Development of a remote laser-induced breakdownspectroscopy system for investigation of calcified

tissue samples

Aleš Hrdlička,1,* Lubomír Prokeš,1 Alice Staňková,1 Karel Novotný,1

Anna Vitešníková,1 Viktor Kanický,1 Vítězslav Otruba,1

Jozef Kaiser,2 Jan Novotný,2 Radomír Malina,2

and Kateřina Páleníková2

1Department of Physical Electronics and Department of Chemistry, Faculty of Science,Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic

2Institute of Physical Engineering, Faculty of Mechanical Engineering, Brno University of Technology,Technická 2896/2, 616 69 Brno, Czech Republic

*Corresponding author: ahrdlicka@chemi.muni.cz

Received 20 October 2009; revised 22 December 2009; accepted 11 January 2010;posted 15 January 2010 (Doc. ID 118774); published 3 February 2010

The development of a remote laser-induced breakdown spectroscopy (LIBS) setup with an off-axisNewtonian collection optics, Galilean-based focusing telescope, and a 532nm flattop laser beam sourceis presented. The device was tested at a 6m distance on a slice of bone to simulate its possible use in thefield, e.g., during archaeological excavations. It is shown that this setup is sufficiently sensitive to bothmajor (P, Mg) and minor elements (Na, Zn, Sr). The measured quantities of Mg, Zn, and Sr correspond tothe values obtained by reference laser ablation–inductively coupled plasma–mass spectrometry (LA-ICP-MS) measurements within an approximately 20% range of uncertainty. A single point calibration wasperformed by use of a bone meal standard . The radial element distribution is almost invariable by use ofLA-ICP-MS, whereas the LIBSmeasurement showed a strong dependence on the sample porosity. Basedon these results, this remote LIBS setup with a relatively large (350mm) collecting mirror is capable ofsemiquantitative analysis at the level of units of mg kg−1. © 2010 Optical Society of America

OCIS codes: 300.6365, 280.1545.

1. Introduction

Laser-induced breakdown spectroscopy (LIBS) hasbeen under development formany years, and itsmainfeatures, pros and cons, are well known [1,2]. The ver-satility of LIBS offers a wide field of applications. Oneof the numerous modifications is the so-called remoteLIBS, which is applicable to stand-off analyses ofsolid, liquid, and gas samples [3]. The stand-off LIBSanalysis can also be combined with Raman spec-troscopy [4–6] and used as a lidar [7] instrument.The remote LIBS analysis focusesmainly on inorgan-

ic samples. Themajority of applications are studies ofgeological samples with the possibility of extraterres-trial use [8,9]. The possibility of explosive detection isalso attractive [10] and logical. Analysis of hot sam-ples as, e.g., hotmelts of liquidmetals over a relativelyshort distance of 1–2 m for remote sensing is also fea-sible [11–13]. In addition to the sample type, the suc-cess of remote sensing depends strongly on theinstrumentation used, which is still not commerciallyproduced. The main feature of this technique is thedecrease of laser-induced plasma (LIP) emission withapproximately a square of the sample distance [14]due to the spherical shape of the microplasma. Thelight that travels several tens or hundreds of metersalso loses intensity because of elastic and inelastic

0003-6935/10/130C16-05$15.00/0© 2010 Optical Society of America

C16 APPLIED OPTICS / Vol. 49, No. 13 / 1 May 2010

scattering. A fraction of the collected LIP emissiondepends on the square of the ratio of the diameterof the collectingmirror or lens to the sample distance.Remote LIBS is not applicable to the trace or localanalysis on the micrometric scale. The detectionlimits of elements are typically approximately 10–100mgkg−1 order of magnitude and crater diametersare approximately 1mm, depending on the focusingoptics and the sample distance [3].An application of remote LIBS to calcified samples

of organic origin has not yet been reported, becausethis type of sample does not generally require thestand-off approach. Spatially resolved analyses of his-tological structures of bones and teeth are routinelyperformed, e.g., by electron microprobe x-ray micr-oanalysis (EPXMA) [15,16], particle-induced x-rayemission (PIXE) spectrometry [17–20], x-ray fluores-cence (XRF) [21,22], or laser ablation–inductively co-upled plasma–mass spectrometry (LA-ICP-MS)[23–25]. The LIBS used in this area was reportedin [26,27].Here we demonstrate that the remote LIBS facility

is sufficiently sensitive for determination of some im-portant minor and major elements that are presentin calcified tissue. The facility offers a large field ofapplications for distant analysis of nontransportablebones or geological samples found in archaeologicalexcavations or set in walls. Thus the results are infact a laboratory simulation and represent prepara-tion for their use in the field. The results obtainedby remote LIBS measurements are validated byLA-ICP-MS analyses.

2. Experimental

A. Remote Laser-Induced Breakdown Spectroscopy

The developed remote LIBS setup is based on aNewtonian off-axis telescope with a 350mm spheri-cal primary mirror on the right-hand side of the laserand a secondary planar mirror reflecting, at a dis-tance of 1:5m, the collected light onto the 0:3mmentrance aperture of an optical bundle LLB592(LOT-Oriel UK, Surrey, England). The light is fo-cused on a LOT Oriel 260i monochromator (Czerny-Turner mounting, 320mm focal length, a 2400mm−1

grating, and a 15 μm entrance slit) coupled to a734iStar intensified CCD detector (Andor Technol-ogy, Belfast, Northern Ireland) (Fig. 1). The integra-tion time was 15 μs.The detector was triggered with an output Q-

switched pulse from a Nd:YAG pulsed laser SolarLQ 916 (Solar Laser Systems, Belarus) with a delayof 5 μs. The laser was operated at 532nm, but theavailable wavelengths are 1064, 532, 355, and266nm. Other parameters are a 9ns pulse duration,a pulse energy range for 532nm of 144–225mJ withthe amplifier on or 33:5–52:3mJ with the amplifieroff, and a pulse repetition rate of 1–10Hz. The outputbeam has an 8mm diameter flattop profile. The laserwas operated in a single pulse mode with an impactpulse energy of 170mJ with respect to the best

signal-to-background ratio. The calculated irradi-ance was 2:4GWcm−2 for the crater size of 1mm.A single element was monitored on each sum often pulses, which was repeated five times to monitorfive elements. The spectral range with the used grat-ing is approximately 19nm.

The focusing unit should be able to focus a laserbeam with a minimum spot size to the intendeddistance of approximately 10m. To match these para-meters a three-lens Galilean telescope was designed.The optical aberrations (particularly spherical aber-ration) of the telescope were minimized by optimiz-ing the parameters of the utilized lenses. Based oncomputer simulations, a good performance and valueoption proved to be the following lens combination: Adivergent element consisting of one plano–concavef ¼ −50mm lens (type BK-7, Thorlabs, Newton,New Jersey, 400–600nm antireflection coated) anda convergence element consisting of two plano–convex f ¼ 500mm lenses (type BK-7, Thorlabs,400–600nm antireflection coated). The sample dis-tance from the focusing objective was 6:0m andthe distance from the collecting mirror was 7:5m.

B. Laser Ablation–Inductively Coupled Plasma–MassSpectrometry

A UP-213 (213nm Nd:YAG, New Wave Research,Fremont, California) laser with a flattop profilecoupled with the ICP-MS 7500ce (Agilent Technolo-gies, Santa Clara, California) was used for this study.The fluence at the ablation crater of 100 μm diameterwas 13 J cm−2. The forward rf power was 1500W.Bone meal pressed pellet National Institute of Stan-dards and Technology (NIST) 1486 was used as thestandard. The standard was ablated in line and onthe spot with a settling time of 60 s. The bone samplewas ablated in two lines of spots also with a settlingtime of 60 s per spot. The dwell time per isotope was0:01 s for Na 23, Mg 24, and P 31 and 0:1 s for Zn 66and Sr 87.

C. Crater Profiles

Profiles of ablation craters were measured with anoptical profilometer, sensor HR 60 from FriesResearch and Technology, Bergisch Gladbach,Germany.

Fig. 1. (Color online) Experimental setup.

1 May 2010 / Vol. 49, No. 13 / APPLIED OPTICS C17

D. Sample

A transverse section of a luetic shinbone (tibia) (17–18th century, ossuary in Lukavice near Kyšperk,Eastern Bohemia) was cut with a diamond saw, em-bedded with epoxy resin, and polished.

3. Results and Discussion

Transverse compact bone sections can be used toprovide a relatively well-defined radial distribut-ion of both major and minor elements, which is asignificant advantage in contrast with the mostly in-homogeneous geological samples. Thus, a long bonesample was chosen as being representative of rela-tively homogeneous calcified tissue. It was firstanalyzed by LA-ICP-MS, which is a well-proved con-ventional technique that can serve as a referencemethod for validation of remote LIBS. However,the poor portability of ICP analyzers discriminatesagainst their application in the field.In our experiment radial profiles of selected ele-

ments were compared at the level of spatial resolu-tion of the remote LIBS facility. This parameter islimited by the yielded crater diameter of 1mm. Fourcircular craters were burned in the radial direction.One crater was burned at the outer end of the com-pact bone, one crater at the inner boundary with thebone spongiosa (the porous inner part of the bone),and two craters in the compact bone (Fig. 2). Evenin the compact part the bone tissue shows naturalporosity. This fact is reflected by the measured radialprofiles in Fig. 3. Herein the zero position corre-sponds to the bone surface and marks the left bound-ary between the compact bone and the pure resin(Fig. 2). The depicted positions correspond to the cra-ter centers so that the first remote LIBS cratertouches the bone surface and its center is locatedin the 0:5mm position. The right end of the compactbone is not well defined and the fourth crater fromthe left is partially stretched over the spongiosa.It is expected that a local relative increase of a par-

ticular element yielded with the remote LIBS willnot be as strong as from the LA-ICP-MS becauseof the 100 μm LA-ICP-MS craters burned at mutualdistances of 150 μm, which depict a radial profilewith seven times better lateral resolution than withthe remote LIBS. The 1mm remote LIBS crateraverages intensities that correspond to seven LA-ICP-MS craters and thus the resulting relative inten-sity increase is seven times lower as a consequence,

provided that the increase is observed for only oneLA-ICP-MS crater. This is, however, an ideal situa-tion that is practically not completely fulfilled. Evenif the scan line in the radial direction of a long bone isreproducible, some local differences are always mea-surable. The two radial scans, even if near each other,will never give identical results.

The strongest changes in the relative concentra-tions of the selected elements are expected just atthe bone surface [28], which are related to growthprocesses that can be characterized by, e.g., Zn accu-mulation. Faster growth of bone tissue also induceshigher Zn accumulation. Most accumulation is thenobserved as a result of regeneration of the bone tis-sue, which could also be the case for some surface lo-cations of this luetic bone. The other measuredelements should be homogeneously distributed.

The measured radial distribution of minor ele-ments (Sr, Zn) corresponds well with the above the-ory (Fig. 3) for both ablation methods. The signals ofelements of medium content such as Mg and Na(Fig. 3) are less uniform for both methods but theirdistributions are apparently similar. In contrast withthe LA-ICP-MS Zn signal, they show lower content atthe outer bone boundary than in other parts of theprofile. Zn content increased slightly in the LA-ICP-MS profile at the surface of the compact bone(Fig. 3), which is also in accordance with the theory.This increase is not observable for remote LIBS be-cause of its strong averaging from its approximately100 times larger crater area. The bone porosity is ea-sily recognizable because of the signal slumps in theLA-ICP-MS profile. The remote LIBS signals of Mgand Na are relatively higher from the left as the cor-responding crater touches only the boundary andalso the distribution itself is a function of local posi-tion. The strictly comparable results could be ob-tained only if the same ablation pulse would servesimultaneously as a source of aerosol for LA-ICP-MS and for spectral emission of LIBS.

The exception from other elements is phosphorus.Its signal from remote LIBS is evidently strongly

Fig. 2. (Color online) Shinbone section with ablation craters fromremote LIBS (four upper holes, 1mm diameter) and LA-ICP-MS(lower dense points, spacing of 0:15mm).

Fig. 3. Radial distribution of important elements in compactbone. The positions of the particular points correspond to the cra-ter centers. The right y axis refers to phosphorus only; the left yaxis refers to other elements. The depicted values of Zn and Sr con-tent are increased by a factor of 10.

C18 APPLIED OPTICS / Vol. 49, No. 13 / 1 May 2010

attributed to the porosity of the bone with the in-creased amount of the epoxy resin in the pores(Figs. 2 and 3). Phosphorus is a matrix elementand its mass content can range from 8 to 12%. Its de-crease in the remote LIBS graph apparently corre-lates with the crater porosity (Figs. 2 and 3). Thehighest content was recorded for the smoothest cra-ter (second from left in Fig. 2). The other (minor) ele-ments do not follow this behavior. The phosphorusbehavior should be addressed in another study deal-ing with matrix effects. Nevertheless, this elementand calcium are not interesting for these studies.The quantification of the elemental content for

both remote LIBS and LA-ICP-MS methods wasdone using a simple single point calibration. The par-ticular signal intensity yielded from ablation of thebone was divided by the intensity from the ablationof the standard and multiplied by the element con-tent in the standard. An evaluation of a calibrationtransfer function for remote LIBS [3] was not neces-sary at the level of these preliminary experiments.The standard was ablated under the same conditionsas the sample, i.e., the distance, pulse energy, focus-ing, and accumulation of signals from ten pulses.Both the sample and the standard distance fromthe setup were maintained at a constant value withapproximately 0:5mm precision, which is a good tol-erance with respect to the focusing length and thedepth of field.The yielded LA-ICP-MS values of calcium were

lower than the expected 25–26.5% content in thebone, and the maximum measured value was 19%.Based on this difference, all the measured elementalcontent was increased by a factor of 0.3. The completeinternal standardization of the calcium signal wasnot done because of the expected loss of spatial infor-mation. Figure 3 shows that the signals of someisotopes are correlative, which can lead to nearlystraight lines instead of depicted decreases in the ra-dial structure.The resultant quantities are also depicted in Fig. 3.

The error bars constructed for remote LIBSmeasure-ments are based on the standard deviation of pulse-to-pulse line intensity variations. The explanation ofsome differences between LA-ICP-MS and remoteLIBS signals has been given above and was verifiedby the profilometric measurement that the averageablation rate (AAR) was 2–2:5 μm=pulse in compactbone and 2:5 μm=pulse in the bone meal standard forLA-ICP-MS and 10 μm=pulse in the compact boneand practically the same in the standard for remoteLIBS. The uncertainty of the AAR determination isestimated to be less than 20%. Thus the AAR can beexcluded from the particular differences between themeasured elemental content with LA-ICP-MS andremote LIBS. The reason could be because of thestrong nonlinear behavior of the calibration depen-dence on remote LIBS measurements and the rela-tively high background. Also, different excitationconditions in the microplasma created above thepressed bone meal and above the real bone in the

epoxy resin could be responsible for this phenome-non. The emission intensities were measured on-peak and no changes in line profiles were observedwithin the framework of the resolution capabilityof the monochromator. The Sr and Zn intensitiesare around the background equivalent levels but stillsufficiently strong for quantification; the Na inten-sity is much stronger (4–6 times higher than thebackground).

Based on the intensity-to-background ratio, thelimits of detection (LOD) are estimated to be approxi-mately 1–2mgkg−1 for Zn and Sr, which here repre-sent the minor elements. These values are sufficientfor calcified tissue analysis, are encouraging in thefield of remote LIBS, and can be attributed to the re-latively large collection mirror with respect to sam-ple distance. The 350mm size of the mirror isapplicable to remote sensing at a distance greaterthan 100m [10,29]. Thus, the radiation loss that isdue to the observed astigmatism of the off-axis con-figuration is also compensated for by the large collec-tion mirror.

4. Conclusion

We proved that remote LIBS has the analytical po-tential for analyses of calcified organic tissues inthe open air over a range of several meters. The sen-sitivity on selected P, Mg, Na, Zn, and Sr emissionlines is sufficient for quantification of these ele-ments. The preliminary results show that the mea-sured quantities of minor elements by LA-ICP-MSand remote LIBS are comparable at a semiquantita-tive level and their improvement by studying the be-havior of used spectral lines and calibration curveswill be the subject of future research. In the first ap-proach, it can be concluded that this remote LIBSsetup with a relatively large collecting mirror is cap-able of analysis at the level of units of mg kg−1.

The authors gratefully acknowledge the Ministryof Education, Youth, and Sports of the Czech Repub-lic for research projects MSM0021630508 andMSM0021622411.

References1. D. A. Cremers and L. J. Radziemski, Handbook of Laser-

Induced Breakdown Spectroscopy (Wiley, 2006).2. A. W. Miziolek, V. Palleschi, and I. Schechter, Laser-Induced

Breakdown Spectroscopy, Fundamentals and Applications(Cambridge U. Press, 2006).

3. B. Sallé, P. Mauchien, and S. Maurice, “Laser-induced break-down spectroscopy in open-path configuration for the analysisof distant objects,” Spectrochim. Acta Part B 62, 739–768(2007).

4. H. W. Hubble, M. Ghosh, S. K. Sharma, K. A. Horton, P. G.Lucey, S. M. Angel, and R. C. Wiens, “A combined remote LIBSand Raman spectroscopic study of minerals,” in Lunar Plane-tary Science Conference, Vol. XXXIII (Lunar and PlanetaryInstitute, 2002), abstract 1935, http://www.lpi.usra.edu/meetings/lpsc2002/pdf/1935.pdf.

5. S. K. Sharma, A. K. Misra, P. G. Lucey, R. C. Wiens, and S. M.Clegg, “Combined remote LIBS and Raman spectroscopy at8:6m of sulfur-containing minerals, and minerals coated with

1 May 2010 / Vol. 49, No. 13 / APPLIED OPTICS C19

hematite or covered with basaltic dust,” Spectrochim. ActaPart A 68, 1036–1045 (2007).

6. S. K. Sharma, A. K. Misra, P. G. Lucey, and R. C. F. Lentz, “Acombined remote Raman, LIBS instrument for characterizingminerals with 532nm laser excitation,” Spectrochim. ActaPart A 73, 468–476 (2009).

7. R. Gronlund, M. Lundqvist, and S. Svanberg, “Remote imag-ing laser-induced breakdown spectroscopy and laser-inducedfluorescence spectroscopy using nanosecond pulses from amobile lidar system,” Appl. Spectrosc. 60, 853–859 (2006).

8. J. R. Thompson, R. C. Wiens, J. E. Barefield, D. T. Vaniman, H.E. Newsom, and S. M. Clegg, “Remote laser induced break-down spectroscopy (LIBS) analyses of Dar al Gani 476 andZagami Martian meteorites,” J. Geophys. Res. Planets 111,E05006 (2006).

9. B. Sallé, J. L. Lacour, P. Mauchien, P. Fichet, S. Maurice, andG. Manhès, “Comparative study of different methodologies forquantitative rock analysis by laser-induced breakdown spec-troscopy in a simulated Martian atmosphere,” Spectrochim.Acta Part B 61, 301–313 (2006).

10. C. López-Moreno, S. Palanco, J. J. Laserna, F. DeLucia Jr., A.W.Miziolek, J. Rose, R. A. Walters, and A. I. Whitehouse, “Testof a stand-off laser-induced breakdown spectroscopy sensor forthe detection of explosive residues on solid surfaces,” J. Anal.At. Spectrom. 21, 55–60 (2006).

11. S. Palanco, S. Conesa, and J. J. Laserna, “Analytical control ofliquid steel in an induction melting furnace using a remotelaser induced plasma spectrometer,” J. Anal. At. Spectrom.19, 462–467 (2004).

12. P. L. Garcia, J. M. Vadillo, and J. J. Laserna, “Real-time mon-itoring of high temperature corrosion in stainless steel byopen-path laser-induced plasma spectrometry,” Appl. Spec-trosc. 58, 1347–1352 (2004).

13. C. López-Moreno, S. Palanco, and J. J. Laserna, “Quantitativeanalysis of samples at high temperature with remote laser-induced breakdown spectrometry using a room-temperaturecalibration plot,” Spectrochim. Acta Part B 60, 1034–1039(2005).

14. A. Ferrero and J. J. Laserna, “A theoretical study of atmo-spheric propagation of laser and return light for stand-off la-ser induced breakdown spectroscopy purposes,” Spectrochim.Acta Part B 63, 305–311 (2008).

15. J.B.Lambert,S.VlasakSimpson,J.E.Buikstra,andD.Hanson,“Electron microprobe analysis of elemental distribution inexcavated human femurs,” Am. J. Phys. Anthropol. 62, 409–423 (1983).

16. R. B. Parker and H. Toots, “Minor elements in fossil bone,”Geol. Soc. Am. Bull. 81, 925–932 (1970).

17. N. Boscher-Barre, P. Trocellier, N. Deschamps, C. Dardenne, J.Blondiaux, and L. Buchet, “Nuclear miroprobe study of traceelement in archaeological bones,” J. Trace Microprobe Tech.10, 77–90 (1992).

18. N. Boscher-Barre and P. Trocellier, “Nuclear microprobe studyof a woman’s skeleton from the sixth century,” Nucl. Instrum.Methods Phys. Res. B 73, 413–416 (1993).

19. R.Brenn,Ch.Haug,U.Klar,S.Zander,K.W.Alt,D.N.Jamieson,K. K. Lee, and H. Schutkowski, “Post-mortem intake of lead in11th century human bones and teeth studied by milli- andmicrobeam PIXE and RBS,” Nucl. Instrum. Methods Phys.Res. B 158, 270–274 (1999).

20. St. Jankuhn, T. Butz, R.-H. Flagmeyer, T. Reinert, J. Vogt,B. Barckhausen, J. Hammerl, R. Protsch von Zieten, D.Grambole, F. Herrmann, and K. Bethge, “Ion microprobe ana-lyses of ancient human bone,” Nucl. Instrum. Methods Phys.Res. B 136–138, 329–333 (1998).

21. P. Voglis, A. Attaelmanan, P. Engström, S. Larsson, A. Rindby,K. Boström, and C. G. Helander, “Elemental mapping of bonetissues by use of capillary focused XRF,” X-Ray Spectrom. 22,229–233 (1993).

22. M. L. Carvalho, A. F. Marques, M. T. Lima, andU. Reus, “Traceelements distribution and post-mortem intake in humanbones from middle age by total reflection x-ray fluorescence,”Spectrochim. Acta Part B 59, 1251–1257 (2004).

23. D. Kang, D. Amarasiriwardena, and A. H. Goodman, “Applica-tion of laser ablation-inductively coupled plasma-mass spec-trometry (LA-ICP-MS) to investigate trace metal spatialdistributions in human tooth enamel and dentine growthlayer and pulp,” Anal. Bioanal. Chem. 378, 1608–1615 (2004).

24. D. J. Bellis, K. M. Hetter, J. Jones, D. Amarasiriwardena, andP. J. Parsons, “Calibration of laser ablation inductivelycoupled plasma mass spectrometry for quantitative measure-ments of lead in bone,” J. Anal. At. Spectrom. 21, 948–954(2006).

25. M. D. Seltzer, V. A. Lance, and R. M. Elsey, “Laser ablationICP-MS analysis of the radial distribution of lead in the femurof Alligator mississippiensis,” Sci. Total Environ. 363, 245–252 (2006).

26. O. Samek, D. C. S. Beddows, H. H. Telle, J. Kaiser, M. Liška, J.O. Cáceres, and A. Gonzáles Ureña, “Quantitative laser-induced breakdown spectroscopy analysis of calcified tissuesamples,” Spectrochim. Acta Part B 56, 865–875 (2001).

27. M. Z. Martin, N. Labbé, N. André, R. Harris, M. Ebinger, S. D.Wullschleger, and A. A. Vass, “High resolution applications oflaser-induced breakdown spectroscopy for environmental andforensic applications,” Spectrochim. Acta Part B 62, 1426–1432 (2007).

28. T. A. Elliott and G. W. Grime, “Examining of diagenetic altera-tion of human bone material from a range of archaeologicalburial sites using nuclear microscopy,” Nucl. Instrum. Meth-ods Phys. Res. B 77, 537–547 (1993).

29. S. Palanco, C. Lopez-Moreno, and J. J. Laserna, “Design, con-struction and assessment of a field-deployable laser-inducedbreakdown spectrometer for remote elemental sensing,” Spec-trochim. Acta Part B 61, 88–95 (2006).

C20 APPLIED OPTICS / Vol. 49, No. 13 / 1 May 2010