Calcified Tissue Research
Calcif. Tiss. Res. 24, 249-251 (1977)
9 by Springer-Verlag 1977
Luminescence Quantum Yields of Sound and Carious Dental Enamel D. Spitzer* and J.J. ten Bosch Laboratory for Materia Technica, University of Groningen, Antillenstraat 11, Groningen, The Netherlands
Summary. The absorption and emission spectra o f slabs o f human and bovine dental enamel were determined. The absorption and scattering coefficients and emission quantum yields were c o m p u t e d according to theoretical models. The samples were gradually demineralized. The absorption, scattering, and emission parameters were determined as a function o f the demineralization time. Using the theoretical models combined with the experimental values, ratio o f the visible and UV luminescence, and the decrease o f visible emission intensity upon demineralization are explained. Key words: Luminescence - - Enamel - - Caries
Introduction In previous papers [11, 12] optical absorption and luminescence phenomena in sound enamel were described. Since dental tissues are turbid (light scattering) materials, special models must be used to quantify these phenomena. F o r the absorption, an extended twoflux model was employed [11]. To determine emission quantum yields, a new model was developed [13] which enables quantitative measurements o f carious dental enamel to be c o m p a r e d with measurements on sound enamel. The theory o f optical absorption and scattering in turbid materials was treated in a previous paper [11]. F o r the luminescence effects in these materials, a fourflux model was developed [13]. F r o m the known refractive index, absorption and scattering coefficients, and emission spectra, the luminescence quantum yields can be computed with Equation 13 [13].
Send offprint requests to J.J. ten Bosch at the above address * Present address: N.I.O.Z., 't Horntje, Texel, The Netherlands
Materials and Methods Plane parallel slabs of sound mature enamel of freshly extracted incisors (eight bovine and four human samples) were prepared as described previously [11]. For demineralization 0.05 M acetate buffer at pH 4.38 [7] was used, because of its low optical absorption in the wavelength range of investigation. The slabs remained in the solution for different periods varying from 8 to 108 h. Thereafter, samples became fragile and no further measurements were possible. Before measurement, the samples were carefully rinsed in distilled water for about 1 h and then dried superficially. Transmission and reflection measurements were carried out in the range 220-250 nm. From these, the true absorption spectra and scattering coefficients were computed [11]. When the absorption to scattering ratio is not small 'enough, a correction is necessary [13]. In this work this situation occurs. We applied this correction. Luminescence measurements were carried out in the range 250-600 nm [12]. True emission spectra, i.e., spectra corrected for scattering, were computed [12]. For each excitation maximum, the complete emission spectrum was integrated for a computation of the quantum yield [13]. No component analysis [9] was performed because of the inaccuracy of the computed spectral data. Reabsorption was estimated at less than 2% and, thus, neglected. Eventual changes [8] in the refractive index during demineralization were also neglected, causing an error of less than 8%.
Experimental Results A. Absorption and Scattering Substantial individual differences in the demineralization rate and, hence, in the measured p a r a m e t e r s of different samples were observed. The transmittance of the samples decreased rapidly upon demineralization, while the reflectance increased. The shape of the absorption spectra did not change substantially upon demineralization. Because of the large errors in the calculated absorption coefficient (~> 40%), originating in the transmittance measurements, no change of absorption coefficient was deduced. As expected, the scattering coefficient, as calculated from transmittance and reflectance, increased with the
250
D. Spitzer and J.J. ten Bosch: Luminescence Quantum Yields of Sound and Carious Dental Enamel
[]
quantum
decad ic scattering coefficient (cm-1)
emission (arb. units) 0.4
yield
_•
1000
0.1
[] ext. 285nm
_
exc. 330 nm c 37Onto
o
500
/
I
demineralization fi me (hours) I
36 e xc.285 m-"~'-, n em.350 nm
0
108
Fig. 2. Quantum yields as function of the demineralization time. Lines are drawn for the quantum yields of the same sample as used in Fig. 1. The excitation wavelengths are indicated. For excitation at 330 nm, values of quantum yields of two other samples are included
exc 330nm,em.410nm~ demineralization time (hours)
36
72
108
Fig. 1. The dashed line represents the increase of the scattering coefficient at 340 nm and refers to the right-hand ordinate. The full lines and the left-hand ordinate show the maxima of the observed emission as function of the demineralization time. Excitation and emission wavelengths are indicated for each line. Estimated errors are indicated
period of treatment in the buffer solution. A n example is shown in Figure 1, A m a x i m u m always developed upon demineralization at about 340 nm. A t 500 rim, the coefficient equals about 40% of the m a x i m u m value [141.
B. Luminescence The shape of the emission and excitation spectra of the enamel [12] did not change upon demineralization. All three observed emission m a x i m a decreased as shown in Figure 1. The q u a n t u m yields o f sound enamel are shown in Table 1. These values decreased upon
demineralization for three samples as shown in Figure 2. The estimated error of the q u a n t u m yield measurements on one sample is about 60%.
Discussion A. Sound Enamel The absorption spectra of the h u m a n enamel samples appeared to agree with spectra previously published [11], when those were also corrected according to reference [13]. However, the absorption m a x i m a of the previously measured bovine samples were about twice as high as those determined for this paper. W e ascribe this discrepancy to biological variations. The emission around 350 nm is ascribed to t r y p t o p h a n [12], and the quantum yields of the 350 nm emission m a y be c o m p a r e d with those of class B proteins [4, 6]. It is also possible that tyrosine absorption causes the t r y p t o p h a n emission. Assuming that enamel contains about 0.6% protein with a concentration of about 60 tyrosine residues per 1000 total residues 110], one can estimate from the true absorption spectra that
Table 1. The luminescence quantum yields of the enamel
Bovine enamel Human enamel
72
Excitation at 285 nm
Excitation at 330 nm
Excitation at 370 nm
0.01-0.05 0.02-0.08
0.01-0.06 0.01-0.03
0.04-0.12 0.01-0.04
D. Spitzer and J.J. ten Bosch: Luminescence Quantum Yields of Sound and Carious Dental Enamel
approximately half of the absorption at 275 nm is caused by tyrosine. The quantum yield of the tryptophan emission in enamel would then be about 0.1, which is a value common for many proteins [5, 6]. On the quantum yields of the visible luminescence of proteins, no review appears available. The values presented here for these emissions in enamel are generally lower than expected from the previous qualitative measurements [ 12]. Present calculations indicate that scattering is the most important factor for the ratio of observed tryptophan and visible effects in the enamel. Since the scattering is lower at longer wavelengths, visible luminescence becomes more significant compared to the UV luminescence of the aromatic amino acids. It follows from the computations of the quantum yields, that if there were no scattering, the observed luminescence maxima would be much lower than the tryptophan luminescence.
B. Carious Enamel
The absorption measurements are not accurate enough to allow conclusions on the loss of protein material. Changes of the scattering coefficient are more significant than changes of the absorption coefficient and are caused by the disintegration of the crystalline structure of the tissue. This means the "white spot" is due to high reflectance caused by high scattering and to the associated low transmittance, which removes the optical influence of the underlying layers. All emission peaks decreased after the demineralization (Fig. 1). This feature was observed by several authors [1, 2, 3] who noted a decrease of visible luminescence in the carious areas of the teeth. This appears to be in disagreement with the previous hypothesis about the predominant role of the organic component in the luminescence of enamel [3, 12]. However, it now appears that the change of quantum yield upon demineralization is an important factor causing decrease of the observed luminescence. The change indicates changes of the organic component upon demineralization. These may be caused by changes in quenching by, e.g., histidyl or cystinyl
251
groups [6] or to conformational changes as recently discussed by Robinson et al. [10]. Acknowledgement. The authors are most indebted to Mr. W.J. van der Veen for his experimental work and to Mr. J.A.D. Schuthof for the preparation of the samples.
References 1. Cooley, R.Q., Hefferren, J.J., Campagne, P.L.: Detectability of dental caries by conventional and ultraviolet-assisted clinical examinations. IADR Abstracts 1974, p. 162 2. Forziati, A.F., Kumpula, J.W., Barone, J.J.: Tooth fluorometer. J.A.D.A. 67, 663-669 (1963) 3. Hefferren, J.J., Cooley, R.Q., Hall, J.B., Olsen, N.H., Lyon, H.W.: Use of ultraviolet illumination in oral diagnosis. J.A.D.A. 82, 1353-1366 (1971) 4. Kronman, M.J., Holmes, L.G.: The fluorescence of native, denatured and reduced - - denatured - - proteins. Photochem. Photobiol. 14, 113-134 (1971) 5. Lehrer, S.S., Fasman, G.D.: Ultraviolet irradiation effects in poly-L-tyrosine and model compounds. Identification of bityrosine as a photoproduct. Biochem. 6, 757-767 (1967) 6. Longworth, J.W.: Luminescence of polypeptides and proteins. In: Excited states of proteins and nucleic acids (Steiner, R.F., Weinryb, I., eds.), pp. 319-474. New York: Plenum Press 1971 7. Moreno, E.C., Zahradnik, R.T.: Chemistry of enamel subsurface demineralization in vitro. J. Dent. Res. Supplement to No. 2 53, 226-235 (1974) 8. Myrberg, N.E.A., Davidson, C.L.: Human Enamel and Polarized Light Microscopy. Abstracts of Res. Rep., Eleventh Annual Meeting of I.A.D.R., C.E.D., Brussels 1974, p. 50 9. Pesce, A.J., Rosen, C.G., Pasby, T.L.: Fluorescence spectroscopy, pp. 182-185. New York: Marcel Dekker 1971 10. Robinson, C., Lowe, N.R., Weatherelt, J.A.: Amino acid composition, distribution and origin of "Tuft" protein in human and bovine dental enamel. Archs. Oral Biol. 20, 29-42 (1975) 11. Spitzer, D., ten Bosch, J.J.: The absorption and scattering of fight in bovine and human dental enamel. Calcif. Tissue Res. 17, 129-137 (1975) 12. Spitzer, D., ten Bosch, J.J.: The total luminescence of bovine and human dental enamel. Calcif. Tissue Res. 20, 201-208 (1976) 13. Spitzer, D., ten Bosch, J.J.: Luminescence of turbid materials. A theoretical model and its comparison with experiment. Appl. Opt. 15, 934-939 (1976) 14. Spitzer, D., Optical properties of turbid materials with reference to dental enamel. Thesis Groningen 1976
ReceivedJanuary 21,1976/AeceptedMareh 24, 1977
Calcif. Tiss. Res. 24, 249-251 (1977)
9 by Springer-Verlag 1977
Luminescence Quantum Yields of Sound and Carious Dental Enamel D. Spitzer* and J.J. ten Bosch Laboratory for Materia Technica, University of Groningen, Antillenstraat 11, Groningen, The Netherlands
Summary. The absorption and emission spectra o f slabs o f human and bovine dental enamel were determined. The absorption and scattering coefficients and emission quantum yields were c o m p u t e d according to theoretical models. The samples were gradually demineralized. The absorption, scattering, and emission parameters were determined as a function o f the demineralization time. Using the theoretical models combined with the experimental values, ratio o f the visible and UV luminescence, and the decrease o f visible emission intensity upon demineralization are explained. Key words: Luminescence - - Enamel - - Caries
Introduction In previous papers [11, 12] optical absorption and luminescence phenomena in sound enamel were described. Since dental tissues are turbid (light scattering) materials, special models must be used to quantify these phenomena. F o r the absorption, an extended twoflux model was employed [11]. To determine emission quantum yields, a new model was developed [13] which enables quantitative measurements o f carious dental enamel to be c o m p a r e d with measurements on sound enamel. The theory o f optical absorption and scattering in turbid materials was treated in a previous paper [11]. F o r the luminescence effects in these materials, a fourflux model was developed [13]. F r o m the known refractive index, absorption and scattering coefficients, and emission spectra, the luminescence quantum yields can be computed with Equation 13 [13].
Send offprint requests to J.J. ten Bosch at the above address * Present address: N.I.O.Z., 't Horntje, Texel, The Netherlands
Materials and Methods Plane parallel slabs of sound mature enamel of freshly extracted incisors (eight bovine and four human samples) were prepared as described previously [11]. For demineralization 0.05 M acetate buffer at pH 4.38 [7] was used, because of its low optical absorption in the wavelength range of investigation. The slabs remained in the solution for different periods varying from 8 to 108 h. Thereafter, samples became fragile and no further measurements were possible. Before measurement, the samples were carefully rinsed in distilled water for about 1 h and then dried superficially. Transmission and reflection measurements were carried out in the range 220-250 nm. From these, the true absorption spectra and scattering coefficients were computed [11]. When the absorption to scattering ratio is not small 'enough, a correction is necessary [13]. In this work this situation occurs. We applied this correction. Luminescence measurements were carried out in the range 250-600 nm [12]. True emission spectra, i.e., spectra corrected for scattering, were computed [12]. For each excitation maximum, the complete emission spectrum was integrated for a computation of the quantum yield [13]. No component analysis [9] was performed because of the inaccuracy of the computed spectral data. Reabsorption was estimated at less than 2% and, thus, neglected. Eventual changes [8] in the refractive index during demineralization were also neglected, causing an error of less than 8%.
Experimental Results A. Absorption and Scattering Substantial individual differences in the demineralization rate and, hence, in the measured p a r a m e t e r s of different samples were observed. The transmittance of the samples decreased rapidly upon demineralization, while the reflectance increased. The shape of the absorption spectra did not change substantially upon demineralization. Because of the large errors in the calculated absorption coefficient (~> 40%), originating in the transmittance measurements, no change of absorption coefficient was deduced. As expected, the scattering coefficient, as calculated from transmittance and reflectance, increased with the
250
D. Spitzer and J.J. ten Bosch: Luminescence Quantum Yields of Sound and Carious Dental Enamel
[]
quantum
decad ic scattering coefficient (cm-1)
emission (arb. units) 0.4
yield
_•
1000
0.1
[] ext. 285nm
_
exc. 330 nm c 37Onto
o
500
/
I
demineralization fi me (hours) I
36 e xc.285 m-"~'-, n em.350 nm
0
108
Fig. 2. Quantum yields as function of the demineralization time. Lines are drawn for the quantum yields of the same sample as used in Fig. 1. The excitation wavelengths are indicated. For excitation at 330 nm, values of quantum yields of two other samples are included
exc 330nm,em.410nm~ demineralization time (hours)
36
72
108
Fig. 1. The dashed line represents the increase of the scattering coefficient at 340 nm and refers to the right-hand ordinate. The full lines and the left-hand ordinate show the maxima of the observed emission as function of the demineralization time. Excitation and emission wavelengths are indicated for each line. Estimated errors are indicated
period of treatment in the buffer solution. A n example is shown in Figure 1, A m a x i m u m always developed upon demineralization at about 340 nm. A t 500 rim, the coefficient equals about 40% of the m a x i m u m value [141.
B. Luminescence The shape of the emission and excitation spectra of the enamel [12] did not change upon demineralization. All three observed emission m a x i m a decreased as shown in Figure 1. The q u a n t u m yields o f sound enamel are shown in Table 1. These values decreased upon
demineralization for three samples as shown in Figure 2. The estimated error of the q u a n t u m yield measurements on one sample is about 60%.
Discussion A. Sound Enamel The absorption spectra of the h u m a n enamel samples appeared to agree with spectra previously published [11], when those were also corrected according to reference [13]. However, the absorption m a x i m a of the previously measured bovine samples were about twice as high as those determined for this paper. W e ascribe this discrepancy to biological variations. The emission around 350 nm is ascribed to t r y p t o p h a n [12], and the quantum yields of the 350 nm emission m a y be c o m p a r e d with those of class B proteins [4, 6]. It is also possible that tyrosine absorption causes the t r y p t o p h a n emission. Assuming that enamel contains about 0.6% protein with a concentration of about 60 tyrosine residues per 1000 total residues 110], one can estimate from the true absorption spectra that
Table 1. The luminescence quantum yields of the enamel
Bovine enamel Human enamel
72
Excitation at 285 nm
Excitation at 330 nm
Excitation at 370 nm
0.01-0.05 0.02-0.08
0.01-0.06 0.01-0.03
0.04-0.12 0.01-0.04
D. Spitzer and J.J. ten Bosch: Luminescence Quantum Yields of Sound and Carious Dental Enamel
approximately half of the absorption at 275 nm is caused by tyrosine. The quantum yield of the tryptophan emission in enamel would then be about 0.1, which is a value common for many proteins [5, 6]. On the quantum yields of the visible luminescence of proteins, no review appears available. The values presented here for these emissions in enamel are generally lower than expected from the previous qualitative measurements [ 12]. Present calculations indicate that scattering is the most important factor for the ratio of observed tryptophan and visible effects in the enamel. Since the scattering is lower at longer wavelengths, visible luminescence becomes more significant compared to the UV luminescence of the aromatic amino acids. It follows from the computations of the quantum yields, that if there were no scattering, the observed luminescence maxima would be much lower than the tryptophan luminescence.
B. Carious Enamel
The absorption measurements are not accurate enough to allow conclusions on the loss of protein material. Changes of the scattering coefficient are more significant than changes of the absorption coefficient and are caused by the disintegration of the crystalline structure of the tissue. This means the "white spot" is due to high reflectance caused by high scattering and to the associated low transmittance, which removes the optical influence of the underlying layers. All emission peaks decreased after the demineralization (Fig. 1). This feature was observed by several authors [1, 2, 3] who noted a decrease of visible luminescence in the carious areas of the teeth. This appears to be in disagreement with the previous hypothesis about the predominant role of the organic component in the luminescence of enamel [3, 12]. However, it now appears that the change of quantum yield upon demineralization is an important factor causing decrease of the observed luminescence. The change indicates changes of the organic component upon demineralization. These may be caused by changes in quenching by, e.g., histidyl or cystinyl
251
groups [6] or to conformational changes as recently discussed by Robinson et al. [10]. Acknowledgement. The authors are most indebted to Mr. W.J. van der Veen for his experimental work and to Mr. J.A.D. Schuthof for the preparation of the samples.
References 1. Cooley, R.Q., Hefferren, J.J., Campagne, P.L.: Detectability of dental caries by conventional and ultraviolet-assisted clinical examinations. IADR Abstracts 1974, p. 162 2. Forziati, A.F., Kumpula, J.W., Barone, J.J.: Tooth fluorometer. J.A.D.A. 67, 663-669 (1963) 3. Hefferren, J.J., Cooley, R.Q., Hall, J.B., Olsen, N.H., Lyon, H.W.: Use of ultraviolet illumination in oral diagnosis. J.A.D.A. 82, 1353-1366 (1971) 4. Kronman, M.J., Holmes, L.G.: The fluorescence of native, denatured and reduced - - denatured - - proteins. Photochem. Photobiol. 14, 113-134 (1971) 5. Lehrer, S.S., Fasman, G.D.: Ultraviolet irradiation effects in poly-L-tyrosine and model compounds. Identification of bityrosine as a photoproduct. Biochem. 6, 757-767 (1967) 6. Longworth, J.W.: Luminescence of polypeptides and proteins. In: Excited states of proteins and nucleic acids (Steiner, R.F., Weinryb, I., eds.), pp. 319-474. New York: Plenum Press 1971 7. Moreno, E.C., Zahradnik, R.T.: Chemistry of enamel subsurface demineralization in vitro. J. Dent. Res. Supplement to No. 2 53, 226-235 (1974) 8. Myrberg, N.E.A., Davidson, C.L.: Human Enamel and Polarized Light Microscopy. Abstracts of Res. Rep., Eleventh Annual Meeting of I.A.D.R., C.E.D., Brussels 1974, p. 50 9. Pesce, A.J., Rosen, C.G., Pasby, T.L.: Fluorescence spectroscopy, pp. 182-185. New York: Marcel Dekker 1971 10. Robinson, C., Lowe, N.R., Weatherelt, J.A.: Amino acid composition, distribution and origin of "Tuft" protein in human and bovine dental enamel. Archs. Oral Biol. 20, 29-42 (1975) 11. Spitzer, D., ten Bosch, J.J.: The absorption and scattering of fight in bovine and human dental enamel. Calcif. Tissue Res. 17, 129-137 (1975) 12. Spitzer, D., ten Bosch, J.J.: The total luminescence of bovine and human dental enamel. Calcif. Tissue Res. 20, 201-208 (1976) 13. Spitzer, D., ten Bosch, J.J.: Luminescence of turbid materials. A theoretical model and its comparison with experiment. Appl. Opt. 15, 934-939 (1976) 14. Spitzer, D., Optical properties of turbid materials with reference to dental enamel. Thesis Groningen 1976
ReceivedJanuary 21,1976/AeceptedMareh 24, 1977
