Multielemental analysis of prehistoric animal teeth by laser-induced breakdown spectroscopy and laser ablation inductively coupled plasma mass spectrometry

Multielemental analysis of prehistoric animal teethby laser-induced breakdown spectroscopy

and laser ablation inductively coupledplasma mass spectrometry

Michaela Galiová,1 Jozef Kaiser,2,* Francisco J. Fortes,3 Karel Novotný,1

Radomír Malina,2 Lubomír Prokeš,1 Aleš Hrdlička,1 Tomáš Vaculovič,1

Miriam Nývltová Fišáková,4 Jiří Svoboda,4 Viktor Kanický,1

and Javier J. Laserna3

1Department of Chemistry, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic2Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology,

Technická 2896/2, 616 69 Brno, Czech Republic3Department of Analytical Chemistry, Faculty of Sciences, University of Malaga,

Campus de Teatinos s/n, 29071 Malaga, Spain4Institute of Archaeology, Academy of Science of the Czech Republic,

Královopolská 147, 612 00 Brno, Czech Republic

*Corresponding author: [email protected]

Received 18 September 2009; revised 31 January 2010; accepted 1 February 2010;posted 2 February 2010 (Doc. ID 117389); published 26 March 2010

Laser-induced breakdown spectroscopy (LIBS) and laser ablation (LA) inductively coupled plasma (ICP)mass spectrometry (MS) were utilized for microspatial analyses of a prehistoric bear (Ursus arctos) toothdentine. The distribution of selected trace elements (Sr, Ba, Fe) was measured on a 26mm× 15mm largeand 3mm thick transverse cross section of a canine tooth. The Na and Mg content together with thedistribution of matrix elements (Ca, P) was also monitored within this area. The depth of the LIBScraters was measured with an optical profilometer. As shown, both LIBS and LA-ICP-MS can be success-fully used for the fast, spatially resolved analysis of prehistoric teeth samples. In addition to micro-chemical analysis, the sample hardness was calculated using LIBS plasma ionic-to-atomic line intensityratios of Mg (or Ca). To validate the sample hardness calculations, the hardness was alsomeasuredwith aVickers microhardness tester. © 2010 Optical Society of America

OCIS codes: 140.3440, 300.6365, 330.6100.

1. Introduction

Teeth and bones consist of an inorganic calcium phos-phate mineral approximated by hydroxylapatiteCa10ðPO4Þ6ðOHÞ2 and matrix proteins. The physicaland chemical properties of these bioapatite crystals

differ from those of geologic hydroxylapatite becauseof the way they are formed. These unique propertiesare required to fulfill the biological functions of bonesand teeth [1]. The chemical constituents of the toothtissue layers (enamel and dentine) are tolerant tosubstitution by a range of trace elements. The enam-el is the hardest and most highly mineralizedsubstance of the body [2], whereas the dentinecomprises the bulk of a tooth. A high content of trace

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elements, including Zn, Sr, Fe, Al, Ba, and Pb, amongothers, in the ≥1000 ppm range [3] have been found intooth tissue.Permanent tooth enamel and dentine begin calci-

fication around birth and continue to calcify into ado-lescence. The composition of subsurface enamel isalready fixed before tooth emergence and is thereforeable to provide a retrospective and relatively perma-nent record of the trace elements absorbed duringthe period of enamel formation [4]. Spatially resolvedanalysis of trace element concentration has a broadrelevance in disciplines ranging from dentistry andhealth care [5] to forensics [6], anthropology [7],zoology [8], and archaeology [9].The trace element concentration of dental tissue is

traditionally studied by a bulk sampling approach[10,11]. Further techniques include the mechanical(grinding, drilling) or chemical (acid dissolution)separation of successive enamel layers and their pos-terior analysis [12,13]. For many years, these layershave been studied by modern analytical techniquessuch as x-ray fluorescence (XRF) [14,15], electronmicroprobe x-ray microanalysis (EPXMA) [16,17],particle (proton)-induced x-ray emission spectro-scopy (PIXE) [18], or secondary ion mass spectrome-try (SIMS) [19,20].The feasibility of LA-based analytical methods for

quantitative and qualitative microspatial analysis ofdifferent samples has been reported by severalauthors [10,21–23]. In particular, LIBS and LA-ICP-MS allow fastmultielemental analysis of a broadvariety of matrices. Furthermore, the excellentspatial resolution of LA-based methods offers thepossibility to study the chemical distribution, namely,chemical mapping, of the elements constituents[24–27]. Additionally, LA-ICP-MS provides highsensitivity and low detection limits.We achieved a microspatial analyses of a 26mm ×

15mm large and 3mm thick transverse cross sectionof a permanent canine tooth of a fossil brown bear(Ursus arctos); see Fig. 1. The investigated sampleconsisted of dentine covered by a thin cementumlayer. The distribution of selected trace (Sr, Ba, Fe)and matrix (Ca, P) elements was monitored. Thespread of further elements (Na, Mg) was also mea-sured. The intertooth distribution of these elementsderived mainly from LA-ICP-MS measurements wasreported in several earlier publications [26–28]. Thechoice of investigated chemical elements was furthermotivated by their importance in paleozoology.

It has been observed that the Sr content decreasesin the dentine toward the pulp [29–31]. Some devia-tions in the Sr=Ca and Sr=Ba ratios indicate popula-tion mobility [32] or social status in childhood [33].The metabolism of Sr and Ba is similar to that ofCa. The Sr=Ca and Ba=Ca ratios generally showon the character of the nutrition [34–37], especiallythe proportion of foodstuff of animal and vegetableorigin in the diet. These ratios can differ in geo-graphic regions with different Sr, Ba, and Ca contentin the environment. The Sr=Ba ratio in calcified tis-sues reflect the Sr=Ba ratio in the environment andthen some deviations of these ratios in incrementsindicate population mobility [32]. In fossil findings,the Ba and Sr content can be increased by postdepo-sition processes [36]. Fe contamination is importantfor archaeological materials [14,38–40]; it is prefer-entially accumulated in cavities or along the rimsof fossilized calcified tissue and can also give evi-dence of postmortem alterations [41,42]. Both the Naand the K content decrease in the dentine [16,43,44].Good indicators of the contamination are also Al andMn [37,40].

Mg plays an important role in tooth mineraliza-tion, mainly in its early phase. It is progressively re-placed with Ca and behaves similar to Sr, i.e., itsdistribution is almost analogical but the metabolismis slightly different. Its content increases slightlyfrom the surface through the dentine. The Mg con-tent in dentine is substantially higher than in enam-el [17,45]. It was verified for macaque, mastodon, andhuman ([16,43,44]), respectively. An increase fromthe apical toward the cervical end of the dentine wasalso reported [46]. The differences among the outer,the inner, and the pulpal parts of the dentine werenegligible. In addition to microchemical analysis,the sample hardness was calculated using LIBSplasma ionic-to-atomic line intensity ratios of Mg.To validate the sample hardness calculations, thehardness was also measured with a Vickers micro-hardness tester. The ablation crater depths were de-termined from the cross sections measured with anoptical profilometer.

2. Material and Sample Preparation

Dolní Věstonice II is one of a series of large settle-ments of mammoth hunters on the loess elevationsat an altitude of approximately 180–240m abovesea level (a.s.l.), rising above the Dyje river and slop-ing further to the top of PavlovskéVrchy (550ma.s.l.).Archaeological excavations at this site were orga-nized by Klíma [47] and Svoboda [48] between 1985and 1989, and additional excavations took place in1991, 1999, and 2005. The site became world famousfor human paleontological finds including the tripleburial (DV 13–15) discovered at the top of the site,the DV 16 burial on the western slope, and individualhuman remains scattered at various places in the cul-tural layer. In terms of chronology, the site probablyresults from repeated occupations extended overthe time span of 28 thousands years before the

Fig. 1. (Color online) Photographs of the studied sample. At left,the investigated cross section of the Ursus arctos canine tooth(rectangle). The bars have a length of 1 cm.

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present (ka) to 24ka 14C BP (all 14C data are uncali-brated). The first series of radiocarbon dates, around29–27ka 14C BP, were obtained from charcoal in theunderlying paleosols, with little or no evidence of hu-man occupation, where only two dates in the lowerpart of the site are associated with artifacts. The sec-ond series of radiocarbon dates, all from clearlyanthropogenic cultural layers and from artificialstructures (settlement units) cluster around 27ka14C BP (Early Pavlovian). A third series of dates fallsinto the time span of 27–25ka 14C BP (Evolved Pav-lovian). This time span is framed by a series of earlierand later luminescence dates frompaleosols and loessbelowand above,whichmakeDolní VěstoniceV II thebest dated Gravettian site in the region.The bear tooth that we analyzed (Fig. 1) originates

from field II, which is a longitudinal zone on top ofthe site, adjacent to the triple burial, and excavatedin 1986. It can be related to the third series ofradiocarbon dates obtained from the same area.The analyzed tooth (canine-C1) belongs to brownbear (Ursus arctos). Abrasion of the tooth’s oclusalarea and increments of cementum of the tooth’s root[49] were studied to determine the age and season-ality. This bear died at the age of 14 and the termof death is possible to estimate from unfinishedsummer increment and absence of winter incrementbetween the summer and the autumn season(August to October). Both LIBS and LA-ICP-MSanalyses were performed on a tooth slice indicatedby the rectangle in Fig. 1.

3. Instrumentation

A. Laser-Induced Breakdown Spectroscopy Device

LIBS analysis was performed in the laser laboratoryat the University of Malaga (Malaga, Spain). In brief,the second harmonic (532nm) of a pulsedQ-switchedNd:YAG laser with homogeneous energy along thebeam cross section (Spectron Model SL 284, pulsewidth 5ns, beam diameter 4mm) was used to gener-ate microplasma on the sample surface in air atatmospheric pressure. The beam was expanded 3×by an optical system consisting of two lenses (a diver-ging BK7 lens with a 25mm focal length and aconverging BK7 lens with a 75mm focal length)and then focused onto the sample surface with a100mm focal length BK7 lens. Plasma emission wascollected by a (5 m long fiber optic, with a 600 μm di-ameter and a 0.22 NA) and guided onto the entranceslit of a 0:5m focal length Czerny–Turner imagingspectrograph (Chromex Model 500 IS, f -number 8,fitted with interchangeable gratings of 300, 1200,and 2400 groovesmm−1). Spectral emission wasdetected by an intensified charge-coupled device(Stanford Computer Optics Model 4Quik 05) with768 × 512pixels, each 7:8 μm × 7:8 μm. This config-uration provides a spectral window of ∼15nm anda spectral resolution of 0:02nmpixel−1 using an en-trance slit width of 50 μm and a 2400 groovesmm−1

grating. Operation of the detector was controlled

with 4Spec software. The sample was positionedon two crossed motorized stages (PI Physik Instru-mente) for both X and Y displacement. Moreover,a viewing system to assist with the examinationand sample positioning was also used.

The LIBS spectra were acquired in appropriatespectral windows from the 253–617 nm region.The following spectral lines were used for the analy-sis: P (I) (253.56 and 255.32 nm); Mg (II) (279.55 and280.27 nm); Mg (I) ð285:21nmÞ; Fe (I) ð302:40nmÞ;Ca (II) (315.89, 317.93, 370.60, 373.69, 393.36, and396.85 nm); Ca (I) (422.67, 518.89, and 616.22nm); Ba (II) ð455:40nmÞ; Sr (I) (460.73 and 407.77nm); and Na (I) (589.00 and 589.59 nm).

Typical single-shot LIBS spectra are shown inFig. 2. For LIBS line scanning, spectral lines routi-nely used in LIBS analysis [50] were chosen. To prop-erly relate the detected emission line intensities withthe species amount, i.e., to avoid self-absorption, themicroplasma emission and the detection temporalinterval were optimized by preliminary measure-ments. The diameter of LIBS ablation craters was∼200 μm, and they were placed at a distance of∼500 μm from each other. After three cleaning shots,the LIBS signal was averaged from seven shots firedto the same sample position. During data analysis,the continuum background determined for each shotfrom five data points on both sides of the monitoredspectral line by a linear background fit method wassubtracted from the intensity value of each datapoint that formed the spectral line.

B. Laser Ablation Inductively Coupled Plasma MassSpectrometry Setup

Instrumentation for LA-ICP-MS consists of a LA UP213 system (NewWave, USA) and an ICP mass spec-trometer, Model 7500 CE (Agilent, Japan). A com-mercial Q-switched Nd:YAG LA device works atthe fifth harmonic frequency (213nm). The ablationdevice is equipped with programmable XY stages tomove the sample along a programmed trajectoryduring ablation. Target visual inspection is accom-plished by means of a built-in microscope/CCD cam-era system. A sample was enclosed in the SuperCell(NewWave, USA) and was ablated by the laser beam,which was focused onto the sample surface through aquartz window. The ablation cell was flushed withhelium (carrier gas), which transported the laser-induced aerosol to the ICP. A sample gas flow ofargon (0.6 l/min) was admixed to the helium carriergas flow (1.0 l/min) behind the LA cell. Therefore, thetotal gas flow was 1:6 l=min. Optimization of LA-ICP-MS conditions (gas flow rates, sampling depth,electrostatic lens voltages of the MS) was performedwith the glass reference material NIST SRM 612with respect to a maximum signal-to-noise ratio(SNR) and a minimum oxide formation (ThOþ=Thþcount ratio 0.2%, Uþ=Thþ count ratio 1.1%).

To analyze specific locations in the sample for LA-ICP-MS line scanning and 2D mapping LA was usedin a hole drilling mode (fixed sample position during

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LA) for 6 s for each spot. The LA-ICP-MS ablationpattern consisted of ablation craters of ∼100 μm di-ameter at a distance of ∼200 μm from each other.Time delay between the end of LA of one spot and theinitiation of LA of the next spot was 10 s. LA was per-formed with a laser spot diameter of 100 μm, a laserfluence of 12 J cm−2, and a repetition rate of 10Hz.The 23Na, 24Mg, 57Fe, 86Sr, 88Sr, and 135Ba isotopeswere measured at an integration time of 0:1 s=isotope; 43Ca, 44Ca, and 31P were measured at anintegration time of 0:05 s=isotope.The LA-ICP-MS quantitative procedure was per-

formed by matrix-matched calibration. As a calibra-tion standard, standard reference material (SRM)NIST 1486 (powdered bone meal) was used becauseits matrix is similar to that of teeth. The powderedbone meal was pressed into 10 mm diameter pellets.Pellet preparation was performed without an addi-tional binder by use of a manual hydraulic press(Mobiko Company, Czech Republic). The influenceof laser beam energy variation on the ablation ratewas minimized using Ca normalization. The Baand Sr elemental content obtained from the NISTstandard were normalized to Ca content:

XðiÞnorm ¼ XðiÞ=XðCaÞ; ð1Þ

where XðiÞnorm is the normalized elemental content(Ba or Sr); XðiÞ is the elemental content in the bearteeth obtained with the NIST standard; XðCaÞ is theCa content in bear teeth obtained using the NISTstandard. The typical error of LA-ICP-MS measure-ments is between 7 and 10%.

C. Measurements of the Laser-Induced BreakdownSpectroscopy Ablation Crater Depths and SampleHardness

The ablation crater depth was determined from thecross sections measured with aMicroProf optical pro-filometer (Fries Research and Technology, Germany).The profilometer utilizes chromatic aberration of thepositive lens of the optical sensor. The white lightfrom the halogen lamp passes through the opticalfiber to the sensor CHR 150N with high chromaticaberration so each chromatic component of the whitelight is focused on a different plane [51]. The sensoris set so that the surface under study would lie in therange of focal planes at a distance of approximately300 μm. The light scattered from the surface is thencollected by the same lens and transported by thesame optical fiber to the spectrometer. The intensityspectral distribution processed by the spectrometerhas a maximum at the wavelength focused ex-actly on the surface. The position of the surface is

Fig. 2. Typical single-shot LIBS spectra of microplasma created by focusing the laser beam with an energy of∼90mJ=pulse. The spectrawere recorded with delay and integration times of 1 and 10 μs, respectively. Ba, Sr, and Ca lines were measured with a 1200 groovemm−1

grating; Na and Fe lines were measured with a 2400groovemm−1 grating.

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computed using the known course of the sensor chro-matic aberration with ∼50nm vertical resolution. Agreater vertical range of the surface is measured inseveral slices, each less than 300 μm thick and con-nected automatically. The lateral resolution is givenby the scanning table motion; the ablation craterswere measured with 1 μm lateral resolution. The var-iation of the sample hardness was measured with aLeco LM247AT microhardness tester.

4. Results and Discussion

A. Line Scans and Mapping

The line scans derived from LIBS and LA-ICP-MSanalysis of macroelement Na and trace element Feare compared in Fig. 3. Both LA-based techniquesrevealed similar accumulation for all the tracked ele-ments across the scanned lines. We observed similarbehavior of elemental distribution inside the dentineto that reported in the literature, i.e., the differencesamong theMg signal in outer, inner, and pulpal partsof the dentine were negligible. The inhomogeneity inthe Na signal can indicate contamination from out-side, which was further confirmed with the lineand area scans of Fe. The Fe=Ca ratio also appar-ently increases, which indicates diagenetic changesin the root canal [Fig. 3(c)] [37,40–42]. The rangeof these changes was further investigated by mea-surements of the sample hardness (Subsection 4.B).The Na content decreases toward the root canal andcorresponds to the common observations [16,43,44].The P distribution derived from line scans was in ac-cordance with the distribution of another studiedmatrix element (Ca).The ∼100 μm ablation crater diameter used for the

LA-ICP-MS technique allowed mapping the samplewith a higher spatial resolution. The results of thiselemental mapping in two different regions of thetooth cross section are shown in Fig. 4. The (normal-ized) variations of Sr=Ca, Ba=Ca, Na=Ca, and Fe=Caare plotted in Fig. 4. The different chemical composi-tion of the cementum (∼1mm thick layer) can beclearly distinguished. The Mg (distribution is notshown) similar to natrium is nearly homogeneouslydistributed (Fig. 4). It does not show an anomalyfrom the theoretical expectation [16,43–46].

B. Sample Hardness and Depth of the Ablation Craters

Beyond the possibility of straightforward elementalmapping, LA-based analytical methods allow direct

estimation of other sample properties. Estimationof the sample hardness by Mg or Ca ionic-to-atomicline intensity ratios is based on the interaction be-tween the laser-induced shock wave and the samplesurface [52]. The recoil force on a softer surface is

Fig. 3. (Color online) LIBS (1) and LA-ICP-MS (2) line scans po-sitioned as shown in the photograph: (a) comparison of the elemen-tal distribution of (b) Na and (c) Fe obtained from the cross sectionsof fossil brown bear (Ursus arctos) canine tooth dentine.

Fig. 4. (Color online) Results of LA-ICP-MS elemental mapping in two different areas of the sample. The bar has a length of 400 μm.

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weaker and reflects the shock wave with lower velo-city in comparison with that of a hard target, result-ing in a reduction of the ionization effectiveness andthen of the ionic-to-neutral intensity ratio.In accordance with the expectations based on

the increased Fe=Ca ratio near the root canal(Subsection 4.A), in the investigated tooth slice thedentine hardness decreases toward the root canal(Fig. 5). This is typical for archaeological teeth sam-ples in which part of the material near the root canalis affected by diagenesis. The estimated hardnesscharacteristic was confirmed by microhardness mea-surements. The sample was repolished before thesemeasurements and the test pattern was placed nearthe LIBS ablation craters for Mg detection. The dis-tance between the Vickers test dents was 100 μm.The ablation crater depths determined from the

cross sections measured with an optical profilometerare shown for comparison in Fig. 5(c).

C. Use of Sr=Ca and Sr=Ba Ratios to Determine the BearMigration

The line and area scans of the sample were used toreconstruct the ethology of this fossil brown bear(Ursus arctos). The seasonal fluctuations of theSr=Ca and Sr=Ba detected by both LA-based techni-ques, indicated the migration of this bear betweenhis hibernaculum location and the place where thefossils were found. As an example, Fig. 6 shows theseratios for both LIBS line scan and LA-ICP-MS map-ping. The incremental strips in the LA-ICP-MSmapsare indicated by dashed curves. The dark areas arewell correlated with the lower Sr=Ba ratio in themap; they are related to the narrow winter stripsthat can be identified in the photographs superim-posed on the Sr=Ba and Sr=Ca line scans above thoseacquired by LIBS. The correlation of the Sr=Ca ratiowith those strips is not so apparent because there areboth higher and lower Sr-doped wide strips in themapped area.

Dentine, similar to bone tissue or cementum, canbe more easily affected by diagenesis in comparisonwith, e.g., enamel [38,54]. In the investigated sam-ple, to minimize the effect of the diagenesis for theoutcomes of the analysis, the area of the sample,where the elemental ratios were mapped (Fig. 6),was carefully selected on the basis of microscopicobservation of the sample structure and samplehardness measurements reported in Subsection 4.B.For further validation of these results, the wholesample area was mapped by quantitative LA-ICP-MS procedure, as described in Subsection 3.B. Onthe basis of this analysis, the average amount ofBa and Sr within the investigated area of the toothdentine was 43 and 688mg=kg, respectively. Thesemeasurements (Fig. 7) proved the correlation be-tween both the Sr=Ba and the Sr=Ca ratios obtainedafter the quantitative LA-ICP-MS procedure andnarrow winter strips. The map of Ca distribution

Fig. 6. (Color online) Sr=Ca and Sr=Ba ratios derived from LIBS line scan and LA-ICP-MS mapping; dotted lines represent the differentregions (white, summer bands; brown, winter bands) of the tooth cross section. The bar has a length of 500 μm.

Fig. 5. (a) Sample hardness obtained withMg ionic-to-atomic lineintensity ratios along the LIBS line scan, (b) results of microhard-nessmeasurements at near proximity of the LIBS ablation craters,and (c) the ablation crater depths calculated from the crosssections as measured with an optical profilometer.

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within this area, expressed in mg=g, is shown forcomparison.The somewhat homogeneous Ca distribution and

the visible change in Sr=Ba and Sr=Ca values inthe middle between the edge of the tooth and the rootcanal indicates that this part was not affected signif-icantly by diagenesis and can be used to reconstructthe ethology of this fossil brown bear (Ursus arctos).The diagenesis of calcified tissue is indicated mainlyby increased amounts of elements such as Fe, Mn,and Al from soil by capillary elevation of water solu-tions, and Ca intensity falls within these profiles.On the basis of these measurements, it can be con-

cluded that the bear probably consumed an animalfood mainly in the hot season because of the Sr=Caincreased ratio that is visible mainly in the LIBSscans and based on parallel results of isotope geo-chemical (86Sr=87Sr, 14N=15N, and 13C=12C) analysis;Sr can be accumulated from the bodies of consumedherbivores. The bear‘s migration is characteristic notonly for the seasons but also for different years,which is visible by comparison of the Sr=Ba ratiosin the particular wide strips from the outer to the in-ner parts of the dentine in the LA-ICP-MS maps andby the Sr=Ba decrease observable in the LIBS scanfrom different soils and food chain. The bear probablychanged its living territory [49,54–56].

5. Conclusion

Laser ablation-based analytical techniques wereused for mapping and line-scanning a fossil animaltooth section. LIBS, similar to LA-ICP-MS, provedto be suitable for fast, spatially resolved analysesof such calcified tissues. Moreover, this techniqueallows straightforward estimation of the sample

hardness. From an archaeological point of view, itwas possible to reconstruct the ethology of the fossilbrown bear, i.e., the nutrition, health, and migrationon the basis of these measurements. The measuredSr=Ca and Sr=Ba profiles across the sample showedseasonal fluctuations and proved the migration ofthis bear between his hibernaculum location andthe place where the fossils were found. Together withthe results from other techniques (i.e., study of ce-mentum increments), we can conclude that this bearspecimen most probably was hunted when it wasforaging before winter dormancy and was migratingnear a human settlement (where the fossils werefound). We have shown that LIBS and LA-ICP-MScan be successfully applied as direct or complemen-tary techniques in spatially resolved microchemicalanalysis of fossil samples.

M. Galiová, J. Kaiser, and R. Malina acknowledgethe Ministry of Education, Youth, and Sports of theCzech Republic for research projects MSM0021630508 and ME09015. K. Novotný, A. Hrdlička.,T. Vaculovič, and V. Kanický acknowledge the Minis-try of Education, Youth, and Sports of the CzechRepublic for research projects MSM 0021622411and ME08002. M. Nývltová Fišáková and J. Svobodaacknowledge funding from the Grant Agency of theAcademy of Sciences of the Czech Republic (GA AVCR), KJB800010701, “Hunting Strategies of UpperPalaeolithic People,” and from the Institute of the Ar-chaeology Academy of Science of the Czech Republic,AVOZ80010507.

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Fig. 7. (Color online) Map of Ca [mg=g] distribution and Sr=Ca, Sr=Ba ratios obtained with quantitative LA-ICP-MSmapping. The Sr=Caand Sr=Ba ratios were normalized. The bar has a length of 500 μm.

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