Microscopy Microanalysis
Microsc. Microanal. 20, 257–267, 2014 doi:10.1017/S1431927613013998
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© MICROSCOPY SOCIETY OF AMERICA 2014
Evaluation of X-Ray Microanalysis for Characterization of Dental Enamel Lisa Melin,1 Jörgen G. Norén,1, * Fabian Taube,2 and David H. Cornell 3 1
Department of Pediatric Dentistry, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, SE-405 30, Gothenburg, Sweden 2 Department of Occupational and Environmental Medicine, Sahlgrenska University Hospital, SE 405 30, Gothenburg, Sweden 3 Department of Earth Sciences, University of Gothenburg, SE 405 30, Gothenburg, Sweden
Abstract: Elemental analysis of dental hard tissues is of importance. The aim of this study is to evaluate X-ray microanalysis ~XRMA! of bovine enamel in a scanning electron microscope ~SEM! with different coatings. The buccal surface of bovine incisors was polished flat, one-third was coated with carbon, one-third with gold, leaving one-third uncoated for XRMA in an SEM equipped with an energy-dispersive microanalysis system. The elements oxygen, sodium, magnesium, phosphorous, chlorine, potassium, and calcium were analyzed using their respective characteristic K X-ray series. Comparisons were made with analyses of glass produced by fusion of the bovine enamel, showing that oxygen analyses using the K X-ray series are reliable and preferable to calculating oxygen by stoichiometry for natural enamel. For the gold-coated and uncoated analyses, carbon was also measured using the K X-ray series. Small area Analyses in small areas ~80 ⫻ 80 mm! in variable pressure-SEM mode with low vacuum ~20 Pa!, without any coating, midway between 40 mm wide gold lines 140 mm apart to avoid build-up of electrostatic charge is the preferred method, especially if carbon is included in the analysis. The analyses of bovine enamel are sufficiently reproducible to be regarded as quantitative for all elements except carbon. Key words: bovine, enamel, scanning electron microscope, elemental analysis, glass, XRMA
I NTR ODUCTION Elemental analysis of dental hard tissues ~enamel, dentin, cementum! has long been of importance. However, due to their brittle and insulating properties, there are few suitable methods. Energy-dispersive spectroscopy ~EDS! or wavelength-dispersive X-ray microanalysis ~XRMA! of spectra excited by electron beams in electron microscopes have often been used ~Mahoney et al., 2004; Weerasinghe et al., 2007; Youravong et al., 2008; Sabel et al., 2009a, 2009b; De Carvalho Filho et al., 2010; De Menezes Oliveira et al., 2010; Fagrell et al., 2010; Souza et al., 2010; Taube et al., 2010; Braga et al., 2011; He et al., 2011!. Analysis of the chemical composition of enamel in teeth with developmental disturbances has been of special interest, especially in relation to the morphology. An important advantage of XRMA in a scanning electron microscope is that measurements can be made with full image control of the morphological location in the analyzed tissue. XRMA analysis is particularly useful for the analysis of Ca, P, and O in dental hard tissues and C is also an element of interest. Several studies have revealed large variations in elemental composition between different samples of normal enamel ~see, e.g., Sabel et al., 2009a!. Dental enamel is said to comprise 96 wt% inorganic mineral and the main elements are Ca, P, and O, which build calcium hydroxyapatite crystals @Ca10 ~PO4 !6 ~OH!2 #. This may vary somewhat in Received July 12, 2013; accepted December 3, 2013 *Corresponding author. E-mail: [email protected]
composition, especially at the surface, depending on reactions with the oral environment ~Nelson et al., 2002!. Dental enamel is far from the idealized form of hydroxyapatite, but biological apatite contains carbonate hydroxyapatite as well as HPO4 , Mg, and Cl ~LeGeros, 1981, 1984, 1991; LeGeros et al., 1995; Takagi et al., 1998!. Other elements of importance are F, Na, and K, which play important roles during the mineralization of enamel. Only 3 wt% of the enamel is organic material in the form of proteins and lipids, with C and N as the main components, whereas another 1 wt% is water, according to Eisenmann ~1994!. In previous analyses of enamel, C has been shown to have a high concentration due to the carbonate content of mature dental enamel ~Sydney-Zax et al., 1991!. The carbonate content of enamel is of specific interest in relation to the caries process, since high levels of carbonate increase susceptibility of enamel to caries ~Hallsworth et al., 1973; Weatherell et al., 1974!. Analysis of carbon in dental enamel is desirable in relation not only to caries but also to reflect differences in the organic content of enamel. Bovine enamel has long been used for in vitro studies because its chemical and morphological properties resemble those of human dental enamel ~e.g., Soares et al., 2012!. Bovine teeth are therefore an excellent proxy for human teeth and also far more easy to handle due to their size. The surface layer of bovine and human enamel after eruption is subjected to the influence of the oral environment and takes up different trace elements that are accumulated in the surface area of the enamel. By removing the surface layer
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down to the bulk of the enamel, the possible variations of morphology and chemical composition can be reduced. A problem with electron beam microanalysis of enamel is that electrostatic charge tends to build up on the nonconducting surface and this has normally been solved by applying thin coatings of conductive material. The aim of this study is to evaluate XRMA analysis of bovine enamel with different coatings using carbon, gold, and without any coating, using a low-vacuum technique. Bovine material provides large homogeneous areas on which to make these tests. The results of this work will be applied to problems in human teeth.
M ATERIALS
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Bovine Tooth Samples Mandibular jaws from ten calves were collected from a slaughterhouse at Skara in western Sweden. The central incisors were cut off from the jaws and after cleaning in tap water stored in 70% ethanol till further preparation. Storage in ethanol enables a better embedding in epoxy resin and has previously been used in several studies employing XRMA ~see, e.g., Sabel et al., 2009a!. In order to eliminate chemical variations in the bovine enamel surface, and to create a flat surface, the buccal surface was ground with SiC paper in a polishing machine ~Struers, Copenhagen, Denmark!. The teeth were placed centrally, with the flat surface on the bottom of 25-mm-diameter mounting cups for epoxy resin ~Struers!. After correct orientation of the teeth the mounting cups were assembled and ID marked with a water-resistant marker pen. The puck diameter fitted the sample holder of the scanning electron microscope ~SEM!. The tooth samples were embedded in epoxy resin for electron microscopy ~Epofix威; Electron Microscopy Sciences, Fort Washington, PA, USA! mixed according to the manufacturer’s instructions. The molds were filled with the mixed epoxy till the teeth were totally embedded. After hardening overnight, the embedded teeth were removed from the molds and the epoxy pucks marked with an ID number. In order to remove any excess epoxy from the enamel surface the embedded samples were polished with abrasive paper with water cooling. This procedure was performed with three different papers ~grit 1,200, 2,400, and 4,000 ⫽ 4 mm! in the polishing machine. Macroscopic photos were taken of all samples in a Leica M80 stereo microscope ~Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany! using a Leica digital camera ~Leica DFC420 C, Leica Mikrosysteme Vertrieb GmbH! with Leica Application Suite LAS V3.7.0 ~Leica Microsystems AG, Heerbrugg, Switzerland!. An overview of the polished surface was taken in a Leica M80 stereo microscope at low magnification ~0.75⫻! in incident light using a Leica digital camera with LAS V3.7.0. In order to have a comparison for the XRMA analysis of natural enamel, powdered samples of the same bovine enamel were also prepared as fused glass samples for comparison. These were weighed after drying at 1108C, then
Figure 1. a: Schematic drawing of the polished flat surface of a calf incisor one-third coated with carbon, one-third uncoated, and one-third coated with gold. ~x ⫽ locations for point and area analyses!. The sample diameter is 25mm. b: Schematic drawing of the polished flat surface of a calf incisor coated with carbon, gold, or uncoated, then coated with 41 mm thick gold lines 140 mm apart to minimize electrostatic charging ~䉺 ⫽ locations for analyses!.
after 30 min ignition at 9808C, giving a consistent loss on ignition value ~volatiles such as water, CO2 , and organic material! of 9.7%. About 0.5 g of ignited powder was fused by electrical heating for about 5 s until seen to be completely fused, in a molybdenum metal boat in an argonfilled pressure chamber with glass observation window, at 5 atmospheres pressure. One fusion was made of the pure ignited material, which melts around 1,6008C, and another using 50% lithium metaborate flux, which lowered the fusion temperature to an estimated 1,1008C. The glass produced was broken out of the boat and mounted in an epoxy puck, then ground and polished with 3 mm diamond to produce a smooth surface for analysis in the SEM.
XRMA Analysis The buccal surface of four bovine tooth samples was divided into three areas, as shown in Figure 1. One area was carbon coated ~'25 nm thick! in a carbon thread vacuum system, the central part was left uncoated, and the third part was coated with gold in a plasma coater ~'5 nm thick!. Thereafter, the samples were placed in a sample holder for SEM. Four analyses were made in the carbon-coated areas, three in the uncoated areas, and three or four in the area coated with gold ~Fig. 1a!. The analyses were carried out using 1,500⫻ magnification as point ~effectively 4 mm diameter analysis points! and also as area analyses using 80 ⫻ 80 mm 2, with a counting time of 40 s. The “All elements” option in the INCA software was used and for carbon-coated enamel the elements oxygen ~O!, sodium ~Na!, magnesium ~Mg!, phosphorous ~P!, chlorine ~Cl!, potassium ~K! and calcium ~Ca! were analyzed using the K X-ray series. For the gold-coated and the uncoated analyses, carbon ~C! was included. Each part of the tooth was analyzed in three different locations, applying both point and area analysis. When analyzing areas coated with carbon or gold, the normal high-vacuum mode for SEM was used for second-
1.205 1.399 1.430
K2O CaO Fe2O3
0.30 0.06 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.06 Na2O MgO SO2 P2O5 SO3 1.348 1.658 2.139 2.291 2.001
~F! 0.30 0.07 0.04 0.06 0.06 0.05 ~Cl! 0.03 0.03 0.04 0.08 MAC JMAC MgO MAC WAC AMAC AnMAC TSl KfMAC WMAC FeMMAC K2O CaO Fe2O3 Total Total-N
0.00 0.55 0.62 0.47 44.59 0.58 ~0.07! 0.06 53.15 0.00 100.00 100.06 Na2O MgO SO2 P2O5 SO3
0.03 0.03 0.01 0.13 0.04 0.02 0.01 0.15 0.00 0.07 0.02 0.01 0.01 0.03 0.01 0.01 0.01 0.04 0.00 0.05
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*Analysis by electron beam EDS X-ray microanalysis. Detection limits are for 400 s live time. Loss on ignition was determined by weighing dry ~1108C! samples after ignition at 9808C. The glass analysis is given as dry element and oxide wt%, then reduced to accommodate the loss on ignition. Loss on ignition includes organic material, crystalline water, CO2 in carbonate, and some Cl and F. SD, standard deviation; SM, stoichiometry; X-RP, using X-ray peak; Total-N, total before normalization; MAC, microanalysis consultants, Cambridge UK; J-MAC, Jadeite Mac; WMAC, wollastonite MAC; AMAC, apatite MAC; AnMAC, anhydrite MAC; TSl, tugtupite Sl; KfMAC, K-feldspar MAC; FeMMAC, Fe metal MAC; LOI, loss on ignition.
Element ~wt%!
0.00 0.41 0.37 0.22 19.46 0.23 0.07 0.05 37.96 0.00 41.25 41.18 0.9046 0.7414 0.7114 0.9836 1.0050 0.8373 1.1028 1.1291 1.0012 0.8141 Stoichiometry K series X-ray F Na Mg Si P S Cl K Ca Fe O O
Oxide
Dry Oxide ~wt%!
Standard
F Na2O MgO SO2 P2O5 SO3 Cl K2O CaO Fe2O3 LOI Total
,0.30 0.49 0.56 0.42 40.27 0.52 0.06 0.05 48.00 ,0.06 9.70 100.07
Element to Oxide Detection Limit Oxide ~%! Natural Material ~wt%! Intensity Correction
In order to evaluate the effect of surface charge on the analysis, a series of measurements were made using a lowvacuum of 20 Pa on uncoated enamel ~Fig. 1b!. The air remaining in the sample chamber is thought to form a surface layer on the sample that conducts away the surface charge, while the tungsten filament in the electron gun is maintained at a much lower pressure to avoid it burning. Two sets of analyses were performed as point analyses with a starting point in the gold-coated area and the remaining points in the uncoated enamel. A further three sets of analyses were performed, one as point analyses and two as area analyses with a square area of 12 ⫻ 12 mm. On one of the specimens a linear gold coating ~line width 41 mm, distance between the lines 140 mm! was made on the specimens with the use of a grid ~Fig. 1b!. Area analyses were performed in four areas with the first starting in the gold-coated part of the enamel.
Element
Control of the Effect of Surface Charging
SD of Three 400 s Counts
The apparent concentrations are corrected by a phi-rho-z iterative procedure described in more detail for glass samples below. For the gold-coated and uncoated areas the analyses were calculated with and without C included in the menu. The mean values for the different elements with carbon coating, gold coating, and without coating are given in Tables 2 and 3. The intensity correction values are given in Table 4.
Counting Error ~wt%!
Presentation of Results by INCA Energy Software
Table 1. Chemical Analysis of Bovine Tooth Sample Bov1, Prepared as Fused Glass, Polished, and Carbon Coated.*
ary electron images. Analyses of areas without coating were performed in low-vacuum mode ~VP-SEM! with a vacuum of 20 Pa using backscattered electron images. The XRMA analysis was performed in a Hitachi S-3400N SEM equipped with an Oxford Instrument EDS microanalysis system and INCA software ~Oxford Instruments, Abingdon, UK!. All analyses were carried out at 20 kV accelerating voltage and the working distance from sample to electron optical column was set to 9.9 mm, with a tolerance of 0.1 mm. The electron beam was aligned in “Microscope setup” using “Wave” in INCA with the beam aimed into a Faraday cage. The specimen current was adjusted to 3.50 6 0.02 nA, and was monitored every 30 min with stability better than 1%. The standard calibration was performed by acquiring a spectrum with 40 s live time on cobalt metal in the sample holder. The standards used are listed in the caption for Table 1. These simple standards are linked to the cobalt standard, which is counted at the beginning of each highor low-vacuum session. The accuracy of mineral analyses is checked periodically using mineral standards from the Smithsonian Institute, Washington. The fused glass samples were analyzed only with high vacuum and carbon coating. Oxygen was analyzed using the spectral peak and also using the oxygen by stoichiometry option. The results are given in Table 2.
Detection Limit Element ~%!
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Lisa Melin et al. Table 2. Mean Values of X-Ray Microanalysis ~XRMA! Area Analysis of Natural Bovine Enamel from Carbon Coated ~C Coat!, Gold Coated ~Au Coat!, Uncoated ~No Coat! Specimens and for Carbon-Coated Bovine Pure Glass and Bovine Fluxed Glass for the Elements O, Na, Mg, P, Cl, K, and Ca.* Natural Bovine Enamel
O Na Mg P Cl K Ca Total
C Coat
Au Coat
No Coat
Pure Glass C Coat
39.99 0.67 0.29 23.48 0.35 0.05 36.53 101.36
40.14 0.70 0.30 22.75 0.26 0.09 35.25 99.49
41.30 0.75 0.33 22.99 0.27 0.05 34.88 100.57
37.21 0.37 0.33 17.57 0.06 0.05 34.30 90.30
Fluxed Glass C Coat 39.96 0.70 0.35 17.00 0.24 0.09 34.46 90.30
Human Neutron Activation
Human EDS XRMA
Primary
Permanent
16.18
21.19
17.23
29.82
52.50
35.11
39.70 0.70 0.28 0.32 37.03
*The values for glass are reduced by 9.70% to compensate for loss of ignition. For comparison some data are shown for human enamel by neutron activation from Retief et al. ~1971!, by XRMA from Fagrell et al. ~2010! and from De Menezes Oliveira et al. ~2010!.
Table 3. Mean Values and Standard Deviations ~SD! of Point and Area Analyses in wt% of C, O, Na, Mg, P, Cl, K, Ca, and Totals for Analyses of Bovine Enamel with Carbon Coating, Gold Coating, and Without Coating.* Carbon Coated Point Element C O - C incl. O - C excl. Na - C incl. Na - C excl. Mg - C incl. Mg - C excl. P - C incl. P - C excl. Cl - C incl. Cl - C excl. K - C incl. K - C excl. Ca - C incl. Ca - C excl. Totals - C incl. Totals - C excl.
Mean
Gold Coated
Area SD
Mean
Point SD
39.59
2.22
39.99
2.32
0.60
0.06
0.67
0.09
0.29
0.05
0.29
0.05
23.61
0.33
23.48
0.38
0.35
0.11
0.35
0.11
0.06
0.02
0.06
0.02
36.59
0.70
36.53
0.84
101.03
2.47
101.31
2.97
No Coating Area
Point
Area
Mean
SD
Mean
SD
Mean
SD
Mean
SD
7.26 40.89 40.67 0.63 0.68 0.30 0.32 22.67 22.86 0.25 0.26 0.06 0.06 35.08 35.32 107.08 100.11
1.20 3.15 2.98 0.09 0.16 0.04 0.07 0.46 0.71 0.04 0.04 0.02 0.02 1.21 1.58 4.37 4.78
7.22 40.49 40.14 0.69 0.70 0.29 0.30 22.72 22.75 0.26 0.26 0.06 0.06 35.31 35.25 106.98 99.40
2.61 3.15 3.13 0.06 0.06 0.03 0.03 0.49 0.48 0.05 0.05 0.01 0.01 1.24 1.25 4.05 4.53
5.70 40.72 40.51 0.70 0.71 0.30 0.30 23.00 23.01 0.27 0.27 0.08 0.08 35.21 35.14 105.90 99.95
0.91 1.07 1.06 0.05 0.06 0.03 0.03 0.59 0.58 0.05 0.05 0.03 0.03 0.92 0.92 2.87 2.45
6.00 41.49 41.30 0.75 0.75 0.33 0.33 22.94 22.99 0.27 0.27 0.09 0.09 34.93 34.88 106.70 100.52
1.91 1.25 1.20 0.06 0.06 0.04 0.04 0.71 0.69 0.05 0.05 0.03 0.03 1.34 1.30 3.26 3.00
*-C incl., values with C included in the analysis; -C excl., values when C is excluded from the analysis.
Statistical Analysis The results were put into a spreadsheet program ~Excel; Microsoft, Seattle, WA, USA! and descriptive statistics were calculated using Statgraphics Plus for Windows ~Statpoint Technologies, Inc., Warrenton, USA!.
R ESULTS A total of 105 measurements were made in the enamel of the four bovine teeth. In the gold-coated enamel 10 point and 10 area measurements were made; in the carbon coated 9 and 9 and in the uncoated enamel 12 and 12 measurements, respectively. Mean values and standard deviations
were calculated for the measurements for each group and the data are given in Table 3.
Surface Roughness of the Polished Specimens One of the epoxy-embedded bovine samples was cut into two halves after XRMA analysis. The sample was positioned in the sample holder for the SEM instrument with the cut surface facing upwards. By tilting the stage holder slightly both the polished and the cut enamel surface could be visualized. The polished surface appeared smooth within about 1 mm and some scratches about 2 mm deep were observed. The surface is thus regarded as satisfactory regarding flatness for XRMA analysis, although the lightest ele-
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Table 4. Mean Values of the Intensity Correction Used in INCA for Point and Area Analysis of C, O, Na, Mg, P, Cl, K, and Ca for Measurements in Bovine Enamel with Carbon Coating, Gold Coating, and Without Coating.* Carbon Coated Element C O - C incl. O - C excl. Na - C incl. Na - C excl. Mg - C incl. Mg - C excl. P - C incl. P - C excl. Cl - C incl. Cl - C excl. K - C incl. K - C excl. Ca - C incl. Ca - C excl.
Point
Area
0.854
0.866
0.764
0.764
0.727
0.726
1.006
1.005
0.761
0.761
1.233
1.230
0.993
0.993
Gold Coated
No Coating
Point
Area
Point
Area
0.256 0.779 0.793 0.727 0.714 0.701 0.691 0.986 0.985 0.737 0.731 1.228 1.235 0.967 0.969
0.256 0.775 0.790 0.728 0.715 0.701 0.691 0.986 0.985 0.737 0.731 1.226 1.234 0.967 0.969
0.333 0.950 0.965 0.783 0.772 0.737 0.729 1.006 1.004 0.765 0.761 1.229 1.238 0.992 0.993
0.335 0.959 0.976 0.782 0.771 0.736 0.727 1.004 1.003 0.765 0.760 1.226 1.234 0.991 0.993
*The apparent concentration is divided by these intensity corrections to produce the wt% values. Thus the closer to unity, the smaller the correction. -C incl., C included in the analysis; -C excl., C is excluded from the analysis.
ments such as C and O might be affected by surface irregularities.
Control of the Effect of Surface Charging (Figs. 2a–2f) In the profiles away from the gold coating, a slight decrease in wt% values from the gold-coated area into the uncoated enamel is seen for all elements and for the totals ~Figs. 2a– 2d!. However, Na surprisingly increased to reach a maximum of 1.5 wt% around 50 mm from the gold coating and then decreased to a slightly variable level around 1.3% from about 60 mm into the uncoated enamel ~Fig. 2a!. Cl also showed this feature to a lesser extent. Similar features were seen in the area analysis, with peaks about 90 mm from the gold ~Fig. 2c!. In both experiments the totals dropped at about 120 mm from the gold, probably reflecting electrostatic charging effects. In the 80 ⫻ 80 mm area measurements of bovine enamel performed between the 41 mm thick gold lines 140 mm apart, the variations of wt% for the different elements were less marked ~Figs. 2e, 2f!.
Point and Area Analysis of Carbon, Gold, and Uncoated Enamel
some details about the correction procedure. The apparent concentration of each element ~not shown! is divided by the intensity correction ~column 2!, to produce the true element wt% ~column 3!. Intensity corrections have three components. The Z or atomic number term models the proportion of electrons that give rise to characteristic X-rays; the absorption term models absorption of the X-rays in their path to leave the sample; the fluorescence term models the lower energy X-rays generated by X-rays moving through the sample, e.g., Oxygen Ka generated by P Ka. The statistical counting error calculated from the number of X-rays counted is also shown, as is the standard deviation of the mean of three replicate 400 s counts. Finally, the detection limits for 400 s live time counts are shown in the last column. The glass analyses are compared with area analyses of carbon, gold, and uncoated bovine specimens in Table 2 and Figure 4. The glass analyses were adjusted down to take the 9.6% measured loss on ignition into account. The glass analyses show good agreement between O by spectrum and by stoichiometry.
D ISCUSSION Area versus Point Analysis
Comparisons of Point and Area Analysis (Figs. 3a–3d) Only minor differences are seen between point and area analyses, however, the differences were more marked for Na. The mean values for area analysis were higher compared with point analysis, with the exception of K and Ca.
Area analyses gave slightly smaller standard errors in Figure 3 and Table 3, probably due to averaging out of aberrant points due to imperfections in the surface, which has a roughness of about 1 mm.
Glass Samples (Figs. 4a, 4b)
Looking at the question of electrostatic charging, the totals shown in Figures 2b and 2d suggest that there is no significant effect up to about 100 mm from the coated surface,
The full results from the chemical analysis of bovine enamel prepared as fused glass are presented in Table 1, including
Coating and Electrostatic Charging
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Figure 2. Wt% values for the X-ray microanalysis point and area measurement profiles in uncoated bovine enamel, starting in a gold-coated area and moving away. a: Values from point measurements for Na, Mg, Cl, K, and Au. b: Values from point measurements for O, P, Ca, and totals. c: Values from area measurement for Na, Mg, Cl, K, and Au. d: Values from area measurements for C, O, P, Ca, and totals. e: Area measurements between the gold lines in uncoated bovine enamel for Na, Mg, Cl, and K. f: Area measurements between the gold lines in uncoated bovine enamel for C, O, P, Ca, and totals.
after which the totals show a significant decrease, probably related to charging. The area analyses within gold lines shown in Figures 2e and 2f show constant Na, but area analyses are higher than points and results with other coatings. Thus there might be a small but constant Na enrichment artifact in the area analyses.
Coating or Not In general, uncoated analyses correspond well to coated ones. All three coating methods agree well for O and Ca.
Uncoated analyses gave slightly lower errors and higher amounts, probably more correct as there is no uncertainty of coating thickness. There seem to have been no problems with charging within about 120 mm of a conducting surface, which would give low values for O and other elements in uncoated samples. P gave similar concentrations within error for all coatings and point and area analyses were also similar. C on uncoated points and areas was about 1 wt% lower than on gold-coated ones, probably reflecting underestimation of the gold-coating thickness. Therefore, the uncoated analyses are to be preferred. The differences in
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Figure 3. Mean values and standard deviations for X-ray microanalysis measurements in bovine enamel in wt% for O, Na, Mg, P, Cl, K, and Ca in point and area analyses with carbon coating, gold coating, and uncoated. ~C-coat point ⫽ carbon coating and point analysis; C-coat area ⫽ carbon coating and area analysis; Au-coat point ⫽ gold coating and point analysis; Au-coat area ⫽ gold coating and area analysis; no-coat point ⫽ no coating and point analysis; no-coat area ⫽ no coating and area analysis!. a: Values for Na, Mg, Cl, and K with C excluded from the analysis. b: Values for O, P, and Ca with C excluded from the analysis. c: Values for Na, Mg, Cl, and K with C included in the analysis. d: Values for C, O, P, and Ca.
Figure 4. Mean values and standard deviations for X-ray microanalysis measurements in natural bovine enamel and bovine pure and fluxed fused glass in wt% for O, Na, Mg, P, Cl, K, and Ca in area analyses with carbon coating, gold coating, and uncoated specimens. C was excluded in the analyses. ~C-coat ⫽ carbon coating; Au-coat ⫽ gold coating; no-coat ⫽ no coating; P-glass ⫽ pure glass; F-glass ⫽ fluxed glass!. a: Values for Na, Mg, Cl, K, pure glass, and fluxed glass. b: Values for O, P, Ca, pure glass, and fluxed glass.
analyses of Na may also reflect uncertainties in coating thickness, which would affect the coated analyses. The thickness of the coating estimated for gold from reference graphs and for carbon from the interference color on a brass surface influences the intensity correction and
calculated wt% values. Since the exact thickness probably has an uncertainty of about 10% it is an uncontrollable source of error. Therefore uncoated specimens analyzed in VP-SEM mode are regarded as being most accurate, provided that electrostatic charging is avoided. Low-vacuum
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analysis at 20 Pa and with gold lines ~measuring ;140 mm between the lines! in the uncoated area is the method of choice.
Control of the Effect of Surface Charging In the experiment using spot analysis at different distances from a gold-coated area, the effect of electrostatic charging seems to become significant at a distance of about 120 mm, as seen from the decrease in totals in Figure 2b. Thus, a distance of ,120 mm from a conductive surface should avoid charging problems for spot analyses at 3.5 nA and 20 Pa pressure, whereas area analyses might be good at greater distances because the charge is spread out. A possible enrichment of Na and Cl was seen in the spot analyses, peaking about 50 mm from the gold coating, and about 100 mm in the area analysis. This might be an artifact created during the gold-coating procedure. In the profile using 12 ⫻ 12 mm area analyses on the same sample profile ~Figs. 2c, 2d!, the peaks were about 100 mm from the gold. However, in the 80 ⫻ 80 mm area analyses parallel to and between gold lines ~Figs. 2e, 2f!, no peaks are seen. Area analysis using a square area between 15 and 80 mm across appears better than point analysis due to better homogeneity and less potential for charging on the larger area. But how far from a coated surface should the area analysis be made? In the area analyses shown in Figures 2c and 2d, the value for the total wt% dropped off dramatically for the first 30 mm, than levelled out. Area analysis with a size between 15 and 80 mm appears better than point analysis due to less charging and more homogeneity. The best position is about 30 mm from gold, using area and not point analysis. The enrichment of Na seen near the gold coating is possibly an artifact created during the gold-coating procedure. The totals when C was included seem reasonable at around 97%. The strategy of coating with gold lines appeared favorable to extend the analyzable region and no gold contamination could be seen. Carbon appeared to vary quite a lot between samples ~Fig. 3d!.
Element by Element Discussion In this section we will discuss the significant aspects of each element in turn, in order of increasing atomic number, except for carbon, which is discussed last. In general the analyses including carbon will be discussed, unless otherwise specified. Oxygen Oxygen is traditionally estimated using stoichiometry in silicate and oxide minerals such as apatite, which are usually fully oxidized. This is because the exact wavelength and peak position of the oxygen K lines can vary considerably, a problem in wavelength-dispersive spectrometers ~WDS!. However, natural dental enamel clearly contains some organic material, which is not stoichiometrically combined with oxygen. Thus, calculating oxygen by stoichiometry is expected to give values that are too high.
EDS spectrometry has lower resolution than WDS and is more tolerant of small wavelength changes, thus quantification using the oxygen K X-ray series is feasible. A test of this is provided by the fused glass samples that had been ignited and had thus lost their nonstoichiometric components, so that the two results for oxygen should be the same. Oxygen wt% for the pure fused material is 41.18 wt% using the K X-ray series and 41.25 wt% by stoichiometry, thus the X-ray quantification seems reliable. A caveat is that the natural enamel surface and body is not as regular as the glass, which could affect the absorption correction and reduce the precision. Another alternative is to calculate “oxygen by difference” in which the total is made up to 100% with oxygen. This is the worst method for oxygen, but may give better values for the other elements if the totals by other methods deviate significantly from 100. Oxygen values shown in Table 3 and Figures 3b and 3d are hardly affected by inclusion or exclusion of carbon. The calculated intensity corrections shown in Table 4 change the apparent oxygen concentration in coated samples by 15 to 20%, whereas only by 3% in uncoated samples, illustrating the major effect of coating. The uncoated area analyses are about 1 wt% higher than the others, possibly reflecting oxygen in the surface layer of gas, which conducts away the electrostatic charge. This effect is minimized in the uncoated point analyses, which are thus considered the most reliable for oxygen. Sodium Area analyses of Na with no coating between gold lines are clearly higher than all other analyses and this is probably due to the sodium enrichment artifact close to gold coating seen in Figure 2a. Point analyses are generally about 0.05% lower than area analyses for Na. This is due either to inhomogeneity in the sample or more likely to heating of the spot during analysis, driving off Na and leading to low values. This phenomenon is common in point analyses of alkali feldspars, which are usually analyzed as areas. C and gold-coated areas agree for Na, are probably most reliable, and are least likely to have problems or artifacts, giving a value of about 0.70 wt%. Uncoated small area ~12 ⫻ 12 mm! analyses between gold lines for Na are probably good if artifacts can be avoided, although this needs further investigation. One way to evaluate artifacts is to compare with small area analyses within the adjacent gold lines. Magnesium and Phosphorous All methods seem comparable, but uncoated areas might have a small artifact near gold. Uncoated point analyses are considered good. There are no significant differences between the methods for phosphorous. Chlorine Chlorine seems high in both carbon-coated methods. The other methods give comparable results, but there is a possible artifact in uncoated analyses near gold coating ~Fig. 2a!. Uncoated point analysis seems good.
Evaluation of XRMA in Bovine Enamel
Potassium K is constant within error in all methods but the low concentrations around 0.06% are close to detection limits for 40 s counts. If K is considered significant, then a longer live time should be used, for example, 400 s giving a lower detection limit of 0.03 wt%, as shown in Table 1. Calcium There are no significant differences between the methods. Uncoated point analyses seem good. Carbon Analysis of C using the K X-ray series has similar problems to oxygen by WDS but is quantifiable by EDS, with caveats related to contamination and uncertainties in the absorption correction related to the heterogeneity of the natural enamel. Contamination may be caused by carbon-bearing material on the surface, or less likely by spectral contamination from other carbon-coated surfaces due to electron scatter in the low-vacuum chamber. Surface contamination is made worse by the correction procedure that incorrectly assumes that the carbon X-rays come from within the material ~Information on contamination from P. Statham, Oxford Instruments, personal communication with DHC 2013!. The measured C concentration is corrected by the software by a factor of about 3.5 ~reciprocal of intensity correction! due to absorption and other matrix effects. The accuracy of carbon analyses cannot be tested using the ignited fused glass samples. Carbon-coated samples can also not be used, thus only gold coated and uncoated analyses can be compared. The mean values in Table 3 show large standard deviations, surprisingly larger for area analyses than point analyses, which might point to surface contamination. The mean values for area and spot analyses agree for each coating, but differ between coatings, gold coating giving about 1.3 wt% higher carbon values than uncoated. This difference may be due to either surface contamination or to uncertainties in the thickness of gold coating, which has a significant effect on the intensity correction, about 0.6 wt% difference for a 10% change in gold thickness. Thus, the uncoated analyses are probably more correct. Area analyses should in principle be less subject to heterogeneity and the large standard deviation of about 2 wt% is probably a good estimate of uncertainty, which could be improved by longer counting times. Uncoated point analyses seem to be equally reliable.
S UMMARY Uncoated point analyses seem a good choice for all elements except Na, for which small area analyses should be used to avoid Na loss. Carbon analyses have the largest uncertainty because of the possibility of surface contamination, which we have not been able to evaluate. The 80 mm areas between gold lines used for this work might be affected by the gold-coating artifact we observed. A good compromise should be to analyze 10 mm 2 areas midway between 41 mm thick gold lines 140 mm apart. These could be combined
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with 10 mm 2 area analyses in the middle of the gold lines, which would provide a check for artifacts. Counting times should be chosen according to the detection limits required ~see Table 1 for 400 s live times!. Detection limits are proportional to the square root of the live time, thus 100 s live times would give a factor of two times worse detection limit than 400 s.
Comparison with Fused Glass The values for the different elements in pure fused glass are generally lower than the mean values for the bovine enamel in Table 2. The most marked differences are found for O, Na, P, and Cl. The corresponding values for the fluxed glass specimens are lower than for pure glass, however, no difference is seen between natural enamel and fluxed glass for Na and Cl whereas P remained at the same low value as for pure glass. The aim of using fused glass analyses was to provide “true” values for reference since there are no reference standards for dental enamel, which is not pure hydroxyapatite. The glass analyses gave totals close to 100% before correction for loss on ignition, thus representing good analyses for the glasses. However, there are a number of problems related to the fusion process of making enamel powder into glass. This can be seen from comparisons of the uncoated sample, taken as the best natural analysis, with pure fused glass and fluxed glass ~corrected for 9.7% loss on ignition! in Table 2. The values for O differ slightly, taking the 1–3% standard deviations of the natural analyses into account. The ignition and fusion process probably decreased O in the pure glass sample as water and CO2 were driven off. P values are as much as 5 wt% lower in the glass samples. This may reflect the loss of organically bound P, which was lost in the ignition and fusion process. The P content in dental enamel is found in the inorganic as well as the organic parts of the enamel. A loss of Na and Cl is also seen in pure glass that is probably related to the higher fusion temperature ~;1,6008C! compared to that for the fluxed glass ~;1,0008C!. The loss of Na, Cl, and predictably K at the higher fusion temperature can be explained by the fact that these elements are more easily vaporized than the other elements. The C-coated analyses of Cl are high and had a higher error, which might reflect concentration of this most mobile element Cl at the surface during carbon coating, as the carbon string radiates heat. Concerning Ca and Mg, all coatings corresponded well with each other and also with the glass analysis. It is concluded that the glass analyses are not directly comparable with those of natural material, but they do provided insights into both the nature of the tooth material and the glass making process.
Totals and Matrix Effects As shown in Table 3, the wt% totals excluding carbon are close to 100% for all methods. For normal inorganic mineral analyses totals within 2 wt% of 100 are regarded as
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acceptable, taking possible crystalline water into account. As shown in Table 3, the totals including carbon are between 105 and 107%, with standard deviations of 2.9 to 4.3 wt%. The high totals and variability of our natural dentine analyses may be ascribed to carbon contamination and other matrix effects. These may include surface roughness, porosity, and inhomogeneity of the hydroxyapatite structure and organic constituents. The light elements C and O are most likely to be affected by matrix effects because of their low energy and long X-ray wavelength. Including C in the analytical menu slightly increased the elements lighter than Mg and decreased those with higher atomic numbers. This is probably due to changes in the fluorescence correction, which may not be correct if some of the carbon analysis is due to surface or spectral contamination. Unless the effects of carbon contamination can be evaluated, analyses without carbon should be regarded as most reliable and carbon analyses should be regarded as semiquantitative. Another factor that may influence results in XRMA analysis of enamel is mineral density in the tissue, which varies between different locations in the enamel and between hypomineralized and normal enamel, mineral hydroxyapatite has a density of 3.16 whereas tooth enamel varies between 3 and 2.84 ~He et al., 2011!. In the present study only the bulk of the enamel was analyzed as the surface layer of the enamel had been ground away. Material with porosity or lower density than solid minerals should generally give lower analytical values ~P. Statham, Oxford Instruments, personal communication with DHC 2013!.
Other Enamel Analyses in the Literature Quantitative studies of the elemental composition of dental enamel are scarce and commonly the measurements are regarded as semi-quantitative or presented as ratios to the Ca content ~Retief et al., 1970, 1971; Shaw & Yen, 1972!. Analyses of human enamel using our method and by neutron activation and EDS ~Retief et al., 1970, 1971; De Menezes Oliveira et al., 2010! shown in Table 2 show values comparable with the bovine enamel results of this study. Other studies of human enamel by X-ray EDS show large deviations from our results and do not describe their methods or discuss their accuracy in any detail ~GotierrezSalazar & Reyes-Gasga, 2003; Mahoney et al., 2004!.
C ONCLUDING R EMARKS Small area ~10 ⫻ 10 mm 2 square! analyses without any coating and midway between with 40 mm gold lines 140 mm apart are preferred, especially if C is included in the analysis. The VP-SEM mode with 20 Pa low vacuum used for calibration and EDS analysis with a stable 3.5 nA specimen current is appropriate for our instrument. Our analyses of bovine enamel are sufficiently reproducible to be regarded as quantitative for all elements except C, for which the potential problem of contamination could not be assessed. There are also probably some unexplained matrix effects
that lead to high totals and suggest that some elements may have systematic errors in the order of 5% relative.
R EFER ENCES Braga, S.R., De Faria, D.L., De Oliveira, E. & Sobral, M.A. ~2011!. Morphological and mineral analysis of dental enamel after erosive challenge in gastric juice and orange juice. Microsc Res Tech 74, 1083–1087. De Carvalho Filho, A.C., Sanches, R.P., Martin, A.A., Do Espírito Santo, A.M. & Soares, L.E. ~2010!. Energy dispersive X-ray spectrometry study of the protective effects of fluoride varnish and gel on enamel erosion. Microsc Res Tech 74, 839–844. De Menezes Oliveira, M.A., Torres, C.P., Gomes-Silva, J.M., Chinelatti, M.A., De Menezes, F.C., Palma-Dibb, R.G. & Borsatto, M.C. ~2010!. Microstructure and mineral composition of dental enamel of permanent and deciduous teeth. Microsc Res Tech 73, 572–577. Eisenmann, D.R. ~1994!. Enamel structure. In Oral Histology— Development, Structure, and Function, Ten Cate, A.R. ~Ed.!, 4th ed., pp. 239–256. St. Louis, MO: Mosby. Fagrell, T.G., Dietz, W., Jälevik, B. & Norén, J.G. ~2010!. Chemical, mechanical and morphological properties of hypomineralized enamel of permanent first molars. Acta Odontol Scand 68, 215–222. Gotierrez-Salazar, M.P. & Reyes-Gasga, J. ~2003!. Microhardness and chemical composition of human tooth. Mat Res 6, 367–373. Hallsworth, A.S., Weatherell, J.A. & Robinson, C. ~1973!. Loss of carbonate during the first stages of enamel caries. Caries Res 7, 345–348. He, B., Huang, S., Zhang, C., Jing, J., Hao, Y., Xiao, L. & Zhou, X. ~2011!. Mineral densities and elemental content in different layers of healthy human enamel with varying teeth age. Arch Oral Biol 56, 997–1004. LeGeros, R.Z. ~1981!. Apatites in biological systems. Prog Crystal Growth Charact 4, 1–45. LeGeros, R.Z. ~1984!. Incorporation of magnesium in synthetic and biological apatite. In Tooth Enamel, Fearnhead, W.R.W. & Suga, S. ~Eds.!, pp. 32–36. Amsterdam: Elsevier Publishers. LeGeros, R.Z. ~1991!. Calcium phosphates in oral biology and medicine. In Monographs in Oral Science, vol. 15, pp. 1–201. Basel: Karger. LeGeros, R.Z., Kijkowska, R., Bautista, C. & LeGeros, J.P. ~1995!. Synergistic effects of magnesium and carbonate on properties of biological and synthetic apatites. Connect Tissue Res 33, 203–209. Mahoney, E.K., Rohanizadeh, R., Ismail, F.S., Kilpatrick, N.M. & Swain, M.V. ~2004!. Mechanical properties and microstructure of hypomineralised enamel of permanent teeth. Biomaterials 25, 5091–5100. Nelson, A.E., Hildebrand, N.K.S. & Major, P.W. ~2002!. Mature dental enamel @calcium hydroxyapatite, Ca10 ~PO4 !6 ~OH!2 # by XPS. Surf Sci Spectra 9, 250–259. Retief, D.H., Cleaton-Jones, P.E. & Turkstra, J. ~1970!. The quantitative determination of Ca, Na, Al, Mg, and Cl in normal enamel and dentin by neutron activation and high resolution gamma spectrometry. J Dent Assoc S Afr 25, 188–192. Retief, D.H., Cleaton-Jones, P.E., Turkstra, J. & De Wet, W.J. ~1971!. The quantitative analysis of sixteen elements in normal human enamel and dentine by neutron activation analysis and
Evaluation of XRMA in Bovine Enamel high-resolution gamma-spectrometry. Arch Oral Biol 16, 1257–1267. Sabel, N., Dietz, W., Lundgren, T., Nietzsche, S., Odelius, H., Rythén, M., Rizell, S., Robertson, A., Norén, J.G. & Klingberg, G. ~2009a!. Elemental composition of normal primary tooth enamel analyzed with XRMA and SIMS. Swed Dent J 33, 75–83. Sabel, N., Klingberg, G., Nietzsche, S., Robertson, A., Odelius, H. & Norén, J.G. ~2009b!. Analysis of some elements in primary enamel during postnatal mineralization. Swed Dent J 33, 85–95. Shaw, J.H. & Yen, P.K. ~1972!. Sodium, potassium, and magnesium concentrations in the enamel and dentin of human and rhesus monkey teeth. J Dent Res 51, 95–101. Soares, L.E., de Oliveira, R., Nahórny, S., Santo, A.M. & Martin, A.A. ~2012!. Micro energy-dispersive X-ray fluorescence mapping of enamel and dental materials after chemical erosion. Microsc Microanal 18, 1112–1117. Souza, R.O., Lombardo, G.H., Pereira, S.M., Zamboni, S.C., Valera, M.C., Araujo, M.A. & Ozcan, M. ~2010!. Analysis of tooth enamel after excessive bleaching: A study using scanning electron microscopy and energy dispersive X-ray spectroscopy. Int J Prosthodont 23, 29–32.
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Sydney-Zax, M., Mayer, I. & Deutsch, D. ~1991!. Carbonate content in developing human and bovine enamel. J Dent Res 70, 913–916. Takagi, T., Ogasawara, T., Tagami, J., Akao, M., Kuboki, Y., Nagai, N. & LeGeros, R.Z. ~1998!. pH and carbonate levels in developing enamel. Connect Tissue Res 38, 181–187. Taube, F., Steiniger, F., Nietzsche, S. & Norén, J.G. ~2010!. Scanning electron microscopic and X-ray micro analysis on tooth enamel exposed to alkaline agents. Swed Dent J 34, 129–137. Weatherell, J.A., Robinson, C. & Hallsworth, A.S. ~1974!. Variations in the chemical composition of human enamel. J Dent Res 53, 180–192. Weerasinghe, D.D., Nikaido, T., Ichinose, S., Waidyasekara, K.G. & Tagami, J. ~2007!. Scanning electron microscopy and energy-dispersive X-ray analysis of self-etching adhesive systems to ground and unground enamel. J Mater Sci Mater Med 18, 1111–1116. Youravong, N., Teanpaisan, R., Norén, J.G., Robertson, A., Dietz, W., Odelius, H. & Dahlén, G. ~2008!. Chemical composition of enamel and dentine in primary teeth in children from Thailand exposed to lead. Sci Total Environ 389, 253–258.
Microsc. Microanal. 20, 257–267, 2014 doi:10.1017/S1431927613013998
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Evaluation of X-Ray Microanalysis for Characterization of Dental Enamel Lisa Melin,1 Jörgen G. Norén,1, * Fabian Taube,2 and David H. Cornell 3 1
Department of Pediatric Dentistry, Institute of Odontology, Sahlgrenska Academy, University of Gothenburg, SE-405 30, Gothenburg, Sweden 2 Department of Occupational and Environmental Medicine, Sahlgrenska University Hospital, SE 405 30, Gothenburg, Sweden 3 Department of Earth Sciences, University of Gothenburg, SE 405 30, Gothenburg, Sweden
Abstract: Elemental analysis of dental hard tissues is of importance. The aim of this study is to evaluate X-ray microanalysis ~XRMA! of bovine enamel in a scanning electron microscope ~SEM! with different coatings. The buccal surface of bovine incisors was polished flat, one-third was coated with carbon, one-third with gold, leaving one-third uncoated for XRMA in an SEM equipped with an energy-dispersive microanalysis system. The elements oxygen, sodium, magnesium, phosphorous, chlorine, potassium, and calcium were analyzed using their respective characteristic K X-ray series. Comparisons were made with analyses of glass produced by fusion of the bovine enamel, showing that oxygen analyses using the K X-ray series are reliable and preferable to calculating oxygen by stoichiometry for natural enamel. For the gold-coated and uncoated analyses, carbon was also measured using the K X-ray series. Small area Analyses in small areas ~80 ⫻ 80 mm! in variable pressure-SEM mode with low vacuum ~20 Pa!, without any coating, midway between 40 mm wide gold lines 140 mm apart to avoid build-up of electrostatic charge is the preferred method, especially if carbon is included in the analysis. The analyses of bovine enamel are sufficiently reproducible to be regarded as quantitative for all elements except carbon. Key words: bovine, enamel, scanning electron microscope, elemental analysis, glass, XRMA
I NTR ODUCTION Elemental analysis of dental hard tissues ~enamel, dentin, cementum! has long been of importance. However, due to their brittle and insulating properties, there are few suitable methods. Energy-dispersive spectroscopy ~EDS! or wavelength-dispersive X-ray microanalysis ~XRMA! of spectra excited by electron beams in electron microscopes have often been used ~Mahoney et al., 2004; Weerasinghe et al., 2007; Youravong et al., 2008; Sabel et al., 2009a, 2009b; De Carvalho Filho et al., 2010; De Menezes Oliveira et al., 2010; Fagrell et al., 2010; Souza et al., 2010; Taube et al., 2010; Braga et al., 2011; He et al., 2011!. Analysis of the chemical composition of enamel in teeth with developmental disturbances has been of special interest, especially in relation to the morphology. An important advantage of XRMA in a scanning electron microscope is that measurements can be made with full image control of the morphological location in the analyzed tissue. XRMA analysis is particularly useful for the analysis of Ca, P, and O in dental hard tissues and C is also an element of interest. Several studies have revealed large variations in elemental composition between different samples of normal enamel ~see, e.g., Sabel et al., 2009a!. Dental enamel is said to comprise 96 wt% inorganic mineral and the main elements are Ca, P, and O, which build calcium hydroxyapatite crystals @Ca10 ~PO4 !6 ~OH!2 #. This may vary somewhat in Received July 12, 2013; accepted December 3, 2013 *Corresponding author. E-mail: [email protected]
composition, especially at the surface, depending on reactions with the oral environment ~Nelson et al., 2002!. Dental enamel is far from the idealized form of hydroxyapatite, but biological apatite contains carbonate hydroxyapatite as well as HPO4 , Mg, and Cl ~LeGeros, 1981, 1984, 1991; LeGeros et al., 1995; Takagi et al., 1998!. Other elements of importance are F, Na, and K, which play important roles during the mineralization of enamel. Only 3 wt% of the enamel is organic material in the form of proteins and lipids, with C and N as the main components, whereas another 1 wt% is water, according to Eisenmann ~1994!. In previous analyses of enamel, C has been shown to have a high concentration due to the carbonate content of mature dental enamel ~Sydney-Zax et al., 1991!. The carbonate content of enamel is of specific interest in relation to the caries process, since high levels of carbonate increase susceptibility of enamel to caries ~Hallsworth et al., 1973; Weatherell et al., 1974!. Analysis of carbon in dental enamel is desirable in relation not only to caries but also to reflect differences in the organic content of enamel. Bovine enamel has long been used for in vitro studies because its chemical and morphological properties resemble those of human dental enamel ~e.g., Soares et al., 2012!. Bovine teeth are therefore an excellent proxy for human teeth and also far more easy to handle due to their size. The surface layer of bovine and human enamel after eruption is subjected to the influence of the oral environment and takes up different trace elements that are accumulated in the surface area of the enamel. By removing the surface layer
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down to the bulk of the enamel, the possible variations of morphology and chemical composition can be reduced. A problem with electron beam microanalysis of enamel is that electrostatic charge tends to build up on the nonconducting surface and this has normally been solved by applying thin coatings of conductive material. The aim of this study is to evaluate XRMA analysis of bovine enamel with different coatings using carbon, gold, and without any coating, using a low-vacuum technique. Bovine material provides large homogeneous areas on which to make these tests. The results of this work will be applied to problems in human teeth.
M ATERIALS
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Bovine Tooth Samples Mandibular jaws from ten calves were collected from a slaughterhouse at Skara in western Sweden. The central incisors were cut off from the jaws and after cleaning in tap water stored in 70% ethanol till further preparation. Storage in ethanol enables a better embedding in epoxy resin and has previously been used in several studies employing XRMA ~see, e.g., Sabel et al., 2009a!. In order to eliminate chemical variations in the bovine enamel surface, and to create a flat surface, the buccal surface was ground with SiC paper in a polishing machine ~Struers, Copenhagen, Denmark!. The teeth were placed centrally, with the flat surface on the bottom of 25-mm-diameter mounting cups for epoxy resin ~Struers!. After correct orientation of the teeth the mounting cups were assembled and ID marked with a water-resistant marker pen. The puck diameter fitted the sample holder of the scanning electron microscope ~SEM!. The tooth samples were embedded in epoxy resin for electron microscopy ~Epofix威; Electron Microscopy Sciences, Fort Washington, PA, USA! mixed according to the manufacturer’s instructions. The molds were filled with the mixed epoxy till the teeth were totally embedded. After hardening overnight, the embedded teeth were removed from the molds and the epoxy pucks marked with an ID number. In order to remove any excess epoxy from the enamel surface the embedded samples were polished with abrasive paper with water cooling. This procedure was performed with three different papers ~grit 1,200, 2,400, and 4,000 ⫽ 4 mm! in the polishing machine. Macroscopic photos were taken of all samples in a Leica M80 stereo microscope ~Leica Mikrosysteme Vertrieb GmbH, Wetzlar, Germany! using a Leica digital camera ~Leica DFC420 C, Leica Mikrosysteme Vertrieb GmbH! with Leica Application Suite LAS V3.7.0 ~Leica Microsystems AG, Heerbrugg, Switzerland!. An overview of the polished surface was taken in a Leica M80 stereo microscope at low magnification ~0.75⫻! in incident light using a Leica digital camera with LAS V3.7.0. In order to have a comparison for the XRMA analysis of natural enamel, powdered samples of the same bovine enamel were also prepared as fused glass samples for comparison. These were weighed after drying at 1108C, then
Figure 1. a: Schematic drawing of the polished flat surface of a calf incisor one-third coated with carbon, one-third uncoated, and one-third coated with gold. ~x ⫽ locations for point and area analyses!. The sample diameter is 25mm. b: Schematic drawing of the polished flat surface of a calf incisor coated with carbon, gold, or uncoated, then coated with 41 mm thick gold lines 140 mm apart to minimize electrostatic charging ~䉺 ⫽ locations for analyses!.
after 30 min ignition at 9808C, giving a consistent loss on ignition value ~volatiles such as water, CO2 , and organic material! of 9.7%. About 0.5 g of ignited powder was fused by electrical heating for about 5 s until seen to be completely fused, in a molybdenum metal boat in an argonfilled pressure chamber with glass observation window, at 5 atmospheres pressure. One fusion was made of the pure ignited material, which melts around 1,6008C, and another using 50% lithium metaborate flux, which lowered the fusion temperature to an estimated 1,1008C. The glass produced was broken out of the boat and mounted in an epoxy puck, then ground and polished with 3 mm diamond to produce a smooth surface for analysis in the SEM.
XRMA Analysis The buccal surface of four bovine tooth samples was divided into three areas, as shown in Figure 1. One area was carbon coated ~'25 nm thick! in a carbon thread vacuum system, the central part was left uncoated, and the third part was coated with gold in a plasma coater ~'5 nm thick!. Thereafter, the samples were placed in a sample holder for SEM. Four analyses were made in the carbon-coated areas, three in the uncoated areas, and three or four in the area coated with gold ~Fig. 1a!. The analyses were carried out using 1,500⫻ magnification as point ~effectively 4 mm diameter analysis points! and also as area analyses using 80 ⫻ 80 mm 2, with a counting time of 40 s. The “All elements” option in the INCA software was used and for carbon-coated enamel the elements oxygen ~O!, sodium ~Na!, magnesium ~Mg!, phosphorous ~P!, chlorine ~Cl!, potassium ~K! and calcium ~Ca! were analyzed using the K X-ray series. For the gold-coated and the uncoated analyses, carbon ~C! was included. Each part of the tooth was analyzed in three different locations, applying both point and area analysis. When analyzing areas coated with carbon or gold, the normal high-vacuum mode for SEM was used for second-
1.205 1.399 1.430
K2O CaO Fe2O3
0.30 0.06 0.03 0.03 0.03 0.03 0.03 0.03 0.03 0.06 Na2O MgO SO2 P2O5 SO3 1.348 1.658 2.139 2.291 2.001
~F! 0.30 0.07 0.04 0.06 0.06 0.05 ~Cl! 0.03 0.03 0.04 0.08 MAC JMAC MgO MAC WAC AMAC AnMAC TSl KfMAC WMAC FeMMAC K2O CaO Fe2O3 Total Total-N
0.00 0.55 0.62 0.47 44.59 0.58 ~0.07! 0.06 53.15 0.00 100.00 100.06 Na2O MgO SO2 P2O5 SO3
0.03 0.03 0.01 0.13 0.04 0.02 0.01 0.15 0.00 0.07 0.02 0.01 0.01 0.03 0.01 0.01 0.01 0.04 0.00 0.05
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*Analysis by electron beam EDS X-ray microanalysis. Detection limits are for 400 s live time. Loss on ignition was determined by weighing dry ~1108C! samples after ignition at 9808C. The glass analysis is given as dry element and oxide wt%, then reduced to accommodate the loss on ignition. Loss on ignition includes organic material, crystalline water, CO2 in carbonate, and some Cl and F. SD, standard deviation; SM, stoichiometry; X-RP, using X-ray peak; Total-N, total before normalization; MAC, microanalysis consultants, Cambridge UK; J-MAC, Jadeite Mac; WMAC, wollastonite MAC; AMAC, apatite MAC; AnMAC, anhydrite MAC; TSl, tugtupite Sl; KfMAC, K-feldspar MAC; FeMMAC, Fe metal MAC; LOI, loss on ignition.
Element ~wt%!
0.00 0.41 0.37 0.22 19.46 0.23 0.07 0.05 37.96 0.00 41.25 41.18 0.9046 0.7414 0.7114 0.9836 1.0050 0.8373 1.1028 1.1291 1.0012 0.8141 Stoichiometry K series X-ray F Na Mg Si P S Cl K Ca Fe O O
Oxide
Dry Oxide ~wt%!
Standard
F Na2O MgO SO2 P2O5 SO3 Cl K2O CaO Fe2O3 LOI Total
,0.30 0.49 0.56 0.42 40.27 0.52 0.06 0.05 48.00 ,0.06 9.70 100.07
Element to Oxide Detection Limit Oxide ~%! Natural Material ~wt%! Intensity Correction
In order to evaluate the effect of surface charge on the analysis, a series of measurements were made using a lowvacuum of 20 Pa on uncoated enamel ~Fig. 1b!. The air remaining in the sample chamber is thought to form a surface layer on the sample that conducts away the surface charge, while the tungsten filament in the electron gun is maintained at a much lower pressure to avoid it burning. Two sets of analyses were performed as point analyses with a starting point in the gold-coated area and the remaining points in the uncoated enamel. A further three sets of analyses were performed, one as point analyses and two as area analyses with a square area of 12 ⫻ 12 mm. On one of the specimens a linear gold coating ~line width 41 mm, distance between the lines 140 mm! was made on the specimens with the use of a grid ~Fig. 1b!. Area analyses were performed in four areas with the first starting in the gold-coated part of the enamel.
Element
Control of the Effect of Surface Charging
SD of Three 400 s Counts
The apparent concentrations are corrected by a phi-rho-z iterative procedure described in more detail for glass samples below. For the gold-coated and uncoated areas the analyses were calculated with and without C included in the menu. The mean values for the different elements with carbon coating, gold coating, and without coating are given in Tables 2 and 3. The intensity correction values are given in Table 4.
Counting Error ~wt%!
Presentation of Results by INCA Energy Software
Table 1. Chemical Analysis of Bovine Tooth Sample Bov1, Prepared as Fused Glass, Polished, and Carbon Coated.*
ary electron images. Analyses of areas without coating were performed in low-vacuum mode ~VP-SEM! with a vacuum of 20 Pa using backscattered electron images. The XRMA analysis was performed in a Hitachi S-3400N SEM equipped with an Oxford Instrument EDS microanalysis system and INCA software ~Oxford Instruments, Abingdon, UK!. All analyses were carried out at 20 kV accelerating voltage and the working distance from sample to electron optical column was set to 9.9 mm, with a tolerance of 0.1 mm. The electron beam was aligned in “Microscope setup” using “Wave” in INCA with the beam aimed into a Faraday cage. The specimen current was adjusted to 3.50 6 0.02 nA, and was monitored every 30 min with stability better than 1%. The standard calibration was performed by acquiring a spectrum with 40 s live time on cobalt metal in the sample holder. The standards used are listed in the caption for Table 1. These simple standards are linked to the cobalt standard, which is counted at the beginning of each highor low-vacuum session. The accuracy of mineral analyses is checked periodically using mineral standards from the Smithsonian Institute, Washington. The fused glass samples were analyzed only with high vacuum and carbon coating. Oxygen was analyzed using the spectral peak and also using the oxygen by stoichiometry option. The results are given in Table 2.
Detection Limit Element ~%!
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Lisa Melin et al. Table 2. Mean Values of X-Ray Microanalysis ~XRMA! Area Analysis of Natural Bovine Enamel from Carbon Coated ~C Coat!, Gold Coated ~Au Coat!, Uncoated ~No Coat! Specimens and for Carbon-Coated Bovine Pure Glass and Bovine Fluxed Glass for the Elements O, Na, Mg, P, Cl, K, and Ca.* Natural Bovine Enamel
O Na Mg P Cl K Ca Total
C Coat
Au Coat
No Coat
Pure Glass C Coat
39.99 0.67 0.29 23.48 0.35 0.05 36.53 101.36
40.14 0.70 0.30 22.75 0.26 0.09 35.25 99.49
41.30 0.75 0.33 22.99 0.27 0.05 34.88 100.57
37.21 0.37 0.33 17.57 0.06 0.05 34.30 90.30
Fluxed Glass C Coat 39.96 0.70 0.35 17.00 0.24 0.09 34.46 90.30
Human Neutron Activation
Human EDS XRMA
Primary
Permanent
16.18
21.19
17.23
29.82
52.50
35.11
39.70 0.70 0.28 0.32 37.03
*The values for glass are reduced by 9.70% to compensate for loss of ignition. For comparison some data are shown for human enamel by neutron activation from Retief et al. ~1971!, by XRMA from Fagrell et al. ~2010! and from De Menezes Oliveira et al. ~2010!.
Table 3. Mean Values and Standard Deviations ~SD! of Point and Area Analyses in wt% of C, O, Na, Mg, P, Cl, K, Ca, and Totals for Analyses of Bovine Enamel with Carbon Coating, Gold Coating, and Without Coating.* Carbon Coated Point Element C O - C incl. O - C excl. Na - C incl. Na - C excl. Mg - C incl. Mg - C excl. P - C incl. P - C excl. Cl - C incl. Cl - C excl. K - C incl. K - C excl. Ca - C incl. Ca - C excl. Totals - C incl. Totals - C excl.
Mean
Gold Coated
Area SD
Mean
Point SD
39.59
2.22
39.99
2.32
0.60
0.06
0.67
0.09
0.29
0.05
0.29
0.05
23.61
0.33
23.48
0.38
0.35
0.11
0.35
0.11
0.06
0.02
0.06
0.02
36.59
0.70
36.53
0.84
101.03
2.47
101.31
2.97
No Coating Area
Point
Area
Mean
SD
Mean
SD
Mean
SD
Mean
SD
7.26 40.89 40.67 0.63 0.68 0.30 0.32 22.67 22.86 0.25 0.26 0.06 0.06 35.08 35.32 107.08 100.11
1.20 3.15 2.98 0.09 0.16 0.04 0.07 0.46 0.71 0.04 0.04 0.02 0.02 1.21 1.58 4.37 4.78
7.22 40.49 40.14 0.69 0.70 0.29 0.30 22.72 22.75 0.26 0.26 0.06 0.06 35.31 35.25 106.98 99.40
2.61 3.15 3.13 0.06 0.06 0.03 0.03 0.49 0.48 0.05 0.05 0.01 0.01 1.24 1.25 4.05 4.53
5.70 40.72 40.51 0.70 0.71 0.30 0.30 23.00 23.01 0.27 0.27 0.08 0.08 35.21 35.14 105.90 99.95
0.91 1.07 1.06 0.05 0.06 0.03 0.03 0.59 0.58 0.05 0.05 0.03 0.03 0.92 0.92 2.87 2.45
6.00 41.49 41.30 0.75 0.75 0.33 0.33 22.94 22.99 0.27 0.27 0.09 0.09 34.93 34.88 106.70 100.52
1.91 1.25 1.20 0.06 0.06 0.04 0.04 0.71 0.69 0.05 0.05 0.03 0.03 1.34 1.30 3.26 3.00
*-C incl., values with C included in the analysis; -C excl., values when C is excluded from the analysis.
Statistical Analysis The results were put into a spreadsheet program ~Excel; Microsoft, Seattle, WA, USA! and descriptive statistics were calculated using Statgraphics Plus for Windows ~Statpoint Technologies, Inc., Warrenton, USA!.
R ESULTS A total of 105 measurements were made in the enamel of the four bovine teeth. In the gold-coated enamel 10 point and 10 area measurements were made; in the carbon coated 9 and 9 and in the uncoated enamel 12 and 12 measurements, respectively. Mean values and standard deviations
were calculated for the measurements for each group and the data are given in Table 3.
Surface Roughness of the Polished Specimens One of the epoxy-embedded bovine samples was cut into two halves after XRMA analysis. The sample was positioned in the sample holder for the SEM instrument with the cut surface facing upwards. By tilting the stage holder slightly both the polished and the cut enamel surface could be visualized. The polished surface appeared smooth within about 1 mm and some scratches about 2 mm deep were observed. The surface is thus regarded as satisfactory regarding flatness for XRMA analysis, although the lightest ele-
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Table 4. Mean Values of the Intensity Correction Used in INCA for Point and Area Analysis of C, O, Na, Mg, P, Cl, K, and Ca for Measurements in Bovine Enamel with Carbon Coating, Gold Coating, and Without Coating.* Carbon Coated Element C O - C incl. O - C excl. Na - C incl. Na - C excl. Mg - C incl. Mg - C excl. P - C incl. P - C excl. Cl - C incl. Cl - C excl. K - C incl. K - C excl. Ca - C incl. Ca - C excl.
Point
Area
0.854
0.866
0.764
0.764
0.727
0.726
1.006
1.005
0.761
0.761
1.233
1.230
0.993
0.993
Gold Coated
No Coating
Point
Area
Point
Area
0.256 0.779 0.793 0.727 0.714 0.701 0.691 0.986 0.985 0.737 0.731 1.228 1.235 0.967 0.969
0.256 0.775 0.790 0.728 0.715 0.701 0.691 0.986 0.985 0.737 0.731 1.226 1.234 0.967 0.969
0.333 0.950 0.965 0.783 0.772 0.737 0.729 1.006 1.004 0.765 0.761 1.229 1.238 0.992 0.993
0.335 0.959 0.976 0.782 0.771 0.736 0.727 1.004 1.003 0.765 0.760 1.226 1.234 0.991 0.993
*The apparent concentration is divided by these intensity corrections to produce the wt% values. Thus the closer to unity, the smaller the correction. -C incl., C included in the analysis; -C excl., C is excluded from the analysis.
ments such as C and O might be affected by surface irregularities.
Control of the Effect of Surface Charging (Figs. 2a–2f) In the profiles away from the gold coating, a slight decrease in wt% values from the gold-coated area into the uncoated enamel is seen for all elements and for the totals ~Figs. 2a– 2d!. However, Na surprisingly increased to reach a maximum of 1.5 wt% around 50 mm from the gold coating and then decreased to a slightly variable level around 1.3% from about 60 mm into the uncoated enamel ~Fig. 2a!. Cl also showed this feature to a lesser extent. Similar features were seen in the area analysis, with peaks about 90 mm from the gold ~Fig. 2c!. In both experiments the totals dropped at about 120 mm from the gold, probably reflecting electrostatic charging effects. In the 80 ⫻ 80 mm area measurements of bovine enamel performed between the 41 mm thick gold lines 140 mm apart, the variations of wt% for the different elements were less marked ~Figs. 2e, 2f!.
Point and Area Analysis of Carbon, Gold, and Uncoated Enamel
some details about the correction procedure. The apparent concentration of each element ~not shown! is divided by the intensity correction ~column 2!, to produce the true element wt% ~column 3!. Intensity corrections have three components. The Z or atomic number term models the proportion of electrons that give rise to characteristic X-rays; the absorption term models absorption of the X-rays in their path to leave the sample; the fluorescence term models the lower energy X-rays generated by X-rays moving through the sample, e.g., Oxygen Ka generated by P Ka. The statistical counting error calculated from the number of X-rays counted is also shown, as is the standard deviation of the mean of three replicate 400 s counts. Finally, the detection limits for 400 s live time counts are shown in the last column. The glass analyses are compared with area analyses of carbon, gold, and uncoated bovine specimens in Table 2 and Figure 4. The glass analyses were adjusted down to take the 9.6% measured loss on ignition into account. The glass analyses show good agreement between O by spectrum and by stoichiometry.
D ISCUSSION Area versus Point Analysis
Comparisons of Point and Area Analysis (Figs. 3a–3d) Only minor differences are seen between point and area analyses, however, the differences were more marked for Na. The mean values for area analysis were higher compared with point analysis, with the exception of K and Ca.
Area analyses gave slightly smaller standard errors in Figure 3 and Table 3, probably due to averaging out of aberrant points due to imperfections in the surface, which has a roughness of about 1 mm.
Glass Samples (Figs. 4a, 4b)
Looking at the question of electrostatic charging, the totals shown in Figures 2b and 2d suggest that there is no significant effect up to about 100 mm from the coated surface,
The full results from the chemical analysis of bovine enamel prepared as fused glass are presented in Table 1, including
Coating and Electrostatic Charging
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Figure 2. Wt% values for the X-ray microanalysis point and area measurement profiles in uncoated bovine enamel, starting in a gold-coated area and moving away. a: Values from point measurements for Na, Mg, Cl, K, and Au. b: Values from point measurements for O, P, Ca, and totals. c: Values from area measurement for Na, Mg, Cl, K, and Au. d: Values from area measurements for C, O, P, Ca, and totals. e: Area measurements between the gold lines in uncoated bovine enamel for Na, Mg, Cl, and K. f: Area measurements between the gold lines in uncoated bovine enamel for C, O, P, Ca, and totals.
after which the totals show a significant decrease, probably related to charging. The area analyses within gold lines shown in Figures 2e and 2f show constant Na, but area analyses are higher than points and results with other coatings. Thus there might be a small but constant Na enrichment artifact in the area analyses.
Coating or Not In general, uncoated analyses correspond well to coated ones. All three coating methods agree well for O and Ca.
Uncoated analyses gave slightly lower errors and higher amounts, probably more correct as there is no uncertainty of coating thickness. There seem to have been no problems with charging within about 120 mm of a conducting surface, which would give low values for O and other elements in uncoated samples. P gave similar concentrations within error for all coatings and point and area analyses were also similar. C on uncoated points and areas was about 1 wt% lower than on gold-coated ones, probably reflecting underestimation of the gold-coating thickness. Therefore, the uncoated analyses are to be preferred. The differences in
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Figure 3. Mean values and standard deviations for X-ray microanalysis measurements in bovine enamel in wt% for O, Na, Mg, P, Cl, K, and Ca in point and area analyses with carbon coating, gold coating, and uncoated. ~C-coat point ⫽ carbon coating and point analysis; C-coat area ⫽ carbon coating and area analysis; Au-coat point ⫽ gold coating and point analysis; Au-coat area ⫽ gold coating and area analysis; no-coat point ⫽ no coating and point analysis; no-coat area ⫽ no coating and area analysis!. a: Values for Na, Mg, Cl, and K with C excluded from the analysis. b: Values for O, P, and Ca with C excluded from the analysis. c: Values for Na, Mg, Cl, and K with C included in the analysis. d: Values for C, O, P, and Ca.
Figure 4. Mean values and standard deviations for X-ray microanalysis measurements in natural bovine enamel and bovine pure and fluxed fused glass in wt% for O, Na, Mg, P, Cl, K, and Ca in area analyses with carbon coating, gold coating, and uncoated specimens. C was excluded in the analyses. ~C-coat ⫽ carbon coating; Au-coat ⫽ gold coating; no-coat ⫽ no coating; P-glass ⫽ pure glass; F-glass ⫽ fluxed glass!. a: Values for Na, Mg, Cl, K, pure glass, and fluxed glass. b: Values for O, P, Ca, pure glass, and fluxed glass.
analyses of Na may also reflect uncertainties in coating thickness, which would affect the coated analyses. The thickness of the coating estimated for gold from reference graphs and for carbon from the interference color on a brass surface influences the intensity correction and
calculated wt% values. Since the exact thickness probably has an uncertainty of about 10% it is an uncontrollable source of error. Therefore uncoated specimens analyzed in VP-SEM mode are regarded as being most accurate, provided that electrostatic charging is avoided. Low-vacuum
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analysis at 20 Pa and with gold lines ~measuring ;140 mm between the lines! in the uncoated area is the method of choice.
Control of the Effect of Surface Charging In the experiment using spot analysis at different distances from a gold-coated area, the effect of electrostatic charging seems to become significant at a distance of about 120 mm, as seen from the decrease in totals in Figure 2b. Thus, a distance of ,120 mm from a conductive surface should avoid charging problems for spot analyses at 3.5 nA and 20 Pa pressure, whereas area analyses might be good at greater distances because the charge is spread out. A possible enrichment of Na and Cl was seen in the spot analyses, peaking about 50 mm from the gold coating, and about 100 mm in the area analysis. This might be an artifact created during the gold-coating procedure. In the profile using 12 ⫻ 12 mm area analyses on the same sample profile ~Figs. 2c, 2d!, the peaks were about 100 mm from the gold. However, in the 80 ⫻ 80 mm area analyses parallel to and between gold lines ~Figs. 2e, 2f!, no peaks are seen. Area analysis using a square area between 15 and 80 mm across appears better than point analysis due to better homogeneity and less potential for charging on the larger area. But how far from a coated surface should the area analysis be made? In the area analyses shown in Figures 2c and 2d, the value for the total wt% dropped off dramatically for the first 30 mm, than levelled out. Area analysis with a size between 15 and 80 mm appears better than point analysis due to less charging and more homogeneity. The best position is about 30 mm from gold, using area and not point analysis. The enrichment of Na seen near the gold coating is possibly an artifact created during the gold-coating procedure. The totals when C was included seem reasonable at around 97%. The strategy of coating with gold lines appeared favorable to extend the analyzable region and no gold contamination could be seen. Carbon appeared to vary quite a lot between samples ~Fig. 3d!.
Element by Element Discussion In this section we will discuss the significant aspects of each element in turn, in order of increasing atomic number, except for carbon, which is discussed last. In general the analyses including carbon will be discussed, unless otherwise specified. Oxygen Oxygen is traditionally estimated using stoichiometry in silicate and oxide minerals such as apatite, which are usually fully oxidized. This is because the exact wavelength and peak position of the oxygen K lines can vary considerably, a problem in wavelength-dispersive spectrometers ~WDS!. However, natural dental enamel clearly contains some organic material, which is not stoichiometrically combined with oxygen. Thus, calculating oxygen by stoichiometry is expected to give values that are too high.
EDS spectrometry has lower resolution than WDS and is more tolerant of small wavelength changes, thus quantification using the oxygen K X-ray series is feasible. A test of this is provided by the fused glass samples that had been ignited and had thus lost their nonstoichiometric components, so that the two results for oxygen should be the same. Oxygen wt% for the pure fused material is 41.18 wt% using the K X-ray series and 41.25 wt% by stoichiometry, thus the X-ray quantification seems reliable. A caveat is that the natural enamel surface and body is not as regular as the glass, which could affect the absorption correction and reduce the precision. Another alternative is to calculate “oxygen by difference” in which the total is made up to 100% with oxygen. This is the worst method for oxygen, but may give better values for the other elements if the totals by other methods deviate significantly from 100. Oxygen values shown in Table 3 and Figures 3b and 3d are hardly affected by inclusion or exclusion of carbon. The calculated intensity corrections shown in Table 4 change the apparent oxygen concentration in coated samples by 15 to 20%, whereas only by 3% in uncoated samples, illustrating the major effect of coating. The uncoated area analyses are about 1 wt% higher than the others, possibly reflecting oxygen in the surface layer of gas, which conducts away the electrostatic charge. This effect is minimized in the uncoated point analyses, which are thus considered the most reliable for oxygen. Sodium Area analyses of Na with no coating between gold lines are clearly higher than all other analyses and this is probably due to the sodium enrichment artifact close to gold coating seen in Figure 2a. Point analyses are generally about 0.05% lower than area analyses for Na. This is due either to inhomogeneity in the sample or more likely to heating of the spot during analysis, driving off Na and leading to low values. This phenomenon is common in point analyses of alkali feldspars, which are usually analyzed as areas. C and gold-coated areas agree for Na, are probably most reliable, and are least likely to have problems or artifacts, giving a value of about 0.70 wt%. Uncoated small area ~12 ⫻ 12 mm! analyses between gold lines for Na are probably good if artifacts can be avoided, although this needs further investigation. One way to evaluate artifacts is to compare with small area analyses within the adjacent gold lines. Magnesium and Phosphorous All methods seem comparable, but uncoated areas might have a small artifact near gold. Uncoated point analyses are considered good. There are no significant differences between the methods for phosphorous. Chlorine Chlorine seems high in both carbon-coated methods. The other methods give comparable results, but there is a possible artifact in uncoated analyses near gold coating ~Fig. 2a!. Uncoated point analysis seems good.
Evaluation of XRMA in Bovine Enamel
Potassium K is constant within error in all methods but the low concentrations around 0.06% are close to detection limits for 40 s counts. If K is considered significant, then a longer live time should be used, for example, 400 s giving a lower detection limit of 0.03 wt%, as shown in Table 1. Calcium There are no significant differences between the methods. Uncoated point analyses seem good. Carbon Analysis of C using the K X-ray series has similar problems to oxygen by WDS but is quantifiable by EDS, with caveats related to contamination and uncertainties in the absorption correction related to the heterogeneity of the natural enamel. Contamination may be caused by carbon-bearing material on the surface, or less likely by spectral contamination from other carbon-coated surfaces due to electron scatter in the low-vacuum chamber. Surface contamination is made worse by the correction procedure that incorrectly assumes that the carbon X-rays come from within the material ~Information on contamination from P. Statham, Oxford Instruments, personal communication with DHC 2013!. The measured C concentration is corrected by the software by a factor of about 3.5 ~reciprocal of intensity correction! due to absorption and other matrix effects. The accuracy of carbon analyses cannot be tested using the ignited fused glass samples. Carbon-coated samples can also not be used, thus only gold coated and uncoated analyses can be compared. The mean values in Table 3 show large standard deviations, surprisingly larger for area analyses than point analyses, which might point to surface contamination. The mean values for area and spot analyses agree for each coating, but differ between coatings, gold coating giving about 1.3 wt% higher carbon values than uncoated. This difference may be due to either surface contamination or to uncertainties in the thickness of gold coating, which has a significant effect on the intensity correction, about 0.6 wt% difference for a 10% change in gold thickness. Thus, the uncoated analyses are probably more correct. Area analyses should in principle be less subject to heterogeneity and the large standard deviation of about 2 wt% is probably a good estimate of uncertainty, which could be improved by longer counting times. Uncoated point analyses seem to be equally reliable.
S UMMARY Uncoated point analyses seem a good choice for all elements except Na, for which small area analyses should be used to avoid Na loss. Carbon analyses have the largest uncertainty because of the possibility of surface contamination, which we have not been able to evaluate. The 80 mm areas between gold lines used for this work might be affected by the gold-coating artifact we observed. A good compromise should be to analyze 10 mm 2 areas midway between 41 mm thick gold lines 140 mm apart. These could be combined
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with 10 mm 2 area analyses in the middle of the gold lines, which would provide a check for artifacts. Counting times should be chosen according to the detection limits required ~see Table 1 for 400 s live times!. Detection limits are proportional to the square root of the live time, thus 100 s live times would give a factor of two times worse detection limit than 400 s.
Comparison with Fused Glass The values for the different elements in pure fused glass are generally lower than the mean values for the bovine enamel in Table 2. The most marked differences are found for O, Na, P, and Cl. The corresponding values for the fluxed glass specimens are lower than for pure glass, however, no difference is seen between natural enamel and fluxed glass for Na and Cl whereas P remained at the same low value as for pure glass. The aim of using fused glass analyses was to provide “true” values for reference since there are no reference standards for dental enamel, which is not pure hydroxyapatite. The glass analyses gave totals close to 100% before correction for loss on ignition, thus representing good analyses for the glasses. However, there are a number of problems related to the fusion process of making enamel powder into glass. This can be seen from comparisons of the uncoated sample, taken as the best natural analysis, with pure fused glass and fluxed glass ~corrected for 9.7% loss on ignition! in Table 2. The values for O differ slightly, taking the 1–3% standard deviations of the natural analyses into account. The ignition and fusion process probably decreased O in the pure glass sample as water and CO2 were driven off. P values are as much as 5 wt% lower in the glass samples. This may reflect the loss of organically bound P, which was lost in the ignition and fusion process. The P content in dental enamel is found in the inorganic as well as the organic parts of the enamel. A loss of Na and Cl is also seen in pure glass that is probably related to the higher fusion temperature ~;1,6008C! compared to that for the fluxed glass ~;1,0008C!. The loss of Na, Cl, and predictably K at the higher fusion temperature can be explained by the fact that these elements are more easily vaporized than the other elements. The C-coated analyses of Cl are high and had a higher error, which might reflect concentration of this most mobile element Cl at the surface during carbon coating, as the carbon string radiates heat. Concerning Ca and Mg, all coatings corresponded well with each other and also with the glass analysis. It is concluded that the glass analyses are not directly comparable with those of natural material, but they do provided insights into both the nature of the tooth material and the glass making process.
Totals and Matrix Effects As shown in Table 3, the wt% totals excluding carbon are close to 100% for all methods. For normal inorganic mineral analyses totals within 2 wt% of 100 are regarded as
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acceptable, taking possible crystalline water into account. As shown in Table 3, the totals including carbon are between 105 and 107%, with standard deviations of 2.9 to 4.3 wt%. The high totals and variability of our natural dentine analyses may be ascribed to carbon contamination and other matrix effects. These may include surface roughness, porosity, and inhomogeneity of the hydroxyapatite structure and organic constituents. The light elements C and O are most likely to be affected by matrix effects because of their low energy and long X-ray wavelength. Including C in the analytical menu slightly increased the elements lighter than Mg and decreased those with higher atomic numbers. This is probably due to changes in the fluorescence correction, which may not be correct if some of the carbon analysis is due to surface or spectral contamination. Unless the effects of carbon contamination can be evaluated, analyses without carbon should be regarded as most reliable and carbon analyses should be regarded as semiquantitative. Another factor that may influence results in XRMA analysis of enamel is mineral density in the tissue, which varies between different locations in the enamel and between hypomineralized and normal enamel, mineral hydroxyapatite has a density of 3.16 whereas tooth enamel varies between 3 and 2.84 ~He et al., 2011!. In the present study only the bulk of the enamel was analyzed as the surface layer of the enamel had been ground away. Material with porosity or lower density than solid minerals should generally give lower analytical values ~P. Statham, Oxford Instruments, personal communication with DHC 2013!.
Other Enamel Analyses in the Literature Quantitative studies of the elemental composition of dental enamel are scarce and commonly the measurements are regarded as semi-quantitative or presented as ratios to the Ca content ~Retief et al., 1970, 1971; Shaw & Yen, 1972!. Analyses of human enamel using our method and by neutron activation and EDS ~Retief et al., 1970, 1971; De Menezes Oliveira et al., 2010! shown in Table 2 show values comparable with the bovine enamel results of this study. Other studies of human enamel by X-ray EDS show large deviations from our results and do not describe their methods or discuss their accuracy in any detail ~GotierrezSalazar & Reyes-Gasga, 2003; Mahoney et al., 2004!.
C ONCLUDING R EMARKS Small area ~10 ⫻ 10 mm 2 square! analyses without any coating and midway between with 40 mm gold lines 140 mm apart are preferred, especially if C is included in the analysis. The VP-SEM mode with 20 Pa low vacuum used for calibration and EDS analysis with a stable 3.5 nA specimen current is appropriate for our instrument. Our analyses of bovine enamel are sufficiently reproducible to be regarded as quantitative for all elements except C, for which the potential problem of contamination could not be assessed. There are also probably some unexplained matrix effects
that lead to high totals and suggest that some elements may have systematic errors in the order of 5% relative.
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