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Quantitative PIXE and PIGME analysis of milligram samples of biomineralized tissue in the limpet Patella vulgata
Nuclear Instruments and Methods North-Holland, Amsterdam
in Physics
Research
B22 (1987)
227-230
227
QUANTITATIVE PIXE AND PIGME ANALYSIS OF MILLIGRAM SAMPLES OF BIOMINERALIZED TISSUE IN THE LIMPET PATELLA VULGATA
K.-S. KIM, J. WEBB and D.J. MACEY School of Mathematical and Physical Sciences and School of Environmental Australia 6150. Australia
and Life Sciences. Murdoch
University. Perth, Western
D.D. COHEN Australian Instituie of Nuclear Science and Engineering,
Private Mail Bag, Sutherland,
New South Wales 2232, Australia
Procedures have been developed to determine, by thick target PIXE and PIGME, the quantitative elemental composition of biological samples with a mass of approximately 1 mg. Systems of particular interest are the biomineralized tissues of chitons and limpets, marine invertebrates of global distribution whose radula teeth and associated tissue contain, variously, inorganic components at different stages of mineralization, e.g. Fe, Ca, P, F, Si. Cu. For the biomineralized teeth and tissue in the limpet Patella vulgata the content of Fe, Ca and P increases rapidly at an early stage of mineralization, while the Si content increases somewhat later. In fully mineralized teeth, the Fe and Si contents are comparable. These data are compared with previous results (Trends Biochem. Sci. 10 (1985) 6) obtained using the Oxford scanning proton microprobe.
1. Introduction Buccal Biological
mineralization
widespread synthesis amorphous
biological and
process
deposition
material
is
[l-3].
now involving
of a variety
recognized the
as
controlled
of crystalline
Radula
and
_
sac
Radula
In the case of iron, biomin-
era1 deposits of goethite (a-FeOOH) and magnetite (Fe,O,) occur in chitons and limpets, marine molluscs found throughout the world. These organisms contain iron biominerals as integral microstructural components of their teeth which form an extensive array along the tongue-like radula. A cross section of the limpet Pntefla vulgata is shown in fig. 1, indicating the anatomical disposition of the radula. The radula teeth are complex structures, containing in general more than one type of biomineral together with an organic matrix. As such, they are biological instances of what are generally known as composite materials. The radula contains teeth at various stages of mineralization and it thus provides a sequence of structures whose composition changes as mineralization proceeds. Consequently, the teeth have received considerable attention in studies of the process of iron biomineralization (4-81 using a variety of instrumental techniques, including electron microscopy, electron and X-ray diffraction, Miissbauer and infrared spectroscopy and the scanning proton microprobe (SPM). Using the Oxford SPM, the elemental composition of several teeth from along the radula of Patella vu/g&a has been reported [7], showing that the com0168-583X/87/$03.50 0 Elsevier Science Publishers (North-Holland Physics Publishing Division)
cavity
a
B.V.
_
Mouth__ Fig.
1. Cross
__
____/ section
(schematic) vulgata.
of
the
limpet
Patella
plex distribution of elements varies as mineralization of iron (as a-FeOOH) and silicon (as SiO,) progresses. The SPM data have now been complemented by quantitative analysis using thick target PIXE and PIGME at Lucas Heights. Evaluation of the role of the various inorganic components of the mineralizing tissue requires that analyses be carried out on segments of the radula that are as small as possible. Here we describe the procedure for analyzing samples consisting of segments of radula tissue of mass as small as 1 mg, and illustrate this with data obtained for the limpet radula of Putella vulgatu. Extensive studies of the radula of the chiton CIuvarizona hirtosa in which magnetite and fluoroapatite occur will be reported elsewhere [9]. II. BIOLOGICAL/MEDICAL
APPLICATIONS
2. tixperimental
Na Al Si P s Cl K Ca V Fe CU Zn
2.1. Samples Specimens of Patella vulgata were collected from the intertidal region at Gower Peninsula, UK, and the radulas with a length of approximately 70 mm removed by dissection. Radulas were cleaned of overlying cellular material by brief washing in an NaOCl solution (5%}. They were then dissected into four sections to provide teeth at different stages of mineralization: I - rows l-20 when a faint yellow colouration was observed in the base of the teeth; II rows 20-32; III-rows 32-62; IV-rows 62 to end. These sections correspond to those used previously for electron microscopy and Mossbauer spectroscopy [5,6]. 2.2.
Sample preparation for PIXE analysis
Several procedures for target preparation were developed and compared since smoothness of the target surface and sample homogeneity are important factors, For the procedure finally adopted, samples of 1 mg were weighed, finely crushed and thoroughly mixed with spectroscopic grade graphite using a boron carbide mortar and pestle. Vanadium had been added to the graphite at 1000 pgig to act as an internal standard. Vanadium was chosen since it does not occur naturally in limpet teeth and radula. Mixing with graphite allows for the handling of small samples of 1 mg and also prevents the formation of an electrostatic charge during target irradiation. The possibilities of loss of trace elements during mixing of the sample with the graphite and of contamination during the mixing process were checked by the preparation of an undiluted sample of teeth that was subsampled and analyzed. The final target composition was one part by weight of crushed sample to two parts by weight of vanadium-spiked graphite in a final mass of 3 mg. After mixing to homogeneity, the sample was pressed into a 3 mm diameter disc, with aluminium foils as support on both sides, using an evacuable die press (Specac, UK). The 3 mm pellet was then mounted using carbon paste onto a 13 mm diameter cellulose disc which had been prepared by compressing cellulose powder (Bio-Rad) into the aluminium cap used in the standard target configuration. The upper aluminium foil coating was removed prior to analysis. 2.3.
PIXE and PIGME analysis
PIXE spectra were recorded on the Lucas Heights facility using a 2.5 MeV proton beam with currents between 1 and 2 nA and a beam size of 1.2 mm in vacuum. An accumulated charge of 0.6 PC was collec-
ELEMENT
0 154 1024 2450 5092 7286 9241 9016 5438 2951 1401 1089
2
Fig. 2. The Kn yield for a graphite matrix for 23 MeV protons as a function of trace element atomic number. ted, requiring some 5-10 min irradiation per sample. Emitted X-rays were detected with a 4mm Si(Li) detector having a 8 pm Be window, set at an angle of 135” to the incident beam and 62 mm from the target. A 125 pm thick Be foil was used to attenuate low energy X-rays and hence only Si and heavier elements could be analyzed. In the case of light elements (F, Na, Mg. Al), analyses were carried out using PIGME spectra. The prompt y-rays were detected with a large Ge(Li) detector and the absolute efficiency measured against a set of standard sources. Beam currents of 30 nA were used with run times of 3-5 min. sufficient for an accumulate charge of 3 PC. The instrumentation has been described in detail elsewhere [lO,ll]. The Ka thick target yields for various elements in a graphite matrix are shown in fig. 2. These yields were obtained for our setup using our thick target yield programs [12]. Normalization to the spiked vanadium peak enabled us to obtain absolute concentrations for trace elements present in our tooth matrix. Data analysis and curve fitting followed procedures established previously for analysis of thick target specimens 112,131.
3. Results and discussion The use of thick targets in PIXE and PIGME analyses for studies of biomineralization offers the distinct advantage of providing quantitative analyses for many elements in a nondestructive manner. Spectra can be obtained routinely for small samples using the described procedures. Typical PIXE spectra of two segments of the radula Patella vulgata are shown in fig. 3. At stage II of mineralization (fig. 3a), the radula contains readily detectable amounts of Si, P, Ca and Fe, four elements whose distribution within teeth was determined previously using the Oxford SPM. The Oxford data, however, were not corrected for internal absorption. As mineralization progresses to stage IV, the elemental composition shows significant changes
K.-S.
h’im et at.
I Analysis of ibsue of Putellu vulgata
DEVELOPMENTAL
229
IV Ill STAGE OF RADULA
Fig. 4. Concentrations of selected components of the radula of the limpet Put& vulgatu at four stages of mineralization.
1.0
3.0
X-RAY
5.0
7.0
9.0
ENERGY (keV)
Fig. 3. PJXE spectra for segments of the radula of the limpet Pate/la vulgara. (a) stage II; (b) stage IV mineralization.
(fig. 3h). In particular, the Si peak has increased in intensity. Further analysis of the spectra allows the changes in elemental composition to be followed quantitatively. These are shown in fig. 4 for the four stages of mineralization. The Fe content of the radula increases considerably early on in mineralization (stages I and II) associated with the deposition of goethite and an unusual superparamagnetic component identified by Mijssbauer spectroscopy [6]. The relative amounts of these two iron components are known to change along the radula, with the superparamagn~tic components contributing 65% of the Massbauer spectral intensity in stage II, but only 12% in stage IV. However, the present PIXE data indicate that the iron content of the radula remains unchanged after stage II, suggesting that the superparamagnetic component may gradually transform into crystalline goethite. Although present at lower levels than Fe, both P and Ca contents also increase from stage I to II. However, the Si content increases later, from II to III, as observed previously with the SPM. The present data indicate further that Fe and Si are the major inorganic components of the mature, fully mineralized
teeth. In these samples. no F was detected by PIGME analysis. However, in the case of the chiton Clavarizona hirtosa, F was detected in the radula and changes in the F concentrations could be readily followed using PIGME spectra. Discrimination in spatial distribution between the elements as mineralization progresses must reflect the underlying biological processes involved. The PIXE and PIGME analyses of small samples provide a valuable analytical probe of the compositional changes associated with mineralization and can reveal significant features of the complex processes involved in biological mineralization. particularly when taken together with other instrumental techniques.
We wish to thank the operating staff of the AAEC’s 3 MeV Van de Graaff accelerator for their assistance. This work was funded by the Australian Institute of Nuclear Science and Engineering and by a Murdoch University Postgraduate Scholarship (to K.-S.K.).
References H.A. Lowenstam, Science 211 (lY81) 1126. S. Mann. Struct. Bonding 54 (1983) 125. .I. Webb. Biomineralization and Biological Metal Accumulation eds., P. Wesbroek and E.W. de Jong (Reidel, Dordrecht. 1983) p. 413. M.A. Burf0rd.D.J. MaceyandJ. Webb.Comp. Biochem. Physiol. X3A ( IYXh) 353. S. Mann, C.C. Perry, J. Webb, B. Luke and R.J.P. Williams, Proc. Roy. Sot. London 227 (19X6) 179. ‘T.G. St Pierre, S. Mann. J. Webb, D.P.E. Dickson, N.W. Runham and R.J.P. Williams. Proc. Roy. Sot. London 228 (1986) 31. II. BIOLOGICAL/MEDICAL
APPLICATIONS
(71 C.W. Grime, F. Watt, S. Mann. C.C. Perry, J. Webb and R.J.P. Wilhams. Trends Biochem. Sot. 10 (1985) 6. 181H.A. Lowenstam and S. Weiner, Science 227 (1985) 51. [9] K.-S. Kim, D.J. Macey, J. Webb and D.D. Cohen. J. Inorg. Biochem. 27 (1986) in press. [lo] D.D. Cohen and P. Duerden, AAEC/E453 (1979).
[ll]
D.D. Cohen, P. Duerden and E. Clayton, AAEC!E468 ( 197Y). [12] D.D. Cohen, E. Clayton and 7. Ainsworth. Nucl. Instr. and Meth. 18X (1981) 203. (131 E. Clayton, D.D. Cohen and P. Duerden. Nucl. Instr. and Meth. 180 (1981) 541.
in Physics
Research
B22 (1987)
227-230
227
QUANTITATIVE PIXE AND PIGME ANALYSIS OF MILLIGRAM SAMPLES OF BIOMINERALIZED TISSUE IN THE LIMPET PATELLA VULGATA
K.-S. KIM, J. WEBB and D.J. MACEY School of Mathematical and Physical Sciences and School of Environmental Australia 6150. Australia
and Life Sciences. Murdoch
University. Perth, Western
D.D. COHEN Australian Instituie of Nuclear Science and Engineering,
Private Mail Bag, Sutherland,
New South Wales 2232, Australia
Procedures have been developed to determine, by thick target PIXE and PIGME, the quantitative elemental composition of biological samples with a mass of approximately 1 mg. Systems of particular interest are the biomineralized tissues of chitons and limpets, marine invertebrates of global distribution whose radula teeth and associated tissue contain, variously, inorganic components at different stages of mineralization, e.g. Fe, Ca, P, F, Si. Cu. For the biomineralized teeth and tissue in the limpet Patella vulgata the content of Fe, Ca and P increases rapidly at an early stage of mineralization, while the Si content increases somewhat later. In fully mineralized teeth, the Fe and Si contents are comparable. These data are compared with previous results (Trends Biochem. Sci. 10 (1985) 6) obtained using the Oxford scanning proton microprobe.
1. Introduction Buccal Biological
mineralization
widespread synthesis amorphous
biological and
process
deposition
material
is
[l-3].
now involving
of a variety
recognized the
as
controlled
of crystalline
Radula
and
_
sac
Radula
In the case of iron, biomin-
era1 deposits of goethite (a-FeOOH) and magnetite (Fe,O,) occur in chitons and limpets, marine molluscs found throughout the world. These organisms contain iron biominerals as integral microstructural components of their teeth which form an extensive array along the tongue-like radula. A cross section of the limpet Pntefla vulgata is shown in fig. 1, indicating the anatomical disposition of the radula. The radula teeth are complex structures, containing in general more than one type of biomineral together with an organic matrix. As such, they are biological instances of what are generally known as composite materials. The radula contains teeth at various stages of mineralization and it thus provides a sequence of structures whose composition changes as mineralization proceeds. Consequently, the teeth have received considerable attention in studies of the process of iron biomineralization (4-81 using a variety of instrumental techniques, including electron microscopy, electron and X-ray diffraction, Miissbauer and infrared spectroscopy and the scanning proton microprobe (SPM). Using the Oxford SPM, the elemental composition of several teeth from along the radula of Patella vu/g&a has been reported [7], showing that the com0168-583X/87/$03.50 0 Elsevier Science Publishers (North-Holland Physics Publishing Division)
cavity
a
B.V.
_
Mouth__ Fig.
1. Cross
__
____/ section
(schematic) vulgata.
of
the
limpet
Patella
plex distribution of elements varies as mineralization of iron (as a-FeOOH) and silicon (as SiO,) progresses. The SPM data have now been complemented by quantitative analysis using thick target PIXE and PIGME at Lucas Heights. Evaluation of the role of the various inorganic components of the mineralizing tissue requires that analyses be carried out on segments of the radula that are as small as possible. Here we describe the procedure for analyzing samples consisting of segments of radula tissue of mass as small as 1 mg, and illustrate this with data obtained for the limpet radula of Putella vulgatu. Extensive studies of the radula of the chiton CIuvarizona hirtosa in which magnetite and fluoroapatite occur will be reported elsewhere [9]. II. BIOLOGICAL/MEDICAL
APPLICATIONS
2. tixperimental
Na Al Si P s Cl K Ca V Fe CU Zn
2.1. Samples Specimens of Patella vulgata were collected from the intertidal region at Gower Peninsula, UK, and the radulas with a length of approximately 70 mm removed by dissection. Radulas were cleaned of overlying cellular material by brief washing in an NaOCl solution (5%}. They were then dissected into four sections to provide teeth at different stages of mineralization: I - rows l-20 when a faint yellow colouration was observed in the base of the teeth; II rows 20-32; III-rows 32-62; IV-rows 62 to end. These sections correspond to those used previously for electron microscopy and Mossbauer spectroscopy [5,6]. 2.2.
Sample preparation for PIXE analysis
Several procedures for target preparation were developed and compared since smoothness of the target surface and sample homogeneity are important factors, For the procedure finally adopted, samples of 1 mg were weighed, finely crushed and thoroughly mixed with spectroscopic grade graphite using a boron carbide mortar and pestle. Vanadium had been added to the graphite at 1000 pgig to act as an internal standard. Vanadium was chosen since it does not occur naturally in limpet teeth and radula. Mixing with graphite allows for the handling of small samples of 1 mg and also prevents the formation of an electrostatic charge during target irradiation. The possibilities of loss of trace elements during mixing of the sample with the graphite and of contamination during the mixing process were checked by the preparation of an undiluted sample of teeth that was subsampled and analyzed. The final target composition was one part by weight of crushed sample to two parts by weight of vanadium-spiked graphite in a final mass of 3 mg. After mixing to homogeneity, the sample was pressed into a 3 mm diameter disc, with aluminium foils as support on both sides, using an evacuable die press (Specac, UK). The 3 mm pellet was then mounted using carbon paste onto a 13 mm diameter cellulose disc which had been prepared by compressing cellulose powder (Bio-Rad) into the aluminium cap used in the standard target configuration. The upper aluminium foil coating was removed prior to analysis. 2.3.
PIXE and PIGME analysis
PIXE spectra were recorded on the Lucas Heights facility using a 2.5 MeV proton beam with currents between 1 and 2 nA and a beam size of 1.2 mm in vacuum. An accumulated charge of 0.6 PC was collec-
ELEMENT
0 154 1024 2450 5092 7286 9241 9016 5438 2951 1401 1089
2
Fig. 2. The Kn yield for a graphite matrix for 23 MeV protons as a function of trace element atomic number. ted, requiring some 5-10 min irradiation per sample. Emitted X-rays were detected with a 4mm Si(Li) detector having a 8 pm Be window, set at an angle of 135” to the incident beam and 62 mm from the target. A 125 pm thick Be foil was used to attenuate low energy X-rays and hence only Si and heavier elements could be analyzed. In the case of light elements (F, Na, Mg. Al), analyses were carried out using PIGME spectra. The prompt y-rays were detected with a large Ge(Li) detector and the absolute efficiency measured against a set of standard sources. Beam currents of 30 nA were used with run times of 3-5 min. sufficient for an accumulate charge of 3 PC. The instrumentation has been described in detail elsewhere [lO,ll]. The Ka thick target yields for various elements in a graphite matrix are shown in fig. 2. These yields were obtained for our setup using our thick target yield programs [12]. Normalization to the spiked vanadium peak enabled us to obtain absolute concentrations for trace elements present in our tooth matrix. Data analysis and curve fitting followed procedures established previously for analysis of thick target specimens 112,131.
3. Results and discussion The use of thick targets in PIXE and PIGME analyses for studies of biomineralization offers the distinct advantage of providing quantitative analyses for many elements in a nondestructive manner. Spectra can be obtained routinely for small samples using the described procedures. Typical PIXE spectra of two segments of the radula Patella vulgata are shown in fig. 3. At stage II of mineralization (fig. 3a), the radula contains readily detectable amounts of Si, P, Ca and Fe, four elements whose distribution within teeth was determined previously using the Oxford SPM. The Oxford data, however, were not corrected for internal absorption. As mineralization progresses to stage IV, the elemental composition shows significant changes
K.-S.
h’im et at.
I Analysis of ibsue of Putellu vulgata
DEVELOPMENTAL
229
IV Ill STAGE OF RADULA
Fig. 4. Concentrations of selected components of the radula of the limpet Put& vulgatu at four stages of mineralization.
1.0
3.0
X-RAY
5.0
7.0
9.0
ENERGY (keV)
Fig. 3. PJXE spectra for segments of the radula of the limpet Pate/la vulgara. (a) stage II; (b) stage IV mineralization.
(fig. 3h). In particular, the Si peak has increased in intensity. Further analysis of the spectra allows the changes in elemental composition to be followed quantitatively. These are shown in fig. 4 for the four stages of mineralization. The Fe content of the radula increases considerably early on in mineralization (stages I and II) associated with the deposition of goethite and an unusual superparamagnetic component identified by Mijssbauer spectroscopy [6]. The relative amounts of these two iron components are known to change along the radula, with the superparamagn~tic components contributing 65% of the Massbauer spectral intensity in stage II, but only 12% in stage IV. However, the present PIXE data indicate that the iron content of the radula remains unchanged after stage II, suggesting that the superparamagnetic component may gradually transform into crystalline goethite. Although present at lower levels than Fe, both P and Ca contents also increase from stage I to II. However, the Si content increases later, from II to III, as observed previously with the SPM. The present data indicate further that Fe and Si are the major inorganic components of the mature, fully mineralized
teeth. In these samples. no F was detected by PIGME analysis. However, in the case of the chiton Clavarizona hirtosa, F was detected in the radula and changes in the F concentrations could be readily followed using PIGME spectra. Discrimination in spatial distribution between the elements as mineralization progresses must reflect the underlying biological processes involved. The PIXE and PIGME analyses of small samples provide a valuable analytical probe of the compositional changes associated with mineralization and can reveal significant features of the complex processes involved in biological mineralization. particularly when taken together with other instrumental techniques.
We wish to thank the operating staff of the AAEC’s 3 MeV Van de Graaff accelerator for their assistance. This work was funded by the Australian Institute of Nuclear Science and Engineering and by a Murdoch University Postgraduate Scholarship (to K.-S.K.).
References H.A. Lowenstam, Science 211 (lY81) 1126. S. Mann. Struct. Bonding 54 (1983) 125. .I. Webb. Biomineralization and Biological Metal Accumulation eds., P. Wesbroek and E.W. de Jong (Reidel, Dordrecht. 1983) p. 413. M.A. Burf0rd.D.J. MaceyandJ. Webb.Comp. Biochem. Physiol. X3A ( IYXh) 353. S. Mann, C.C. Perry, J. Webb, B. Luke and R.J.P. Williams, Proc. Roy. Sot. London 227 (19X6) 179. ‘T.G. St Pierre, S. Mann. J. Webb, D.P.E. Dickson, N.W. Runham and R.J.P. Williams. Proc. Roy. Sot. London 228 (1986) 31. II. BIOLOGICAL/MEDICAL
APPLICATIONS
(71 C.W. Grime, F. Watt, S. Mann. C.C. Perry, J. Webb and R.J.P. Wilhams. Trends Biochem. Sot. 10 (1985) 6. 181H.A. Lowenstam and S. Weiner, Science 227 (1985) 51. [9] K.-S. Kim, D.J. Macey, J. Webb and D.D. Cohen. J. Inorg. Biochem. 27 (1986) in press. [lo] D.D. Cohen and P. Duerden, AAEC/E453 (1979).
[ll]
D.D. Cohen, P. Duerden and E. Clayton, AAEC!E468 ( 197Y). [12] D.D. Cohen, E. Clayton and 7. Ainsworth. Nucl. Instr. and Meth. 18X (1981) 203. (131 E. Clayton, D.D. Cohen and P. Duerden. Nucl. Instr. and Meth. 180 (1981) 541.