Journal of Dentistry (2005) 33, 7378

www.intl.elsevierhealth.com/journals/jden

A preliminary investigation of a spectroscopic technique for the diagnosis of natural caries lesions
Adriana Ribeiroa, Christel Rousseaub, John Girkinb, Andrew Hallc,*, Ronald Strangc, C. John Whittersc, Stephen Creanorc, Anderson S.L. Gomesd
Departmento de Endodontia, Universidade de Sao Paulo, Sao Paulo, Brazil Institute of Photonics, University of Strathclyde, Glasgow, UK c Glasgow Dental Hospital and School, University of Glasgow, 378 Sauchiehall Street, Glasgow G2 3JZ, Scotland, UK d Department of Physics, UFPE, Recife, Brazil
b a

Received 12 February 2004; received in revised form 30 July 2004; accepted 10 August 2004

KEYWORDS
Spectroscopy; Caries; Teeth; Laser; Diagnosis

Summary Objective. To report the use of spectroscopic analysis of dental uorescence excited with a blue InGaN laser diode operating at 405 nm. Method. The spectra resulting from three classications of smooth surface noncavitated caries lesions (dull, shiny, brown) with 20 samples in each group were examined using the ratio of integrated uorescence intensity in two spectral bands. Results. All lesions demonstrated spectra which were signicantly different from sound tooth structure. As expected, the brown lesions demonstrated a signicantly different spectral prole from the two white spot lesion classications. Dull and shiny lesions had signicantly different spectral measurements when examining the ratio of the integrated uorescence in spectral bands between 480500 and 620640 nm. Conclusion. This method has application for detection of dental caries as well as demonstrating potential application to evaluate lesions which may represent different degrees of caries activity. q 2004 Elsevier Ltd. All rights reserved.

Introduction
Recent discussions about caries detection and diagnosis have stressed that diagnosis is a multicomponent event encompassing the detection of

* Corresponding author. Tel.: C44 141 211 9778; fax: C44 141 331 2798. E-mail address: a.hall@dental.gla.ac.uk (A. Hall).

caries, assessment of activity and informed prescription of suitable treatment.1 While many papers have been published on the subject of caries detection there is, by comparison, less information available about diagnosis. In vivo, the clinical appearance of lesion development has been described by Backer-Dirks2 and von der Fehr.3 However, recently it has been suggested that descriptions of the visual appearance of smooth surface caries may be indicative of lesion activity.46

0300-5712/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jdent.2004.08.006

74 The features of naturally occurring caries are particularly relevant 5,6 Specically, an active smooth surface enamel lesion with an intact surface may present as being, whitish/yellowish opaque with loss of luster. Conversely, an inactive smooth surface enamel lesion with an intact surface may present as being, shiny and feels hard and smooth when the tip of the probe is moved gently across the surface. A proliferation of optical methods for detection and monitoring of dental caries has been seen for the past 20 years. These optical methods have included measurement of light scattering7 and the use of uorescence in combination with various ltering systems.8 Fluorescence has been used to observe tooth structure for the past 75 years.9 However, Alfano and Yao10 were among the rst to use uorescence to differentiate between sound and carious tooth structure using excitation wavelengths between 400 and 700 nm. Additional work by Sundstrom et al.11 also demonstrated differences between sound and carious tooth structure using excitation wavelengths of 337633 nm. This work led to the establishment of a quantitative relationship between gross scattering of uorescent light and mineral loss,12 from which the technique of Quantitative Light Fluorescence (QLF) was developed.13 Similarly, Hibst and Gall14 described the development of a diode laser-based caries detector operating at 655 nm. This detector was subsequently developed and clinically evaluated as the Diagnodent.15 The two systems operate on different principles. The QLF system determines the relative loss of uorescence within a carious lesion due to increased scatter, whereas the Diagnodent measures the level/quantity of uorescence from the carious lesion due to the presence of bacterial by-products such as porphyrins. The readings from both systems may be inuenced by the presence of plaque, calculus and agents which cause tooth staining. Much of the above work has been based on earlier studies to ascertain the optical properties of tooth structure. Among these studies, several workers have used spectroscopy to describe the spectral properties of tooth structure at different excitation wavelengths.1618 To the authors knowledge, spectroscopic analysis has only been applied to natural caries lesions in a very limited capacity. Both QLF and the Diagnodent compare the relative uorescence of sound and test tooth structure over a narrow range of wavelengths to detect dental caries. Evaluations of dental caries in this manner may also be used to determine lesion activity.19 The aim of this study was to select extracted teeth with smooth surface, non-cavitated natural

A. Ribeiro et al. caries lesions based on their visual appearance, and ascertain if the spectroscopic analysis of specic wavelength ranges could be related to clinical visual evaluation of the lesion.

Method and materials

Dental specimen selection
An experienced dental practitioner (SC) examined a large selection of extracted human teeth collected at Glasgow Dental Hospital. All of the teeth had been stored in a saturated solution of thymol (0.12%). The clinical history of each sample was unknown. Using clinical judgement, 20 samples of dull, shiny and brown, non-cavitated caries lesions were selected using the criteria outlined by Thylstrup et al.4 and Nyvad et al.5 A total of 60 lesions were therefore evaluated. These samples were then randomly assigned an identication mark, which was subsequently used as the reference for each experiment.

Optical system
Initial optical system In a preliminary evaluation, spectra from the 60 teeth which were excited by a 405 nm laser diode permitted plots to be made of normalised average spectra for each of the lesion types described.20 Normalisation was undertaken by rst subtracting from each spectral point the average (background) uorescence within the range 740750 nm. Each point was then normalised such that the maximum uorescence value was a nominal one unit by dividing the uorescence at each point by the maximum uorescence value. Comparison of these average spectra was undertaken to determine if and where optical spectral differences between the three lesion types may occur. This allowed us to direct efforts for further analysis of un-normalised data from individual teeth to specic spectral regions. The spectral regions of interest were 480500 and 620640 nm. Our optical system was then improved to permit easier, more repeatable and rapid acquisition of accurate spectral data. Improved optical system A diagram for the optical arrangement of the probe used is shown in Fig. 1. The light from a 30 mW blue, 405 nm, InGaN blue laser diode (Nichia, Inc., Japan) was collected and then imaged into a 1 mm, 0.37 NA optical bre using a fZ4.5 mm, 0.45 NA lens (Olympus, Inc., Japan). The bre formed part of

Spectroscopy for natural caries

75 the later analysis and were therefore ignored. We were thus certain that the origin of the uorescence we were examining was from the dental tissue under investigation. All the spectra were later analysed using computer software (Origin, Origin Lab. Corp., USA, Version 7) to determine the area under the un-normalised spectral curve in both the 480500 and 620640 nm spectral regions.

Experimental procedure
Prior to conducting spectroscopic tests on natural lesions, tests were made to determine the laser conditions used did not photo-bleach the specimens and also that there were no saturation effects resulting from the laser excitation. This was done by varying the laser power and the time over which the light was collected (integration time). Initially, ve samples of each lesion type were selected at random and spectra recorded at four power levels (0.25, 0.75, 1.0 and 1.25 mW) and ve integration times (2, 4, 6, 8 and 10 s). Each lesion was on a separate specimen. Each spectral prole was recorded three times and then the average prole used for subsequent comparisons. Averaging of spectral proles was undertaken in real time using the integrated Avantes control software. Measurements with different powers (0.25 1.25 mW) and different integration times (210 s) did not show any inuence on the uorescence ratios calculated by comparing the uorescence intensities in the 480500 and 620640 nm regions. Thus, the data demonstrated no saturation or bleaching effects using the blue laser diode as the excitation source. For ease of data smoothing, and to prevent very long integration times, a power at the sample of 1 mW and a 4 s integration time were chosen as these parameters generated, clean spectra, with no risk of saturation in the spectrometer. Using the selected laser conditions (1 mW, 4 s integration time) spectra from 60 natural caries lesions were recorded. All specimens, in each of the three groups (dull, brown and shiny), were stored in a humid environment. Prior to collection of spectra, specimens were each dried for 10 s using compressed air. This was slightly longer that some clinical protocols which have been advocated for detection of caries lesions.5 Drying of lesions will exchange uid within the lesion for air. Air has a lower refractive index than dental tissue and water and will therefore increase the light scattering. This may change the observed uorescence due to a slight change in the level of scattered excitation light in the vicinity of the uorophore. An increase

Figure 1 A schematic diagram of the apparatus used to collect the spectral data.

the handpiece from a Diagnodent caries detection monitor (KaVo, GmbH., Germany). The bre had previously been disconnected from Diagnodent control box to expose the input end of the excitation bre. The A tip was tted to the end of the handpiece. The A tip is one of the interchangeable tips used for the end of the Diagodent handpiece. It is the smaller of two tips available and was considered most suitable for analysis of small caries lesions. Light output from the tip was measured using a photodiode power meter (Melles Griot, Inc., USA) to be 1 mW. The light was then directed onto the sample under investigation and the resulting uorescence collected back through the A-tip and handpiece via the Diagnodent collection bre bundle (six, 400 mm bres arranged around the central exciting bre). The collected uorescent light was then sent into the spectrometer by butt-coupling the bre optic input of the spectrometer to the Diagnodent collection bre bundle. The spectra were taken using an Avantes single grating spectrometer (Avantes, Inc., USA) and the results recorded on a PC. This type of spectrometer is a non-scanning spectrometer. Incoming light is directed onto a xed diffraction grating and the resultant spectra recorded directly onto a linear photodiode array. The integration time is the time taken to record a complete spectrum on the array. The spectral range was 350950 nm with a resolution of 1 nm. Initially, the probe was directed into an empty sample holder in a darkened room and a background spectrum recorded. In addition, the probe was directed onto a sample of Barium Sulphate (Macam Instruments Ltd, Livingston UK) in order to determine if any uorescence was excited within the probes optical bres by the 405 nm excitation source. Some uorescence was seen below 460 nm and several peaks were noticed. These peaks were outside the spectral region of interest used for

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Figure 2 A schematic diagram to illustrate the method by which the spectral ratio for demineralised tissue was determined. A similar calculation was performed for the sound tooth tissue spectrum. These calculations were performed for all teeth in the study.

in scattering increases the probability of photon absorption and uorescence, thereby, increasing the uorescence yield by the ratio of the refractive index of air divided by water when the lesion geometry is constant. Spectra were recorded from an adjacent region of sound enamel and the caries lesion. Spectroscopy data were collected from 480 to 780 nm and imported into Excel (Microsoft Corp., USA). No normalisation was undertaken as all of the

evaluations were based upon a ratio of the area under the two wavelength regions of interest (480 500 and 620640 nm) under one and the same curve (Fig. 2). This method was adopted in an attempt to prevent noise on the data from affecting the results due to spurious divisions between a noise peak in one curve and a trough on another. The spectral intensity values in the wavelength regions of interest were then imported into a suitable computer program (Origin). The areas under the curve were obtained by

Figure 3 Typical spectra from sound and demineralised tissue of extracted teeth previously evaluated as having shiny (a) or dull (b) white spot lesions or a brown (c) spot lesion.

Spectroscopy for natural caries

Table 1 Table of mean (SD) spectral ratio values for sound tooth and lesion for each of the three groups. Group (nZ20 per group) Dull Shiny Brown Sound tooth mean spectral ratio (SD) 13.3 (1.2) 14.0 (1.8) 10.7 (1.4) Lesion mean spectral ratio (SD) 7.0 (0.9) 8.9 (1.1) 2.4 (0.5)

77 at the 5% probability level. The 95% condence intervals for these differences are shown in Table 2.

Discussion
It has been demonstrated that sound and carious areas on dental tissue can be discriminated through the use of uorescence excited using a blue InGaN laser diode conrming previous results.21,22 We have demonstrated that by using the integrated uorescence ratio in two widely separated wavelength regions it is possible to classify lesion types both between white spot and brown spot and more signicantly, for the rst time dull from shiny lesions. It was observed that the uorescence ratio in the sound samples was signicantly lower in the samples with brown lesions than in those exhibiting white lesions of both types. This may indicate that a staining element present in the diet of these patients led to the staining of the lesions and sound structure or that there may be some intrinsic colouring caused through cross-linking of structural proteins. As the area from which the spectral ratio for sound tooth tissue was quite close to the brown lesion, this spectral ratio difference between teeth with brown spot lesions and teeth with white spot lesions could be due to colour transmission through the sound tooth structure from the stained lesion. One way in which this could be assessed would be to evaluate the spectral ratios of apparently sound enamel and determine the effect of placing coloured structures adjacent to the site from which the spectral ratio had been determined. In the QLF system such an effect would not be seen as it works on the ratio of sound structure adjacent to the lesions. Although the equipment used for the work was built around a spectroscopic system, a practical instrument could be built using dielectric lters to select the uorescence in the two wavelength regions and direct ratio-metric measurements would then be possible using simple photodiodes as the detection system.

integrations. Subsequently, the areas obtained by this method were divided (480520/620640 nm) to obtain a spectral ratio. This procedure was completed on each tooth for the spectral curve obtained for sound enamel and again for the spectral curve obtained for the caries lesion. Comparison of the ratios of areas under the spectral curve between sound and carious tooth structure was made for each group of lesions using a paired t-test. For the comparison of the ratios of areas under the spectral curve for different lesion types, a one-way analysis of variance was performed and 95% condence intervals calculated of differences between groups. Specimens were not sectioned for histology or microradiography as we did not consider lesion spectra to be related to lesion depth.

Results
Fig. 3ac illustrates typical spectra from sound and demineralised tissue for each of the three groups. For all teeth, the sound surfaces generated greater uorescence radiance compared to the lesions. Spectral ratios for sound tooth structure and caries lesion for groups of teeth are shown in Table 1 and demonstrate signicant differences (p!0.001) between sound tooth structure and caries lesions for each lesion type. A one-way analysis of variance to compare the spectral ratios from lesions in the three groups demonstrated signicant differences (p!0.001). A Tukey pairwise analysis indicated each of the groups was signicantly different from the other
Table 2 95% Condence intervals for comparison of differences in spectral ratios for lesions by pairs of lesion type. Comparison Dull and shiny Dull and brown Shiny and brown 95% Condence interval 1.252.67 3.845.26 5.817.22

Conclusion
This method has application for detection of dental caries as well as demonstrating potential application to evaluate lesions, which may represent different degrees of caries activity and hence a potential method for caries diagnosis.

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12. Hafstrom-Bjorkman U, Sundstrom F, de Josselin de Jong E, Oliveby A, Angmar-Mansson B. Comparison of laser uor escence and longitudinal microradiography for quantitative assessment of in vitro caries. Caries Research 1992;26: 2417. 13. De Josselin de Jong E, Sundstrom F, Westerling H, Traneus S, ten Bosch JJ, Angmar-Mansson B. A new method for in vivo quantication of mineral changes in initial enamel caries with laser uorescence. Caries Research 1995;29:27. 14. Hibst R, Gall R. Development of a diode laserbased uorescence caries detector. Caries Research 1998; 32:294. 15. Lussi A, Megert B, Longbottom C, Reich E, Francescut P. Clinial performance of a laser uorescence device for detection of occlusal caries lesions. European Journal of Oral Sciences 2001;109:1419. 16. Spitzer D, ten Bosch JJ. Total luminescence of bovine and human dental enamel. Calcied Tissue Research 1976;20: 2018. 17. Alfano RR, Lam W, Zarrabi HJ, Alfano MA, Cordero J, Tata DB. Human teeth with and without caries studied by laser scattering, uorescence and absorption spectroscopy. Institution of Electrical Engineers Journal of Quantum Electronics 1984;8:15126. 18. Konig K, Flemming G, Hibst R. Laser-induced autouores cence spectroscopy of dental caries. Cellular and Molecular Biology 1998;44:1293300. 19. Al-Khateeb S, Forsberg C-M, de Josselin de Jong E, AngmarMansson B. A longitudinal laser uorescence study of white spot lesions in orthodontic patients. American Journal of Orthodontics and Dentofacial Orthopedics 1998;113: 595602. 20. Rousseau C. Development and application of novel optical techniques for the early detection of dental decay. PhD thesis, University of Strathclyde, UK; 2003. 21. Al-Khateeb S, ten Cate JM, Angmar-Mansson B, de Josselin de Jong E, Sundstrom G, Exterkate RA, Oliveby A. Quanti cation of formation and remineralization of articial enamel lesions with a new portable uorescence device. Advances in Dental Research 1997;11:5026. 22. Angmar-Mansson B, ten Bosch JJ. Quantitative light-induced uorescence (QLF): a method for assessment of incipient caries lesions. Dento-Maxillo-Facial Radiology 2001;30: 298307.

Acknowledgements
The authors wish to acknowledge the nancial assistance of the EPSRC and the British Council.

References
1. Featherstone JDB. Clinical implications: caries prevention strategies. In: Stookey GK, editor. Proceedings of the 1st annual Indiana conference, early detection of caries. Indiana University School of Dentistry; 1996. p. 28796. 2. Backer-Dirks O. Posterruptive changes in dental enamel. Journal of Dental Research 1966;45:50311. 3. von der Fehr FR. A study of carious lesions produced in vivo in unabraded, abraded, exposed and uoride treated human enamel surfaces, with emphasis on the x-ray dense outer layer. Archives of Oral Biology 1967;12:797814. 4. Thylstrup A, Bruun C, Holmen L. In vivo caries models mechanisms for initiation and assessment. Advances in Dental Research 1994;14457. 5. Nyvad B, Machiulskiene V, Baelum V. Reliability of a new caries diagnostic system differentiation between active and inactive caries lesions. Caries Research 1999; 33:25260. 6. Nyvad B, Machiulskiene V, Baelum V. Construct and predictive validity of clinical caries diagnostic criteria assessing lesion activity. Journal of Dental Research 2003; 83:11722. 7. ten Bosch JJ, van der Mei HC, Borsboom PCF. Optical monitor of in vitro caries. Caries Research 1984;18:5407. 8. de Josselin de Jong E, Sundstrom F, Westerling H, Traneus S, ten Bosch JJ, Angmar-Mansson B. A new method for in vivo quantication of changes in initial enamel caries with laser uorescence. Caries Research 1995;29:27. 9. Benedict HC. A note on the uorescence of teeth in ultraviolet rays. Science 1928;67:442. 10. Alfano RR, Yao SS. Human teeth with and without caries, studied by visible luminescent spectroscopy. Journal of Dental Research 1981;60:1202. 11. Sundstrom F, Fredriksson KK, Montan SSS, Hafstrom Bjorkman U, Strom J. Laser induced uorescence from sound and carious tooth substance: spectroscopic studies. Swedish Dental Journal 1985;9:7180.