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Elemental mapping of teeth using μSRXRF
Nuclear Instruments and Methods in Physics Research B 213 (2004) 569–573 www.elsevier.com/locate/nimb
Elemental mapping of teeth using lSRXRF M.J. Anjos a,b,*, R.C. Barroso b, C.A. Perez c, D. Braz a, S. Moreira d, K.R.H.C. Dias e, R.T. Lopes a a
Nuclear Instrumentation Laboratory/COPPE/UFRJ, P.O. Box 68509, 21945-970 Rio de Janeiro, Brazil b Universidade do Estado do Rio de Janeiro, Physics Institute, 20550-900, Rio de Janeiro, RJ, Brazil c Laborat orio Nacional de Luz Sıncrotron-LNLS/CNPq/MCT, 13084-971, P.O. Box 6192, Campinas, SP, Brazil d Universidade de Campinas, Faculdade de Engenharia Civil, 13084-971, P.O. Box 6021, Campinas, SP, Brazil e Universidade do Estado do Rio de Janeiro, Faculdade de Odontologia, 20550-900, Rio de Janeiro, RJ, Brazil
Abstract Human teeth were analysed by X-ray microfluorescence analysis using synchrotron radiation (lSRXRF). The aim of this work was to study the elemental distribution for Ca, Zn and Sr along the dental regions, enamel, dentine and pulp from patterns of relative fluorescence intensities. The measurements were performed in standard geometry of 45 incidence, exciting with a white beam and using a conventional system collimation (orthogonal slits) in the XRF beamline at the Synchrotron Light National Laboratory (Campinas, Brazil). The results show that Ca distribution is quite constant and it is independent of the tooth type and individuals characteristics. An increase of the Zn concentration was found for the pulp region and for untreated carious areas. Ca and Sr distributions show a similar behavior. 2003 Elsevier B.V. All rights reserved. PACS: 87.64.N; 41.60.Ap; 28.41.Rc Keywords: X-ray fluorescence; Elemental distribution; Human teeth; Synchrotron radiation
1. Introduction Human tooth is a complex system of specialized tissues: enamel, dentin, cementum and pulp. Each tooth is basically made up of two parts: the crown and the root. Enamel serves to protect the underlying tooth, the next layer of which is dentine. The bulk of the pulp is similar in composition to connective tissue, containing various types of cells,
* Corresponding author. Address: Universidade de Estado do Rio de Janeiro Instituto de Fisica, Rio de Janeiro, Brazil. Fax: +55-21-25628444/2906626. E-mail address: [email protected] (M.J. Anjos).
collagen fibers, nerve trunks, lymphatic and blood vessels. The tooth is anchored to the bone at its root by means periodontal ligament. The outer face of the root dentine is covered with a thin layer of cementum [1]. Tooth tissues are similar to those materials making up bone. Since bone accumulates a variety of trace elements, it is very interesting to study the elemental distribution in human teeth to evaluate biological processes. The knowledge of the spatial distribution of trace elements in tissues is involved in many biological functions of living organisms. In this way, elemental distribution in teeth can provide information about physiology of elements, environmental influence, dietary habits,
0168-583X/$ - see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-583X(03)01673-2
570
M.J. Anjos et al. / Nucl. Instr. and Meth. in Phys. Res. B 213 (2004) 569–573
or contamination by metallic amalgams used as restorative material [2]. Also, chemical composition and local variations in elemental contents were correlated with mechanical characteristics of enamel [3]. Micro X-ray fluorescence using synchrotron radiation (lSRXRF) is a multielementar analytical technique. The intrinsic characteristics of synchrotron radiation permit to implement spectrochemical analysis with spatial resolution on the micrometer scale, high efficiency for trace elements determination and short time of analysis requirement. lSRXRF technique is an useful tool for the study of elemental distribution in biological samples such as human teeth [4–6]. In this paper, the spatial sensitivity of lSRXRF technique was used to study local variations in the elemental distribution for Ca, Zn and Sr over the axial crosssection of four human teeth.
2. Materials and methods 2.1. Instrumentation The Synchrotron Light National Laboratory (Laborat orio Nacional de Luz Sıncrotron, LNLS) is a national research center located in Campinas, Brazil. The electron energy inside the storage ring is 1.37 GeV with a dipole magnetic field of 1.65 T, which produces a critical photon energy of 2.08 keV. The synchrotron radiation source for the XRF beamline is the D09B (15) bending magnet of the storage ring [7]. The sample was positioned in the image plane within an accuracy of 0.5 lm with a 3 axis (x, y, z) remote-controlled stage. A video microscope (magnification 500·) was used to position the sample precisely in the beam. The measurements were performed in standard geometry (45 + 45), exciting with a white beam and using orthogonal slits (300 lm · 300 lm). In this way, pixels of 300 lm · 300 lm were obtained keeping a high flux of photons on the sample. The fluorescence spectrum was recorded with a Si(Li) detector of 165 eV FWHM at 5.9 keV in air atmosphere positioned at 90 of the incidence direction. To determine the chemical elements in teeth, Ka -line of Ca
(3.691 keV), Zn (8.631 keV) and Sr (14.142 keV) were used. All the spectra were analyzed using the Quantitative X-ray Analysis Software (QXAS) package, which is a conventional program for spectrum analysis [8]. 2.2. Sample preparation Human teeth samples were obtained from adults donors aged 23–70 years whose extractions were a result of periodontal diseases. The samples consisted of one canine, two first premolars and one first molar collected from three subjects, one of them was a male. Sample #4 has a small carious region and the other ones are healthy. Once teeth were extracted, they were immediately rinsed in tap water, disinfected and placed in an individual container. The teeth were cut with a diamond saw so that the teeth were divided into two halves, with the cut lying perpendicular to the bucolingual division line. The cut surfaces of each half were polished in order to obtain a smooth and plane surface, of 1 mm thickness. After this procedure the slices were rinsed in distilled water, dried in a clean environment at room temperature and stored in an individual container. Ultrasonic cleaning was avoided because it tended to crack the samples. In this analysis only one slice of each tooth was analyzed. Selected areas of the crown of each sample were analyzed by bidimensional (x, y) scanning. The scan pattern adopted was the same for all samples: from left to right for x-axis and from upper crown surface (enamel) to root for yaxis. The counting live-time for each pixel (300 lm · 300 lm) was 7s/step and the step size was 300 lm/step in both directions.
3. Results and discussion The elemental variations as function of position (x, y) within the crown of the human teeth are shown in Figs. 1–3. The local distribution of Ca, Zn and Sr in all analyzed samples named #1, #2, #3 and #4 are presented in Figs. 1–3, respectively. The relative fluorescence intensities were normalized using the maximum value found for each
M.J. Anjos et al. / Nucl. Instr. and Meth. in Phys. Res. B 213 (2004) 569–573
571
Fig. 1. Elemental distribution for Ca in (a) sample #1, (b) sample #2, (c) sample #3 and (d) sample #4.
Fig. 2. Elemental distribution for Zn in (a) sample #1, (b) sample #2, (c) sample #3 and (d) sample #4.
element. For samples #1, #2 and #3, the spacing between consecutive patterns was 1.5 mm beginning at 1.5 mm from the upper crown surface. For
sample #4, the first pattern was obtained at 0.9 mm from the upper crown surface and the spacing between consecutive patterns was 0.6 mm.
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M.J. Anjos et al. / Nucl. Instr. and Meth. in Phys. Res. B 213 (2004) 569–573
Fig. 3. Elemental distribution for Sr in (a) sample #1, (b) sample #2, (c) sample #3 and (d) sample #4.
The spatial distribution of Ca (Fig. 1) showed a significant decrease on moving towards the enamel–dentin junction. There are no significant Ca levels changes at the dentin region. This behavior was similar for all analyzed samples, independently of the tooth types and of the donors characteristics. It can be observed that the Zn content increases from the upper enamel surface reaching the highest content in pulp region for all analyzed samples (Fig. 2). In Fig. 2(d), it can be noted a high Zn content in the enamel region (left side) at 2.4 mm from the upper crown surface and it is due to the presence of an untreated carious lesion. In Fig. 3, it can be noted a slightly higher Sr level at the external enamel region in all samples. There is no significant variation on the Sr level in whole dentin region. The similar behavior observed in the distribution of Ca and Sr is expected taking into account the chemical affinity of theses elements.
adult individuals were evaluated. Elemental fluorescence intensities as a function of position (x, y) within teeth showed the variations of Ca, Zn and Sr levels with respect of enamel, dentin and pulp regions in tooth. The scanning experimental results showed that the distribution map of Ca, Zn and Sr in all analyzed samples were very similar. The highest levels of Zn accumulated in the pulp may be explained by the Zn absorption through the nutrients transported by the blood. The most interesting finding is an increase of the Zn level in regions adjacent to untreated caries suggesting that variations in the Zn distributions were restricted to the surrounding of carious region. These results demonstrate that lSRXRF technique can be used successfully to know the spatial distributions and the local elemental levels in human teeth.
Acknowledgements 4. Conclusions The continuos elemental distribution pattern of Ca, Zn and Sr in canine, premolar and molar, maxillary and mandibular permanent teeth of
The authors would like to thank Dr. Mauro Say~ao de Miranda for his help in preparing samples. Research partially supported by LNLS – National Synchrotron Light Laboratory.
M.J. Anjos et al. / Nucl. Instr. and Meth. in Phys. Res. B 213 (2004) 569–573
References [1] F.H. Jones, Surf. Sci. Rep. 42 (2001) 75. [2] T. Pinheiro, M.L. Carvalho, C. Casaca, M.A. Barreiros, A.S. Cunha, P. Chevallier, Nucl. Instr. and Meth. B 158 (1999) 393. [3] J.L. Cuy, A.B. Mann, K.J. Livi, M.F. Teaford, T.P. Weihs, Arch. Oral Biol. 47 (2002) 281. [4] M.L. Carvalho, C. Casaca, J.P. Marques, T. Pinheiro, A.S. Cunha, X-ray Spectr. 30 (2001) 190.
573
[5] H.J. Sanches, C.A. Perez, M. Gren on, Nucl. Instr. and Meth. A 170 (2000) 211. [6] V. Zaichick, N. Ovchjarenko, S. Zaichick, Appl. Radiat. Isot. 50 (1999) 283. [7] C.A. Perez et al., X-ray Spectr. 28 (1999) 320. [8] G. Bernasconi, A. Tajani, Quantitative X-ray Analysis System (QXAS) Software, Package: Documentation Version 1.2, International Atomic Energy Agency, Vienna, 1996.
Elemental mapping of teeth using lSRXRF M.J. Anjos a,b,*, R.C. Barroso b, C.A. Perez c, D. Braz a, S. Moreira d, K.R.H.C. Dias e, R.T. Lopes a a
Nuclear Instrumentation Laboratory/COPPE/UFRJ, P.O. Box 68509, 21945-970 Rio de Janeiro, Brazil b Universidade do Estado do Rio de Janeiro, Physics Institute, 20550-900, Rio de Janeiro, RJ, Brazil c Laborat orio Nacional de Luz Sıncrotron-LNLS/CNPq/MCT, 13084-971, P.O. Box 6192, Campinas, SP, Brazil d Universidade de Campinas, Faculdade de Engenharia Civil, 13084-971, P.O. Box 6021, Campinas, SP, Brazil e Universidade do Estado do Rio de Janeiro, Faculdade de Odontologia, 20550-900, Rio de Janeiro, RJ, Brazil
Abstract Human teeth were analysed by X-ray microfluorescence analysis using synchrotron radiation (lSRXRF). The aim of this work was to study the elemental distribution for Ca, Zn and Sr along the dental regions, enamel, dentine and pulp from patterns of relative fluorescence intensities. The measurements were performed in standard geometry of 45 incidence, exciting with a white beam and using a conventional system collimation (orthogonal slits) in the XRF beamline at the Synchrotron Light National Laboratory (Campinas, Brazil). The results show that Ca distribution is quite constant and it is independent of the tooth type and individuals characteristics. An increase of the Zn concentration was found for the pulp region and for untreated carious areas. Ca and Sr distributions show a similar behavior. 2003 Elsevier B.V. All rights reserved. PACS: 87.64.N; 41.60.Ap; 28.41.Rc Keywords: X-ray fluorescence; Elemental distribution; Human teeth; Synchrotron radiation
1. Introduction Human tooth is a complex system of specialized tissues: enamel, dentin, cementum and pulp. Each tooth is basically made up of two parts: the crown and the root. Enamel serves to protect the underlying tooth, the next layer of which is dentine. The bulk of the pulp is similar in composition to connective tissue, containing various types of cells,
* Corresponding author. Address: Universidade de Estado do Rio de Janeiro Instituto de Fisica, Rio de Janeiro, Brazil. Fax: +55-21-25628444/2906626. E-mail address: [email protected] (M.J. Anjos).
collagen fibers, nerve trunks, lymphatic and blood vessels. The tooth is anchored to the bone at its root by means periodontal ligament. The outer face of the root dentine is covered with a thin layer of cementum [1]. Tooth tissues are similar to those materials making up bone. Since bone accumulates a variety of trace elements, it is very interesting to study the elemental distribution in human teeth to evaluate biological processes. The knowledge of the spatial distribution of trace elements in tissues is involved in many biological functions of living organisms. In this way, elemental distribution in teeth can provide information about physiology of elements, environmental influence, dietary habits,
0168-583X/$ - see front matter 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0168-583X(03)01673-2
570
M.J. Anjos et al. / Nucl. Instr. and Meth. in Phys. Res. B 213 (2004) 569–573
or contamination by metallic amalgams used as restorative material [2]. Also, chemical composition and local variations in elemental contents were correlated with mechanical characteristics of enamel [3]. Micro X-ray fluorescence using synchrotron radiation (lSRXRF) is a multielementar analytical technique. The intrinsic characteristics of synchrotron radiation permit to implement spectrochemical analysis with spatial resolution on the micrometer scale, high efficiency for trace elements determination and short time of analysis requirement. lSRXRF technique is an useful tool for the study of elemental distribution in biological samples such as human teeth [4–6]. In this paper, the spatial sensitivity of lSRXRF technique was used to study local variations in the elemental distribution for Ca, Zn and Sr over the axial crosssection of four human teeth.
2. Materials and methods 2.1. Instrumentation The Synchrotron Light National Laboratory (Laborat orio Nacional de Luz Sıncrotron, LNLS) is a national research center located in Campinas, Brazil. The electron energy inside the storage ring is 1.37 GeV with a dipole magnetic field of 1.65 T, which produces a critical photon energy of 2.08 keV. The synchrotron radiation source for the XRF beamline is the D09B (15) bending magnet of the storage ring [7]. The sample was positioned in the image plane within an accuracy of 0.5 lm with a 3 axis (x, y, z) remote-controlled stage. A video microscope (magnification 500·) was used to position the sample precisely in the beam. The measurements were performed in standard geometry (45 + 45), exciting with a white beam and using orthogonal slits (300 lm · 300 lm). In this way, pixels of 300 lm · 300 lm were obtained keeping a high flux of photons on the sample. The fluorescence spectrum was recorded with a Si(Li) detector of 165 eV FWHM at 5.9 keV in air atmosphere positioned at 90 of the incidence direction. To determine the chemical elements in teeth, Ka -line of Ca
(3.691 keV), Zn (8.631 keV) and Sr (14.142 keV) were used. All the spectra were analyzed using the Quantitative X-ray Analysis Software (QXAS) package, which is a conventional program for spectrum analysis [8]. 2.2. Sample preparation Human teeth samples were obtained from adults donors aged 23–70 years whose extractions were a result of periodontal diseases. The samples consisted of one canine, two first premolars and one first molar collected from three subjects, one of them was a male. Sample #4 has a small carious region and the other ones are healthy. Once teeth were extracted, they were immediately rinsed in tap water, disinfected and placed in an individual container. The teeth were cut with a diamond saw so that the teeth were divided into two halves, with the cut lying perpendicular to the bucolingual division line. The cut surfaces of each half were polished in order to obtain a smooth and plane surface, of 1 mm thickness. After this procedure the slices were rinsed in distilled water, dried in a clean environment at room temperature and stored in an individual container. Ultrasonic cleaning was avoided because it tended to crack the samples. In this analysis only one slice of each tooth was analyzed. Selected areas of the crown of each sample were analyzed by bidimensional (x, y) scanning. The scan pattern adopted was the same for all samples: from left to right for x-axis and from upper crown surface (enamel) to root for yaxis. The counting live-time for each pixel (300 lm · 300 lm) was 7s/step and the step size was 300 lm/step in both directions.
3. Results and discussion The elemental variations as function of position (x, y) within the crown of the human teeth are shown in Figs. 1–3. The local distribution of Ca, Zn and Sr in all analyzed samples named #1, #2, #3 and #4 are presented in Figs. 1–3, respectively. The relative fluorescence intensities were normalized using the maximum value found for each
M.J. Anjos et al. / Nucl. Instr. and Meth. in Phys. Res. B 213 (2004) 569–573
571
Fig. 1. Elemental distribution for Ca in (a) sample #1, (b) sample #2, (c) sample #3 and (d) sample #4.
Fig. 2. Elemental distribution for Zn in (a) sample #1, (b) sample #2, (c) sample #3 and (d) sample #4.
element. For samples #1, #2 and #3, the spacing between consecutive patterns was 1.5 mm beginning at 1.5 mm from the upper crown surface. For
sample #4, the first pattern was obtained at 0.9 mm from the upper crown surface and the spacing between consecutive patterns was 0.6 mm.
572
M.J. Anjos et al. / Nucl. Instr. and Meth. in Phys. Res. B 213 (2004) 569–573
Fig. 3. Elemental distribution for Sr in (a) sample #1, (b) sample #2, (c) sample #3 and (d) sample #4.
The spatial distribution of Ca (Fig. 1) showed a significant decrease on moving towards the enamel–dentin junction. There are no significant Ca levels changes at the dentin region. This behavior was similar for all analyzed samples, independently of the tooth types and of the donors characteristics. It can be observed that the Zn content increases from the upper enamel surface reaching the highest content in pulp region for all analyzed samples (Fig. 2). In Fig. 2(d), it can be noted a high Zn content in the enamel region (left side) at 2.4 mm from the upper crown surface and it is due to the presence of an untreated carious lesion. In Fig. 3, it can be noted a slightly higher Sr level at the external enamel region in all samples. There is no significant variation on the Sr level in whole dentin region. The similar behavior observed in the distribution of Ca and Sr is expected taking into account the chemical affinity of theses elements.
adult individuals were evaluated. Elemental fluorescence intensities as a function of position (x, y) within teeth showed the variations of Ca, Zn and Sr levels with respect of enamel, dentin and pulp regions in tooth. The scanning experimental results showed that the distribution map of Ca, Zn and Sr in all analyzed samples were very similar. The highest levels of Zn accumulated in the pulp may be explained by the Zn absorption through the nutrients transported by the blood. The most interesting finding is an increase of the Zn level in regions adjacent to untreated caries suggesting that variations in the Zn distributions were restricted to the surrounding of carious region. These results demonstrate that lSRXRF technique can be used successfully to know the spatial distributions and the local elemental levels in human teeth.
Acknowledgements 4. Conclusions The continuos elemental distribution pattern of Ca, Zn and Sr in canine, premolar and molar, maxillary and mandibular permanent teeth of
The authors would like to thank Dr. Mauro Say~ao de Miranda for his help in preparing samples. Research partially supported by LNLS – National Synchrotron Light Laboratory.
M.J. Anjos et al. / Nucl. Instr. and Meth. in Phys. Res. B 213 (2004) 569–573
References [1] F.H. Jones, Surf. Sci. Rep. 42 (2001) 75. [2] T. Pinheiro, M.L. Carvalho, C. Casaca, M.A. Barreiros, A.S. Cunha, P. Chevallier, Nucl. Instr. and Meth. B 158 (1999) 393. [3] J.L. Cuy, A.B. Mann, K.J. Livi, M.F. Teaford, T.P. Weihs, Arch. Oral Biol. 47 (2002) 281. [4] M.L. Carvalho, C. Casaca, J.P. Marques, T. Pinheiro, A.S. Cunha, X-ray Spectr. 30 (2001) 190.
573
[5] H.J. Sanches, C.A. Perez, M. Gren on, Nucl. Instr. and Meth. A 170 (2000) 211. [6] V. Zaichick, N. Ovchjarenko, S. Zaichick, Appl. Radiat. Isot. 50 (1999) 283. [7] C.A. Perez et al., X-ray Spectr. 28 (1999) 320. [8] G. Bernasconi, A. Tajani, Quantitative X-ray Analysis System (QXAS) Software, Package: Documentation Version 1.2, International Atomic Energy Agency, Vienna, 1996.