranging from 80 to 160 GPa, depending on the different crystallographic directions.27 The ER values of aragonite nanograins from this study agree well with this range. However, the lowest ER value, which is expected to be mainly contributed by the intracrystalline biopolymers, is still approximately 70% of that of aragonite nanograins. It appears much stiffer than is expected for the biopolymers. Besides the limitation of the BE method as depicted before, this is also likely to be due to interference from the subsurface or neighbouring minerals. The thickness of the intracrystalline biopolymer was reported to be about 4 nm.5 The extent of stress eld in the CR-FM measurement can be larger than the thickness of the biopolymers, and it also depends on the loading force and tip– sample contact. A smaller loading force helps to better preserve the tip radius and probe the elasticity closer to the sample surface. However, the loading force needs to be large enough to
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overcome the adhesion force, which was found to increase with tip radius in this experiment. Thus, a moderate loading force needs to be maintained during the measurements, and hence it is not easy to determine the elastic property of the pure biopolymers from this in situ experiment. On the other hand, the value of tan(d) can also be used to complement the understanding of the location and behavior of biopolymers. As mentioned before, tan(d) can be quantied for all microconstituents in the abalone shell. Tan(d) reveals the degree of damping, and is equivalent to the ratio between the loss modulus and the storage modulus.26 A higher value of tan(d) indicates a larger damping of the tip–sample interaction. The image contrast of the tan(d) mapping can be correlated to that of the modulus mapping. The highest damping (dark red-color) occurs at the boundaries between the regions with the highest and the lowest ER, which is expected to be the transition between the nanograins and biopolymers. This nding is consistent with our previous results by using the piezoresponse force microscopy on mollusk shell, in which the regions of highest Q-factor always appear at the transition between the high and low contact resonance frequencies.28 Thus, the elastic modulus changes in the shell structure can lead to a sudden increase of tip–sample energy dissipation. The width of the high values of tan(d) regions [red curves in Fig. 1(d)] may indicate the extent of the elasticity transition regions, which are about 30–40 nm. In addition, for the rest part of region (green- and blue-color), the value of tan(d) does not differ much. However, lower tan(d) usually maps with the regions having a relatively high elastic modulus. Another nding is that, aer the BE-CR-FM measurement, the topography of the scanned surface is slightly distorted and the nanograins appear much larger than those before the scanning [Fig. 1(a) vs. (b)]. This may be a true response from the sample or be caused by tip blunting. To verify this, AFM images with the image size of 1 1 mm2 were scanned at the same location with the same tip under tapping mode before and aer the BE-CR-FM observation, as illustrated in Fig. 2. It shows much larger size of nanograins and less void spaces aer the BE-CR-
Fig. 2 AFM topography (a and c) and phase images (b and d) of the same area before (a and b) and after (c and d) the BE-CR-FM imaging (1 1 mm2). the enclosed area is where the BE-CR-FM has been scanned.
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FM scan. The tip may be gradually blunted during the BE-CR-FM scanning, which may cause the nanograins to appear larger. However, it is obvious that the larger grain size is not solely due to tip blunting, which is clearly shown in Fig. 2(c) and (d). The size of the nanograins that have been scanned by BE-CR-FM is larger than that of the other nanograins surrounding them. The loading force in the vertical direction may slightly press the nanograins into the sample surface, and the intracrystalline biopolymer matrix may be squeezed out of the surface via a certain path, preferably the void spaces. If the bonding between the nanograins and biopolymers is strong enough, the movement of biopolymers may cause rotation or displacement of nanograins. Therefore, aer being scanned by the BE-CR-FM method, the surface may have new spatial arrangement of certain micro-constituents. The surface property change is revealed clearly by the phase images [Fig. 2(b) vs. (d)]. The entire BE-CR-FM scanned surface shows higher degree of phase lag under the repulsive regime, which indicates more energy dissipation between the tip–sample interactions. The phase contrast of grain boundaries is clearly different from that of the nanograins, and this contrast difference is expected to be caused by the intracrystalline biopolymers. In addition, it seems that the effects of the rearrangement of the micro-constituents has an extent much larger than the scan size of BE-CR-FM measurement. The size and shape of grains far from the affected regions are also altered, for example, the two grains in red circles. The upper one is rotated, and the lower one is slightly pushed out of the surface and the original void space appears smaller. It indicates the interconnectivity of the micro-constituents within a nacre platelet. Additional measurement further conrms this phenomenon is caused primarily by the material response during BE-CR-FM scanning rather than the tip induced artifact, and this can be proven by Fig. S1† which shows the AFM images including the BE-CR-FM scanned area. Lastly, the parallelogram shape of the BE-CR-FM scanned area as shown in the AFM images (Fig. 2) is expected to be due to the thermal dri during the scanning by BE technique with a small scanning area. As indicated before, choice of loading force can affect the values of elastic modulus. Thus, another nacre platelet is scanned by BE-CR-FM with two different loading forces, 36 nN and 72 nN, at the same location. As illustrated in Fig. 3, a higher loading force causes the average value of ER of the same scanned surface to be reduced by about 7 GPa, but the value of tan(d) does not change signicantly. Thus, the tan(d) value is not sensitive to the loading force. In other words, it is only sensitive to the changes on the very surface layer. A larger loading force leads to a larger contact radius, which theoretically should result in a higher contact resonance frequency and a higher contact elasticity. Thus, the reduction of elastic modulus should originate mainly from the difference in sample properties. The contrast in Fig. 3(b) and (d) is nearly identical, except for a few locations, such as the place highlighted by the oval circles. The relative difference of the elastic moduli between the neighbouring points are changed due to the larger loading force. The originally so location appears stiffer, which indicates that the subsurface mineral grains may impose more signicant effects under the higher loading force at this particular location.
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Fig. 3 BE-CR-FM images of the same location with different loading force (500 300 nm2): 36 nN (b and c), and 72 nN (d and e). (a) Topographic image; (b) and (d) mapping of reduced elastic modulus; (c) and (e) mapping of loss tangent, tan d.
Generally speaking, under a higher loading force, a large amount of the subsurface organic matrix may be included in the extended stress eld, which leads to the overall reduction of ER. However, the reduction is not uniform. At some locations, the relative stiffness with respect to the neighbouring regions is changed more signicantly. Furthermore, ER and tan(d) mappings of the nacre on the cross-sectional surface have also been obtained (Fig. 4). On this surface, not only the intracrystalline biopolymers, but also the interlamellar biopolymers are exposed to the surface, and they can be differentiated by their locations. In addition, the aragonite nanograins are orientated in a different crystallographic direction at this surface. As illustrated in Fig. 4(a), bulging layers are usually observed at the boundaries between the mineral layers and interlamellar biopolymers on the ne polished cross-sectional surface of nacre. Their height and thickness depend on the polishing directions as explained in the previous study.29 Similarly, the smallest value of ER should be associated with the highest biopolymer concentration in the
scanned region. The interlamellar biopolymer is located between the mineral layers and should be the so region next to the bulging layer [blue-color in Fig. 4(b)]. The layer of the interlamellar biopolymers can be displaced due to the polishing process, thus it does not show as a perfect straight line. The tan(d) value of interlamellar biopolymer is found to be about 0.0190. In addition, similar to the platelet surface, the interior of the cross-sectional platelet also shows diverse values of elastic modulus. The stiff aragonite nanograins should have the highest elastic modulus (red-color), whose average ER is about 87 GPa, and the corresponding tan(d) is about 0.0104. The soer intracrystalline biopolymers may reduce the detected stiffness. The difference of elastic modulus between the regions of nanograins and the regions of intracrystalline biopolymers is approximately 15%, which is similar to that observed on the platelet surface, with the same reason as discussed previously. In addition, the interlamellar biopolymers appear soer than that of the intracrystalline biopolymers. Thus, the interlamellar biopolymers should deform prior to any other deformations in
Fig. 4 Topography and corresponding BE-CR-FM images of cross-sectional nacre (500 500 nm2, 64 64 pixels, Fn ¼ 72 nN). (a) AFM topography obtained after the BE-CR-FM scanning; (b) mapping of reduced elastic modulus; and (c) mapping of loss tangent, tan d.
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the nacre structure. In general, the average value of elastic modulus on the cross-sectional surface of nacre is about 10 GPa smaller than that on the platelet surface when observed under the same loading force. Thus, when experiencing an external force, nacre tends to deform in the direction parallel to the shell surface rst, rather than perpendicular to it, even at the nanoscale.
Elasticity and loss tangent mappings of natural calcite composite Calcite is another important part of the abalone shell. It locates at the outermost shell, covers the nacre section, and works as the rst shield for all kinds of external forces. However, the elastic properties of the natural calcite composite in abalone shell have not been well studied, especially at the nanoscale. Under the optical microscope, different large prism crystals can be clearly visualized on the nely polished surface of the abalone shell. Each prism is found to be composed by a higher order of hierarchical nanostructures. Three typical kinds of nanostructures have been identied, which are foliated-/needle, granular- (round shape) and block-like (facet shape) structures/particles, as shown in Fig. 5(a), (d) and (g) respectively, depending on the observation locations and orientations. Examples of each structure have been highlighted by red shapes
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in the topographic images. The ER and tan(d) mappings associated with these structures are also illustrated in Fig. 5. Each of the BE-CR-FM mappings is acquired from a single prism. The wide range of elastic moduli in each mapping [Fig. 5(b), (e) and (h)] indicates that a calcite prism is not a simple single crystal as reported in the literature.10 Same as the case of nacre, the calcite prism is also a nanocomposite, which is composed by biopolymers and nanograins in different shapes. The location of concentrated biopolymers can be identied as the soest regions in these mappings (blue- and purple-color). On the cross-sectional surface of the abalone shell [Fig. 5(a)–(c)], a needle-like structure is typically shown in the calcite section. The average ER and tan(d) of the calcite nanograins (red-color) is about 101.15 GPa and 0.0108 respectively. There are also regions with intermediate elasticity (greencolor). This is either because the extent of the stress eld may enclose both nanograins and biopolymers, or may be due to the slightly different crystal orientations of the needles at these scanned locations. On the other hand, both granular-like and block-like shape of nanograins can be observed on the polished outer surface of calcite [Fig. 5(d)–(i)]. The elastic moduli of calcite nanograins are very similar between these two structures. Compared to that of nacre, the variation of elastic modulus at different locations is less dramatic for calcite, especially for the block-like nanostructure. Large areas of the
Fig. 5 Topography and corresponding BE-CR-FM images of calcite (500 500 nm2, 64 64 pixels) observed from: (a–c) cross-sectional surface (Fn ¼ 72 nN) with needle shaped features; (d–f) outer surface with granular (round shape particles) feature in topography (Fn ¼ 36 nN); (g–i) outer surface with block (facet shape particles) feature in topography (Fn ¼ 36 nN). (a), (d) and (g) are AFM topography images; (b), (e) and (h) are reduced elastic modulus mappings; (c), (f) and (i) are mapping of the loss tangent, tan d.
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Table 3 Summary of the quantified reduced elastic moduli and loss tangent of nanograins in nacre and calcite structures based on the nanoscale mappings
Shell surface (36 nN)
Cross-sectional surface (72 nN) Tan d (10 )
ER/GPa
Tan d (103)
Mean values of the entire scanned area (500 500 nm2) Nacre 97.63 5.31 Calcite (needle) — Calcite (granular) 83.85 1.06 Calcite (block) 85.33 4.13
12.2 2.9 — 23.3 9.7 16.0 3.0
79.75 1.35 95.98 3.71 — —
10.8 5.9 11.3 1.8 — —
Average values for each type of mineral nanograins Nacre nanograin 103.1 Calcite grain (needle) — Calcite grain (granular) 92.45 Calcite grain (block) 89.77
11.3 — 20.1 14.0
87.00 101.15 — —
10.4 10.8 — —
Regions
R
E /GPa
nanograins exhibit a very similar elasticity, and the biopolymers are mainly located at the void spaces and boundaries between the calcite blocks. Thus, the mineral nanograins and biopolymers in calcite are not interwoven as intensively as that in the nacre region, and the amount of biopolymers seems less in the calcite nanostructure compared to that in the nacre region. Generally-speaking, the average elastic modulus of these surfaces is less than that of the cross-sectional calcite surface, and higher energy dissipation is also accompanied with a lower elastic modulus. Lastly, the average values of ER and tan(d) from the BE-CR-FM mappings, as well as the values of mineral nanograins for both nacre and calcite are summarized in Table 3. In most cases, the external force may act in the direction normal to the outer shell surface, in which the calcite structure has much smaller elastic modulus than that of the nacre structure. Therefore, calcite structure can work as a buffer layer to discharge the external force and to better protect the internal structures and the living mollusk. On the cross-sectional surface, however, the calcite structure shows a much higher stiffness than the nacre structure. If any resolved force is applied in the direction parallel to the shell surface, the calcite structure may be stiff enough to resist the force. If the force is delivered to the nacre structure, more deformation is allowed within the elastic range. As a result, the living mollusk can be well protected by such a mechanism. Furthermore, from the nanoscale BE-CR-FM mappings, the relative stiffness of each micro-constituent in abalone shell can be ranked and the elastic modulus of mineral nanograins are quantied. In the direction normal to the shell surface, the calcite biopolymers are generally soer than those of the intracrystalline biopolymers in nacre. All the biopolymers are denitely soer than the nanograins, and the calcite nanograins are less stiff than the nacre nanograins. On the other hand, the interlamellar biopolymers show the lowest stiffness among all the components on the cross-sectional surface of abalone shell. The intracrystalline biopolymers are stiffer than the interlamellar ones on this surface, and the calcite biopolymers are stiffer than those of both biopolymers in nacre. In addition, the needle-shaped calcite nanograins are also stiffer than those of the nacre nanograins.
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Conclusions This study has presented and discussed the nanoscale elasticity and loss tangent mappings using the BE-CR-FM method for both nacre and calcite structures in abalone shell. The elastic properties of the micro-constituents have been extracted from these mappings and the relative relations among them are presented on different surfaces of the shell structures. In summary, both nacre and calcite structures can be considered as highly non-homogeneous nanocomposites in terms of their elastic properties. The variations of the elastic modulus can reach over tens of GPa within a hundreds of nanometers area. These variations are caused by the different crystal orientations of the nanograins and the different types and concentrations of the biopolymers as well as their interactions with the mineral grains. The quantied values of the elastic moduli of different mineral nanograins are reported in this work (Table 3). The observed elasticity of the biopolymers may be affected by the subsurface mineral nanograins, and the value of the elasticity may be restricted by the limited width of the frequency band in the BE method. Overall-speaking, these elasticity mappings can provide more insight into the nanoscale structure, the distribution of micro-constituents and the resultant variations of elasticity at the nanoscale, and to contribute further to the better understanding of the extraordinary mechanical behavior and mechanisms of the abalone shell.
Acknowledgements The authors would like to present their sincere gratitude to all the fellows who have contributed to this study. This work was supported by Ministry of Education (Singapore) through National University of Singapore (NUS) under Academic Research Fund (R-265-000-406-112).
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3 K. S. Katti, D. R. Katti and B. Mohanty, in Biomimetics, Learning from Nature, ed. A. Mukherjee, InTech, India, 2010, pp. 193–216. 4 X. Li, W.-C. Chang, Y. J. Chao, R. Wang and M. Chang, Nano Lett., 2004, 4, 613–617. 5 P. Stemp´ e, O. Pantal´ e, M. Rousseau, E. Lopez and X. Bourrat, Mater. Sci. Eng., C, 2010, 30, 715–721. 6 F. D. Fleischli, M. Dietiker, C. Borgia and R. Spolenak, Acta Biomater., 2008, 4, 1694–1706. 7 R. Menig, M. H. Meyers, M. A. Meyers and K. S. Vecchio, Acta Mater., 2000, 48, 2383–2398. 8 R. Z. Wang, Z. Suo, A. G. Evans, N. Yao and I. A. Aksay, J. Mater. Res., 2001, 16, 2485–2493. 9 F. Barthelat and H. D. Espinosa, Exp. Mech., 2007, 47, 311–324. 10 F. Nudelman, H. H. Chen, H. A. Goldberg, S. Weiner and L. Addadi, Faraday Discuss., 2007, 136, 9–25. 11 Crystran, Calcite (CaCO3) Data Sheet http://www. crystran.co.uk/userles/les/calcite-caco3-data-sheet.pdf, accessed 26 September 2013. 12 Z. H. Xu, Y. Yang, Z. Huang and X. Li, Mater. Sci. Eng., C, 2011, 31, 1852–1856. 13 F. Barthelat, C.-M. Li, C. Comi and H. D. Espinosa, J. Mater. Res., 2006, 21, 1977–1986. 14 H. Moshe-Drezner, D. Shilo, A. Dorogoy and E. Zolotoyabko, Adv. Funct. Mater., 2010, 20, 2723–2728. 15 D. Katti and K. Katti, J. Mater. Sci., 2001, 36, 1411–1417. 16 P. Stempe, O. Pantale, R. K. Njiwa, M. Rousseau, E. Lopez and X. Bourrat, Int. J. Nanotechnol., 2007, 4, 712–729. 17 D. C. Hurley, in Scanning Probe Microscopy of Functional Materials: Nanoscale Imaging and Spectroscopy, ed. S. V.
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Kalinin and A. Gruverman, Springer, New York, 2010, pp. 95–124. J. P. Killgore, R. H. Geiss and D. C. Hurley, Small, 2011, 7, 1018–1022. D. G. Yablon, A. Gannepalli, R. Proksch, J. Killgore, D. C. Hurley, J. Grabowski and A. H. Tsou, Macromolecules, 2012, 45, 4363–4370. A. U. Kareem and S. D. Solares, Nanotechnology, 2012, 23, 015706. S. Jesse and S. V. Kalinin, J. Phys. D: Appl. Phys., 2011, 44, 464006. S. Jesse, A. Kumar, S. V. Kalinin, A. Gannepali and R. Proksch, Band excitation scanning probe microscopies: travelling through the fourier space, http://www.asylumresearch.com/ Applications/BandExcitation/BandExcitation.shtml, accessedSeptember 20, 2013. S. Jesse, S. V. Kalinin, R. Proksch, A. P. Baddorf and B. J. Rodriguez, Nanotechnology, 2007, 18, 435503. R. Proksch, T. E. Sch¨ affer, J. P. Cleveland, R. C. Callahan and M. B. Viani, Nanotechnology, 2004, 15, 1344–1350. J. P. Killgore, D. G. Yablon, A. H. Tsou, A. Gannepalli, P. A. Yuya, J. A. Turner, R. Proksch and D. C. Hurley, Langmuir, 2011, 27, 13983–13987. S. E. Campbell, V. L. Ferguson and D. C. Hurley, Acta Biomater., 2012, 8, 4389–4396. J. D. Bass, in Mineral Physics & Crystallography: A Handbook of Physical Constants, American Geophysical Union, 1995, pp. 45–63. T. Li and K. Zeng, J. Struct. Biol., 2012, 180, 73–83. T. Li and K. Zeng, J. Appl. Phys., 2013, 113, 187202.
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Nanoscale elasticity mappings of micro-constituents of abalone shell by band excitation-contact resonance force microscopy† Tao Li and Kaiyang Zeng* The macroscopic mechanical properties of the abalone shell have been studied extensively in the literature, but the in situ nanoscale elasticity of various micro-constituents in the shell have not been characterized and reported yet. In this study, the nanoscale elasticity mappings including different micro-constituents in abalone shell were observed by using the Contact Resonance Force Microscopy (CR-FM) technique. CR-FM is one of the advanced scanning probe microscopy techniques that is able to quantify the local elastic moduli of various materials in a non-destructive manner. Instead of an average value, an elasticity mapping that reveals the nanoscale variations of elastic moduli with location can be extracted and
Received 4th October 2013 Accepted 14th November 2013
correlated with the topography of the structure. Therefore in this study, by adopting the CR-FM technique that is incorporated with the band excitation technique, the elasticity variations of the abalone
DOI: 10.1039/c3nr05292c
shell caused by different micro-constituents and crystal orientations are reported, and the elasticity
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values of the aragonite and calcite nanograins are quantified.
Introduction Mollusk shell is a typical natural nanocomposite and also an armour which has protected the host mollusk from predators over billions of years of evolution. The shell is generally composed by more than 95 wt% of calcium carbonate (CaCO3) and less than 5 wt% of biopolymers (mainly chitin and various proteins). The two micro-constituents together form multiple levels of hierarchical structures via a biomineralization process under ambient conditions.1,2 The mollusk shell is well-known for its outstanding mechanical properties (in terms of toughness and strength) compared to those of their individual microconstituents. Amongst various shell structures, the mechanical properties of nacre have been most extensively studied due to its better toughness behavior compared to others.3 The nacre structure is found in many species of mollusk shells, including abalone shell. The nacre structure is composed of CaCO3 mineral layers (around 500 nm in thickness) joined by thin layers of biopolymers, which are usually called interlamellar biopolymers (thickness range within 5 to 20 nm).4 Thus, when viewed on the cross-section, it shows a “bricks-and-mortar” type of structure. In addition, each mineral layer is constructed by closelypacked polygonal CaCO3 (aragonite) platelets (size range within 5 to 10 mm) that are separated by intertabular biopolymers.1 Department of Mechanical Engineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576. E-mail: [email protected] † Electronic supplementary 10.1039/c3nr05292c
information
(ESI)
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available.
See
DOI:
Furthermore, each platelet is composed of a large number of nanograins (38 21 nm of diameter) that are embedded in an intracrystalline biopolymer matrix (4 nm thick).5 There are many reports presenting the average value of the elastic moduli of the entire nacre structure and the micro-constituents, which were usually measured by traditional mechanical testing methods, such as micro-/nano-indentation with numerical modelling.5–9 Table 1 summarizes some of the reported elastic modulus values of the nacre and its constituents. Depending on the testing methods, large deviations of the elasticity values are reported, especially for the biopolymers. This is due to the difficulties of measuring the value of elastic modulus of the biopolymers in situ, especially for the intracrystalline biopolymers. Thus, many of the reported values are obtained by using nite element (FE) modelling. Furthermore, in abalone shell, there is an outermost shell section which works as the rst shield when mollusk is under attack, and this section consists mainly of CaCO3 in the calcite polymorph. In this shell section, large calcite prisms of tens of micrometers are enveloped in the interprismatic biopolymers.10 Each of these prisms is reported as a single crystal. The reported mechanical properties of the calcite section in abalone shell is scarce. The elastic modulus of pure calcite crystals was reported to be about 72 and 88 GPa, depending on the crystal orientations.11 Generally-speaking, the traditional mechanical testing methods are usually destructive, and by these only the average values can be obtained. These methods are not very suitable to depict the elasticity of a nanocomposite, especially the complex hierarchical structures, such as those in the abalone shell. In this case, contact resonance force microscopy (CR-FM) shows
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Paper
Elastic moduli of different component in abalone shell that reported in the literatures
E/GPa
Method
Shell species
Overall nacre section 72.6 5.8 69 7 79 15 62.5 11.2
Nanoindentation Nanoindentation Nanoindentation Nanoindentation
Haliotis rufescens Haliotis rufescens Haliotis rufescens Pinctada maxima
Biopolymers 40 11 3 15 2.84 0.27 6.3 0.56 3.81 0.41 49 3
CR-FM & FE analysis Inverse FE analysis 3D FE modelling Nanoindentation Modelling Mori–Tanaka's model Nanoindentation
Perna canaliculus Haliotis rufescens Haliotis rufescens Haliotis rufescens Pinctada maxima Pinctada maxima Haliotis rufescens
Interlamellar, b-chitin Interlamellar polymer strand Interlamellar Interlamellar Interlamellar Intracrystalline Interlamellar
14 12 15 13 16 5 12
Aragonite Nanograin 82.7 96.75 5.67
FE modelling Sharp nanoindentation
Haliotis rufescens Pinctada maxima
Single-crystal Nanograins
13 5
advantages to study the local elasticity of the nanocomposite structure. Moshe-Drezner et al. have demonstrated the usage of CR-FM in mollusk shell.14 In their study, the elastic modulus of b-chitin in nacre was reported to be 40 GPa based on experimental observation and nite element analysis. However, it did not provide any high spacial resolution mapping of the nanograins and biopolymers. CR-FM is a general name of the dynamic-approach atomic force microscopy (AFM) method, which is the most promising method for quantitative measurements of the elasticity of relatively stiff materials. Instead of an averaged value over the entire sample surface, CR-FM shows a high-resolution image (usually at the nanoscale) of elasticity mapping of the scanned surface. For highly nonhomogeneous materials, such as mollusk shell, CR-FM can provide more meaningful moduli mappings rather than a single averaged modulus value. During the CR-FM measurement, a cantilever is vibrated at its contact resonant frequency.17 Its working principle is based on the accurate measurements of the frequencies and quality factors at which the free and contact resonances occur. During the postmeasurement analysis, the mechanical properties of samples can be deduced based on two classical models: the dynamic motion of the cantilever beam and the tip–sample contact mechanics.17 In the CR-FM technique, a tested sample is welladhered on top of a resonator in order to initiate the ultrasonic oscillation of sample. Such oscillations, in terms of contact resonance frequency ( fc) and quality factor (Qc), are detected by an AFM probe in contact with the sample surface with a normal static loading force (Fn). Experimentally, the CR-FM measurement involves many precautions and procedures. First, the cantilever has to be calibrated to determine the free resonance frequency ( f0) and quality factor (Q0), as well as the sensitivity and spring constant. Therefore, the exact value of Fn can be determined (in Newtons). Second, a reference sample with a known elastic modulus, which needs to be similar to that of the tested sample, is required in order to quantify the elastic
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Ref.
6 12 13 5
modulus of the tested sample. Finally, due to the relatively large contact force, the AFM tip may be blunted during the scanning processes, in which case the contact radius between the tip and sample may vary with the scanning process.18 Thus, the reference material needs to be examined again, i.e., during the measurement, the reference material was examined at least twice, before and aer the sample was tested. CR-FM can be conducted at a single point or over a pre-dened area. Because of the analysis relies on the accuracy of the measured values of fc and Qc at each data point, the tracking of contact resonance frequency is necessary. Thus, the band excitation (BE) technique is used for frequency tracking. BE is one of the newlydeveloped multifrequency SPM methods to trace shis of fc caused by surface roughness and property difference.19–22 The BE method allows a decoupling between conservative and dissipative tip–sample interactions and identifying the non-linear responses. It can also eliminate topographic crosstalk.22 This technique excites and detects responses at all frequencies simultaneously within a specied frequency band, within which the contact resonance frequency is roughly located in the center.21 During the operation of BE, cantilever oscillations are recorded at hundreds of frequency intervals within the frequency band, and the responsive amplitude and phase data can be tted within the whole frequency spectrum. Thus, the fc can be identied unambiguously for each scanned data point. Any change of the fc can be detected within this band. Most importantly, because of the usage of multiple frequencies, more data can be recorded and used for tting with the high-quality damped harmonic oscillation (DHO) model, i.e., tting the cantilever amplitude response to a Lorentzian curve,20 so that the real sample deformation can be extracted. Aer tting the raw data obtained by the Band Excitation CR-FM (BE-CR-FM) measurements, the fc and Qc images can be generated.21 For elasticity data analysis, the value of the storage modulus depends on the value of fc, while the loss modulus is more dependent on both the fc and Qc of the resonance
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spectrum.19 More detailed working principles of the BE technique can be found in some recent publications.21,23
Experimental Sample preparation Fresh abalones were purchased from a seafood market. The shucked shell was cut into small fragments using a water-cooled rotating diamond blade. The shell fragments were ground with SiC papers, in a sequence of #1200, #2400 and #4000, and then polished with 0.3 mm and 0.05 mm Al2O3 powder solution. Finally, the samples were cleaned by sonication in deionized water and blow-dried in air. Two types of samples were prepared for CR-FM studies. One was the cross-sectional abalone shell where both calcite and nacre sections were exposed. The crosssectional nacre can be clearly identied by its “bricks-andmortar” structure. The other type of sample was polished from the outer surface. Due to the uneven surface features, both calcite and nacre sections were able to be exposed in this surface. Nacre sections can be identied easily by their closely packed polygons or platelets. This type of sample is referred to as the nacre platelet surface or calcite outer surface in the following text. The polished samples have a surface roughness smaller than 6 nm (RMS value as measured by AFM), except for the cross-sectional nacre, whose RMS value can reach to about 35 nm due to the presence of bulging layers (this will be explained in the Results section). All of the polished samples were stored under ambient conditions for further examination. BE-CR-FM method In this study, CR-FM was conducted by using a commercial AFM (MFP-3D, Asylum Research, USA) with a contact resonance module. Each sample was adhered rmly onto the piezoelectric actuator of the CR-FM module by using a fast-setting epoxy adhesive. The elasticity mapping of these samples were examined by two individual rectangular AFM probes (AC200TS, Olympus, Japan), whose tip was located right at the end of the cantilever, and the nominal tip radius was 9 2 nm. The 1st eigenmode free resonance properties of both probes were summarized in Table 2. The cantilever spring constant was calibrated by the thermal noise method.24 To quantify the elastic modulus of the abalone shell, a reference sample with a similar stiffness to that of the abalone samples needed to be examined as well. In this work, SiO2, whose elastic modulus is around 72 GPa, was chosen as the reference sample. The exact elastic moduli of SiO2 associated with different ranges of loading force were characterized by nanoindentation tests with
Table 2 Calibrated cantilever properties by force curve and thermal methods, including free resonance frequency f0, free resonance quality factor Q0, and cantilever spring constant kc
Cantilever
f0/kHz
Q0
kc/N m1
#1 #2
126.96 131.01
237.30 216.40
5.84 6.56
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continuous stiffness measurement (Nanoindenter XP, MTS, USA). The fc and Qc of the reference sample were examined before and aer the CR-FM scanning of the abalone shell samples, with the identical cantilever, laser positioning, and loading force. Samples were scanned under two different loading forces, 36 and 72 nN, which were ensured to be larger than the tip–sample adhesion force (about 10 to 20 nN), and small enough to preserve the tip radius and also to avoid plastic deformation of the shell sample. As mentioned above, the BE method is used to track closely the frequency shi during the scanning process. Instead of dragging from one point to another point, the BE method allows the movement of tip in a way that lis up rst and then moves to the next data location. Thus, it greatly slows down tip wear and minimizes topographic cross-talk due to the surface roughness. In this study, a dynamic frequency band with a band width of 40 kHz was used for fc tracking under the chirp drive signal.20 For proper working of the BE method, small amplitude of cantilever tip oscillation is ensured (much smaller than 1 nm in the experiment).20 The area associated with each pixel in the mapping is less than 8 8 nm2. The method to obtain the elasticity mappings in this study followed strictly that reported by Killgore et al.25 In addition, the loss tangent [tan(d)] mappings are also calculated using the method proposed by Campbell, Ferguson and Hurley.26 tan(d) is a value which measures the sample damping and is equivalent to the ratio between the loss modulus and storage modulus. The analysis methods are briey described in the following. Input data to calculate the elasticity and dissipation mappings are: (i) total length of cantilever (L); (ii) tip location with respect to the free end of cantilever (L0 ); (iii) f0 and Q0 as illustrated in Table 2; and (iv) fc and Qc which are generated by tting the raw BE-CRFM experimental data with the DHO model. For the Kelvin– Voigt contact model, the cantilever dynamics of the 1st exural mode can be described with a complex normalized wave number l1L ¼ a1 + ib1. The values of a1, b1, and l1L can be determined from the measurements of the values of f0, Q0, fc, and Qc. In this study, small damping is ensured by satisfying the condition of a1 [ b1. Aerwards, the normalized tip–sample contact stiffness (a) and damping coefficient (b) can be calculated from l1L. This method applies to both the reference and tested samples. With the known elastic modulus of the reference sample, Eref, and by assuming a Hertzian contact mode, the reduced elastic modulus of the tested sample (ER) can be extracted from the equation of ER ¼ Eref(a/aref)3/2. Thus, by characterizing the reference sample under the same loading force, the determination of the tip–sample contact radius can be omitted. Furthermore, the true sample modulus can be extracted from the reduced modulus by reasonably assuming the Si cantilever properties and sample Poisson's ratio with the formula 1/ER ¼ (1 y2)/E + (1 ycant2)/Ecant, where y and E are the Poisson's ratio and elastic modulus of the sample and ycant and Ecant are the corresponding values for the cantilever. In this paper, all of the data points in the mappings presented in the results have been put through these calculations to determine the corresponding values of ER. The calculations for each data point in elasticity mappings were accomplished by a
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home-made computational routine using commercial soware (Matlab). In addition, the loss tangent can be expressed as tan(d) ¼ (bfcl2)/(af0), where l ¼ 1.8751 for the 1st exural mode. Based on this formula, tan(d) does not rely on a referencing or calibration technique, but only on the dissipative and conservative interactions of the tip–sample surface. Thus, the value of tan(d) for both mineral and biopolymer phases can be quantied unambiguously. However, BE-CR-FM imposes certain intrinsic limitations on quantication of the elasticity of different microconstituents in the nanocomposite if the elasticity values of the microconstituents differ by several orders of magnitude. As a result, the contact resonance frequencies for these components, for example mineral and biopolymer phases in abalone shell, can be far-separated, in such cases the 40 kHz frequency band may not be wide enough to capture the large frequency shis caused by these microconstituents. In addition, the selection of cantilever is also based on the approximate range of the elastic modulus of the tested sample. For so biopolymers, a soer cantilever should be used, while stiff mineral nanograins need to be examined by a stiffer cantilever. The dilemma restricts the quantication of elasticity in abalone shell on either nanograins or biopolymers, but not on both of them at the same time. Thus, in this study, the quantication of elastic moduli of various mineral nanograins is the major concern. The reference sample, (SiO2), is also selected based on the elasticity of the mineral phase. Under such circumstances, the observed values may not describe the true elastic moduli for pure biopolymers, thus they will not be reported explicitly. Although only one type of component can be quantied, the relative relations revealed by the BE-CR-FM mappings still provide valuable insights into the in situ elasticity variations and arrangement of different microconstituents in the abalone shell.
Results and discussions Nanoscale elasticity and loss tangent mappings of nacre The BE-CR-FM was rstly conducted on the surface of a random platelet in nacre. The mappings of the reduced elastic modulus (ER) and loss tangent [tan(d)] of this platelet surface are shown in Fig. 1, with approximately a 4 4 nm2 area associated with each pixel. The topography of the same platelet is also scanned by AFM tapping mode before and aer the BE-CR-FM imaging. Due to the long acquisition time (usually 1–2 hours), thermal dri cannot be avoided, especially for such small scan size. Thus, there is possible mis-alignment between the topography image and the BE-CR-FM mappings, but surface features can still be clearly identied and correlated. The values of ER in this particular region are found within the range of 85 to 110 GPa [Fig. 1(c)]. This implies a highly non-uniform nature of the nacre platelet at the nanoscale. Thus, an average value obtained over the sample surface (such as those by nanoindentation) cannot describe accurately the elasticity variations of the nacre platelets. Furthermore, the image contrast is similar among the mappings of contact resonance frequency, reduced modulus and sample modulus. In this scanned region, aragonite nanograins and intracrystalline biopolymers are the main
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Fig. 1 Topography and corresponding BE-CR-FM images of nacre platelet surface (500 500 nm2, 128 128 pixels, Fn ¼ 36 nN). (a) AFM topography obtained before the BE-CR-FM scanning; (b) AFM topography image obtained after the BE-CR-FM scanning; (c) mapping of reduced elastic modulus; (d) mapping of loss tangent, tan d.
components. Thus, the locations exhibiting the lowest stiffness are expected to be associated with high concentrations of intracrystalline biopolymers, while the highest stiffness should be contributed by the nanograins. By examining the elasticity mapping together with the topographic image, the average ER and tan(d) for pure aragonite nanograins (red-colour regions) can be quantied as about 103.10 GPa and 0.0113 respectively. The blue- and purple-color regions should have a higher concentration of the intracrystalline biopolymers due to the greatly reduced elasticity. These biopolymers are not evenly distributed over the sample surface and do not surround every nanograin. They are usually trapped at the relatively large void spaces. Furthermore, a bunch of nanograins usually form an aggregate and exhibit a similar elastic modulus, but not all of the nanograins have the same values. The subsurface biopolymers, if they are reached by the stress eld created due to the contact with the AFM tip, may slightly reduce the values of the observed elastic moduli of nanograins. It was reported that the pure geological aragonite crystal had an elastic modulus ranging from 80 to 160 GPa, depending on the different crystallographic directions.27 The ER values of aragonite nanograins from this study agree well with this range. However, the lowest ER value, which is expected to be mainly contributed by the intracrystalline biopolymers, is still approximately 70% of that of aragonite nanograins. It appears much stiffer than is expected for the biopolymers. Besides the limitation of the BE method as depicted before, this is also likely to be due to interference from the subsurface or neighbouring minerals. The thickness of the intracrystalline biopolymer was reported to be about 4 nm.5 The extent of stress eld in the CR-FM measurement can be larger than the thickness of the biopolymers, and it also depends on the loading force and tip– sample contact. A smaller loading force helps to better preserve the tip radius and probe the elasticity closer to the sample surface. However, the loading force needs to be large enough to
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overcome the adhesion force, which was found to increase with tip radius in this experiment. Thus, a moderate loading force needs to be maintained during the measurements, and hence it is not easy to determine the elastic property of the pure biopolymers from this in situ experiment. On the other hand, the value of tan(d) can also be used to complement the understanding of the location and behavior of biopolymers. As mentioned before, tan(d) can be quantied for all microconstituents in the abalone shell. Tan(d) reveals the degree of damping, and is equivalent to the ratio between the loss modulus and the storage modulus.26 A higher value of tan(d) indicates a larger damping of the tip–sample interaction. The image contrast of the tan(d) mapping can be correlated to that of the modulus mapping. The highest damping (dark red-color) occurs at the boundaries between the regions with the highest and the lowest ER, which is expected to be the transition between the nanograins and biopolymers. This nding is consistent with our previous results by using the piezoresponse force microscopy on mollusk shell, in which the regions of highest Q-factor always appear at the transition between the high and low contact resonance frequencies.28 Thus, the elastic modulus changes in the shell structure can lead to a sudden increase of tip–sample energy dissipation. The width of the high values of tan(d) regions [red curves in Fig. 1(d)] may indicate the extent of the elasticity transition regions, which are about 30–40 nm. In addition, for the rest part of region (green- and blue-color), the value of tan(d) does not differ much. However, lower tan(d) usually maps with the regions having a relatively high elastic modulus. Another nding is that, aer the BE-CR-FM measurement, the topography of the scanned surface is slightly distorted and the nanograins appear much larger than those before the scanning [Fig. 1(a) vs. (b)]. This may be a true response from the sample or be caused by tip blunting. To verify this, AFM images with the image size of 1 1 mm2 were scanned at the same location with the same tip under tapping mode before and aer the BE-CR-FM observation, as illustrated in Fig. 2. It shows much larger size of nanograins and less void spaces aer the BE-CR-
Fig. 2 AFM topography (a and c) and phase images (b and d) of the same area before (a and b) and after (c and d) the BE-CR-FM imaging (1 1 mm2). the enclosed area is where the BE-CR-FM has been scanned.
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FM scan. The tip may be gradually blunted during the BE-CR-FM scanning, which may cause the nanograins to appear larger. However, it is obvious that the larger grain size is not solely due to tip blunting, which is clearly shown in Fig. 2(c) and (d). The size of the nanograins that have been scanned by BE-CR-FM is larger than that of the other nanograins surrounding them. The loading force in the vertical direction may slightly press the nanograins into the sample surface, and the intracrystalline biopolymer matrix may be squeezed out of the surface via a certain path, preferably the void spaces. If the bonding between the nanograins and biopolymers is strong enough, the movement of biopolymers may cause rotation or displacement of nanograins. Therefore, aer being scanned by the BE-CR-FM method, the surface may have new spatial arrangement of certain micro-constituents. The surface property change is revealed clearly by the phase images [Fig. 2(b) vs. (d)]. The entire BE-CR-FM scanned surface shows higher degree of phase lag under the repulsive regime, which indicates more energy dissipation between the tip–sample interactions. The phase contrast of grain boundaries is clearly different from that of the nanograins, and this contrast difference is expected to be caused by the intracrystalline biopolymers. In addition, it seems that the effects of the rearrangement of the micro-constituents has an extent much larger than the scan size of BE-CR-FM measurement. The size and shape of grains far from the affected regions are also altered, for example, the two grains in red circles. The upper one is rotated, and the lower one is slightly pushed out of the surface and the original void space appears smaller. It indicates the interconnectivity of the micro-constituents within a nacre platelet. Additional measurement further conrms this phenomenon is caused primarily by the material response during BE-CR-FM scanning rather than the tip induced artifact, and this can be proven by Fig. S1† which shows the AFM images including the BE-CR-FM scanned area. Lastly, the parallelogram shape of the BE-CR-FM scanned area as shown in the AFM images (Fig. 2) is expected to be due to the thermal dri during the scanning by BE technique with a small scanning area. As indicated before, choice of loading force can affect the values of elastic modulus. Thus, another nacre platelet is scanned by BE-CR-FM with two different loading forces, 36 nN and 72 nN, at the same location. As illustrated in Fig. 3, a higher loading force causes the average value of ER of the same scanned surface to be reduced by about 7 GPa, but the value of tan(d) does not change signicantly. Thus, the tan(d) value is not sensitive to the loading force. In other words, it is only sensitive to the changes on the very surface layer. A larger loading force leads to a larger contact radius, which theoretically should result in a higher contact resonance frequency and a higher contact elasticity. Thus, the reduction of elastic modulus should originate mainly from the difference in sample properties. The contrast in Fig. 3(b) and (d) is nearly identical, except for a few locations, such as the place highlighted by the oval circles. The relative difference of the elastic moduli between the neighbouring points are changed due to the larger loading force. The originally so location appears stiffer, which indicates that the subsurface mineral grains may impose more signicant effects under the higher loading force at this particular location.
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Fig. 3 BE-CR-FM images of the same location with different loading force (500 300 nm2): 36 nN (b and c), and 72 nN (d and e). (a) Topographic image; (b) and (d) mapping of reduced elastic modulus; (c) and (e) mapping of loss tangent, tan d.
Generally speaking, under a higher loading force, a large amount of the subsurface organic matrix may be included in the extended stress eld, which leads to the overall reduction of ER. However, the reduction is not uniform. At some locations, the relative stiffness with respect to the neighbouring regions is changed more signicantly. Furthermore, ER and tan(d) mappings of the nacre on the cross-sectional surface have also been obtained (Fig. 4). On this surface, not only the intracrystalline biopolymers, but also the interlamellar biopolymers are exposed to the surface, and they can be differentiated by their locations. In addition, the aragonite nanograins are orientated in a different crystallographic direction at this surface. As illustrated in Fig. 4(a), bulging layers are usually observed at the boundaries between the mineral layers and interlamellar biopolymers on the ne polished cross-sectional surface of nacre. Their height and thickness depend on the polishing directions as explained in the previous study.29 Similarly, the smallest value of ER should be associated with the highest biopolymer concentration in the
scanned region. The interlamellar biopolymer is located between the mineral layers and should be the so region next to the bulging layer [blue-color in Fig. 4(b)]. The layer of the interlamellar biopolymers can be displaced due to the polishing process, thus it does not show as a perfect straight line. The tan(d) value of interlamellar biopolymer is found to be about 0.0190. In addition, similar to the platelet surface, the interior of the cross-sectional platelet also shows diverse values of elastic modulus. The stiff aragonite nanograins should have the highest elastic modulus (red-color), whose average ER is about 87 GPa, and the corresponding tan(d) is about 0.0104. The soer intracrystalline biopolymers may reduce the detected stiffness. The difference of elastic modulus between the regions of nanograins and the regions of intracrystalline biopolymers is approximately 15%, which is similar to that observed on the platelet surface, with the same reason as discussed previously. In addition, the interlamellar biopolymers appear soer than that of the intracrystalline biopolymers. Thus, the interlamellar biopolymers should deform prior to any other deformations in
Fig. 4 Topography and corresponding BE-CR-FM images of cross-sectional nacre (500 500 nm2, 64 64 pixels, Fn ¼ 72 nN). (a) AFM topography obtained after the BE-CR-FM scanning; (b) mapping of reduced elastic modulus; and (c) mapping of loss tangent, tan d.
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the nacre structure. In general, the average value of elastic modulus on the cross-sectional surface of nacre is about 10 GPa smaller than that on the platelet surface when observed under the same loading force. Thus, when experiencing an external force, nacre tends to deform in the direction parallel to the shell surface rst, rather than perpendicular to it, even at the nanoscale.
Elasticity and loss tangent mappings of natural calcite composite Calcite is another important part of the abalone shell. It locates at the outermost shell, covers the nacre section, and works as the rst shield for all kinds of external forces. However, the elastic properties of the natural calcite composite in abalone shell have not been well studied, especially at the nanoscale. Under the optical microscope, different large prism crystals can be clearly visualized on the nely polished surface of the abalone shell. Each prism is found to be composed by a higher order of hierarchical nanostructures. Three typical kinds of nanostructures have been identied, which are foliated-/needle, granular- (round shape) and block-like (facet shape) structures/particles, as shown in Fig. 5(a), (d) and (g) respectively, depending on the observation locations and orientations. Examples of each structure have been highlighted by red shapes
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in the topographic images. The ER and tan(d) mappings associated with these structures are also illustrated in Fig. 5. Each of the BE-CR-FM mappings is acquired from a single prism. The wide range of elastic moduli in each mapping [Fig. 5(b), (e) and (h)] indicates that a calcite prism is not a simple single crystal as reported in the literature.10 Same as the case of nacre, the calcite prism is also a nanocomposite, which is composed by biopolymers and nanograins in different shapes. The location of concentrated biopolymers can be identied as the soest regions in these mappings (blue- and purple-color). On the cross-sectional surface of the abalone shell [Fig. 5(a)–(c)], a needle-like structure is typically shown in the calcite section. The average ER and tan(d) of the calcite nanograins (red-color) is about 101.15 GPa and 0.0108 respectively. There are also regions with intermediate elasticity (greencolor). This is either because the extent of the stress eld may enclose both nanograins and biopolymers, or may be due to the slightly different crystal orientations of the needles at these scanned locations. On the other hand, both granular-like and block-like shape of nanograins can be observed on the polished outer surface of calcite [Fig. 5(d)–(i)]. The elastic moduli of calcite nanograins are very similar between these two structures. Compared to that of nacre, the variation of elastic modulus at different locations is less dramatic for calcite, especially for the block-like nanostructure. Large areas of the
Fig. 5 Topography and corresponding BE-CR-FM images of calcite (500 500 nm2, 64 64 pixels) observed from: (a–c) cross-sectional surface (Fn ¼ 72 nN) with needle shaped features; (d–f) outer surface with granular (round shape particles) feature in topography (Fn ¼ 36 nN); (g–i) outer surface with block (facet shape particles) feature in topography (Fn ¼ 36 nN). (a), (d) and (g) are AFM topography images; (b), (e) and (h) are reduced elastic modulus mappings; (c), (f) and (i) are mapping of the loss tangent, tan d.
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Table 3 Summary of the quantified reduced elastic moduli and loss tangent of nanograins in nacre and calcite structures based on the nanoscale mappings
Shell surface (36 nN)
Cross-sectional surface (72 nN) Tan d (10 )
ER/GPa
Tan d (103)
Mean values of the entire scanned area (500 500 nm2) Nacre 97.63 5.31 Calcite (needle) — Calcite (granular) 83.85 1.06 Calcite (block) 85.33 4.13
12.2 2.9 — 23.3 9.7 16.0 3.0
79.75 1.35 95.98 3.71 — —
10.8 5.9 11.3 1.8 — —
Average values for each type of mineral nanograins Nacre nanograin 103.1 Calcite grain (needle) — Calcite grain (granular) 92.45 Calcite grain (block) 89.77
11.3 — 20.1 14.0
87.00 101.15 — —
10.4 10.8 — —
Regions
R
E /GPa
nanograins exhibit a very similar elasticity, and the biopolymers are mainly located at the void spaces and boundaries between the calcite blocks. Thus, the mineral nanograins and biopolymers in calcite are not interwoven as intensively as that in the nacre region, and the amount of biopolymers seems less in the calcite nanostructure compared to that in the nacre region. Generally-speaking, the average elastic modulus of these surfaces is less than that of the cross-sectional calcite surface, and higher energy dissipation is also accompanied with a lower elastic modulus. Lastly, the average values of ER and tan(d) from the BE-CR-FM mappings, as well as the values of mineral nanograins for both nacre and calcite are summarized in Table 3. In most cases, the external force may act in the direction normal to the outer shell surface, in which the calcite structure has much smaller elastic modulus than that of the nacre structure. Therefore, calcite structure can work as a buffer layer to discharge the external force and to better protect the internal structures and the living mollusk. On the cross-sectional surface, however, the calcite structure shows a much higher stiffness than the nacre structure. If any resolved force is applied in the direction parallel to the shell surface, the calcite structure may be stiff enough to resist the force. If the force is delivered to the nacre structure, more deformation is allowed within the elastic range. As a result, the living mollusk can be well protected by such a mechanism. Furthermore, from the nanoscale BE-CR-FM mappings, the relative stiffness of each micro-constituent in abalone shell can be ranked and the elastic modulus of mineral nanograins are quantied. In the direction normal to the shell surface, the calcite biopolymers are generally soer than those of the intracrystalline biopolymers in nacre. All the biopolymers are denitely soer than the nanograins, and the calcite nanograins are less stiff than the nacre nanograins. On the other hand, the interlamellar biopolymers show the lowest stiffness among all the components on the cross-sectional surface of abalone shell. The intracrystalline biopolymers are stiffer than the interlamellar ones on this surface, and the calcite biopolymers are stiffer than those of both biopolymers in nacre. In addition, the needle-shaped calcite nanograins are also stiffer than those of the nacre nanograins.
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Conclusions This study has presented and discussed the nanoscale elasticity and loss tangent mappings using the BE-CR-FM method for both nacre and calcite structures in abalone shell. The elastic properties of the micro-constituents have been extracted from these mappings and the relative relations among them are presented on different surfaces of the shell structures. In summary, both nacre and calcite structures can be considered as highly non-homogeneous nanocomposites in terms of their elastic properties. The variations of the elastic modulus can reach over tens of GPa within a hundreds of nanometers area. These variations are caused by the different crystal orientations of the nanograins and the different types and concentrations of the biopolymers as well as their interactions with the mineral grains. The quantied values of the elastic moduli of different mineral nanograins are reported in this work (Table 3). The observed elasticity of the biopolymers may be affected by the subsurface mineral nanograins, and the value of the elasticity may be restricted by the limited width of the frequency band in the BE method. Overall-speaking, these elasticity mappings can provide more insight into the nanoscale structure, the distribution of micro-constituents and the resultant variations of elasticity at the nanoscale, and to contribute further to the better understanding of the extraordinary mechanical behavior and mechanisms of the abalone shell.
Acknowledgements The authors would like to present their sincere gratitude to all the fellows who have contributed to this study. This work was supported by Ministry of Education (Singapore) through National University of Singapore (NUS) under Academic Research Fund (R-265-000-406-112).
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