Avaliar e caracterizar a aplicabilidade de técnicas ópticas para qualificar e quantificar a perda mineral por cárie ou erosão dentária, elucidando a importância do conhecimento integrado das propriedades ópticas da interação laser-tecido (in vitro).
Objetivos Específicos
Estabelecer comparações quanto aos métodos de QLF, com a diminuição de fluorescência, e da OCT, com o aumento do coeficiente de atenuação, na análise de detecção de cárie precoce no esmalte dentário (artigo I);
Analisar o sinal de decaimento da luz (A-Scan) segundo o coeficiente de decaimento da luz para diferenciar o comportamento da luz em áreas sadias e cariadas quando analisadas pela Tomografia por coerência optica (artigo I);
Caracterizar a superfície erodida de esmalte através da microscopia confocal com escaneamento a laser (artigo II);
Quantificar o efeito protetor do fluoreto de estanho e o fluoreto de sódio, e da caseína fosfopeptidea associada ao fluoreto de sódio, contra a erosão dentária através de mensurações de batentes entre a área sadia e erodida, utilizando as imagens seccionais da microscopia confocal com escaneamento a laser (artigo II);
Aplicar a tomografia por coerência optica como método para análise de perfil ótico, a ser comparado com a perfilometria de contato, na mensuração de perda mineral entre região sadia e erodida (artigo III);
Comparar protocolos de múltipla exposição previamente realizados em ensaios erosivos para estabelecer parâmetros de julgamento quanto ao protocolo adequado para visualizar o dano real e o efeito protetor do fluoreto testado (artigo III);
ARTIGO I
ARTIGO I
ARTIGO I
ARTIGO I
Dental caries assessment by two optical techniques: Quantitative Light Induced Fluorescence (QLF) and Optical Coherence Tomography (OCT)
Ana Marly Araújo Maia 1, Anderson Zanardi de Freitas2, Sergio de L. Campello3, Lena Karlsson4, Anderson Steven Leônidas Gomes5
1
Post-graduate Program in Dentistry, Universidade Federal de Pernambuco – UFPE, Recife, PE, Brazil.
2
Nuclear Energy Research Institute, IPEN-CNEN/SP, São Paulo, SP, Brazil.
3
Post-graduate Program in Material Science, Universidade Federal de Pernambuco – UFPE, Recife, PE, Brazil.
4
Departament of Dental Medicine, Karolinska Institutet, Sweden.
5
Physics Department, Universidade Federal de Pernambuco – UFPE, Recife, PE, Brazil.
Short title: Dental Caries detection: QLF and OCT
Key words: Dental Caries, Quantitative Light Fluorescence, Optical Coherence
Tomography.
Corresponding Author: Msc. Ana Marly Araujo Maia, Universidade Federal de Pernambuco, Physics Departament, Av. Prof Luis Moraes Rego, S/N, Cidade Universitária, Recife, Pernambuco, Brazil. CEP 50670-901; e-mail: anamarlyamaia@gmail.com; Fax: +55- 81-32710359
Abstract
A conservative, non-invasive, or minimally invasive approach to management of caries lesions requires diagnostic methods which can quantify very small changes in enamel structure. In this context, optical techniques seem to promote advantages to minimize subjective diagnostic by the clinical dentist and promoting early interceptive methods to control caries progression. The aim of this work was to exploit two important photonics based techniques to characterize alterations between sound
dental structure and artificially induced caries lesions in human teeth, through the loss of fluorescence by Quantitative Light-Induced Fluorescence (QLF) and the alterations of attenuation coefficient of light signal by Optical Coherence Tomography (OCT). Six vestibular surfaces of premolar teeth were submitted to demineralization cycle to develop artificial caries lesions. Then, each tooth was analyzed by QLF and OCT techniques, detecting average changes and lesion area. Lesion severity in terms of fluorescence loss and backscattered increase were calculated by commercial and home-made software, respectively. Samples were then sectioned in slices (~200µm), and analyzed transversally by optical microscope. Carious damage showed correlation between images of each system, although comparing the percentage of alteration, the attenuation of light calculated by OCT image processing showed difference with high intensity. QLF images are easily obtained, however OCT images processed by tomographic sections showed higher differences of optical characteristics between sound and caries regions.
Keywords: Optical Coherence Tomography, Fluorescence, Dental Caries Introduction
Caries-related clinical decision-making remains a centrepiece of clinical dentistry. To arrest or reverse the disease process and to intervene before operative restorative dentistry is needed requires most often an early detection of the carious lesion. Clinically applicable methods for detection of a very early phase of mineral loss and quantification of caries lesions have therefore emerged [Featherstone, 2008].
The evolution of biomedical research and industry has developed several optical techniques that exploit different interactions between light and dental hard tissue. This development has been possible due to the evolution in the knowledge about optical properties and interactions inherent to the complex inhomogeneous dental structures [Darling, 2006]. Enamel consists of approximately 96% inorganic material, constituting biological hydroxyapatite crystals. Remnants of organic matter (proteins 0.6%) from the period of development and water (3,5%) are also found in the enamel [Ten Cate 1994; Ehrlich et al. 2009]. The crystals are clustered together and roughly perpendicularly to the tooth surface, due to the scattering distributions
are generally anisotropic and depend on tissue orientation relative to the irradiating light source in addition to the polarization of the incident light [Fried, 1995; Zijp, Ten Bosch, 1993; Zijp, Ten Bosch, 1998].
One optical method for an objective assessment of early incipient changes in the enamel mineral content is the quantitative light-induced fluorescence (QLF). The method is based on a visible blue-green light system with an excitation wavelength of 370 nm that is applied to the enamel. The resultant auto-fluorescence is detected by filtering out the excitation light using bandpass filter at >540nm by a small intra-oral camera [Pretty, 2006]. The high sensitivity of QLF has been confirmed in several studies [Tranæus et al., 2002; Gmur et al., 2006] and the method has been rapidly adopted as a standard reference measure in clinical tests of the efficacy of preventive measures [Pitts and Stamm, 2004], with established correlation between the mineral loss and the fluorescence loss in enamel demineralized.
The other system evaluated, Optical Coherence Tomography (OCT) consists of an emerging diagnostic method for creating nondestructive cross-sectional imaging of internal biological structures due to the scattering and absorption of laser light [Huang et al., 1991; Fujimoto, 2008]. The light source used are near-IR lasers or broadband incoherent radiation sources from 780 to 1550nm that offer a great potential for optical imaging modalities also in dentistry due to the weak scattering and absorption in dental hard tissue [Hall and Girkin, 2004].
Optical coherence Tomography allows several comparisons between sound and caries lesions enamel. For instance, caries lesions can be detected by the reduction in enamel reflectivity [Amaechi et al., 2003]; by the increase of scattering in the analyzed image [Maia et al., 2010]; by the mineral loss correlation with the increase in reflectivity [Douglas, Fried, Darling, 2010]; or by the refractive index alterations [Hariri et al, 2013]. It also has been demonstrated that PS OCT, which stands for polarization sensitive OCT, can be more efficient than conventional OCT, even though the last one has also potential to detect early demineralization [Douglas, Fried, Darling, 2010]. Furthermore, other researchers have compared carious surfaces by changes of attenuation coefficient of signal light exponentially decay [Popescu et al., 2008; Cara et al., 2012].
The aim of the present study was to analyze the correlation between the loss of fluorescence by QLF and the alterations of attenuation coefficient of light signal by OCT, in artificially caries lesions. OCT cross sectional images were analyzed by attenuation coefficient and a new map transverse to caries lesions were generated promoting comparison between QLF and OCT images.
Materials and Methods
Ethics
As the biological material comprising the study sample could not be traced to an individual donor, the regional Ethics Committee in Stockholm, Sweden determined that the study was not subject to the law of ethical approval (2006/3:4). Eight intact premolar teeth, extracted for orthodontic reasons, were collected and stored in saturated thymol saline under refrigeration before the experiment. Extrinsic deposits were gently removed with a soft toothbrush and water, and the teeth were thereafter photographed with a digital camera (COOLPIX 4500, Japan), to detect cracks or other inhomogeneity on the buccal surface. Two of them were excluded from the study because of crack findings.
Demineralisation procedure
Each of the 06 samples were embedded in wax leaving a 2x3 mm open window on the buccal surface, and artificial caries lesions were created on all samples using a demineralising solution (pH=5,0) described by Buskes et al. [1985]. The solution also contained protective agents as 2–50 µM MHDP (methanehydroxydiphosphonate), which leads to the formation of subsurface lesions and inhibits demineralization in vitro. The waxed teeth were placed in separate small tins filled with a demineralising solution and placed in an incubator (Electrolux, Sweden) in 37°C. The solution was replaced every th ird day for 9 days, but due to natural anatomical structure, different stages of non-cavitated artificial incipient carious lesion on smooth surfaces was produced. The teeth were rinsed with ionized water at the occasion of replacing the solution.
After the artificial caries induction procedure, the wax was removed from the teeth and cleaned with deionized water. The artificial white-spot lesions were investigated by optical techniques quantifying: visible light reflected and trans- illuminated by an stereomicroscope, levels of fluorescence by Quantitative Light- induced Fluorescence (QLF™) and intensity of backscattering by the Optical Coherence Tomography (OCT). In the final step each sample was transversally sectioned and evaluated by polarized optical Microscopy to confirm lesion depth.
Experimental set up
Quantitative Light Induced Fluorescence (QLF)
The artificial caries lesions were examined automatically by the QLF commercial software. The sample was illuminated by violet-blue light (wavelengths 290-450 nm, average pick 380 nm) from a handpiece, and image was obtained using a camera fitted with a yellow 520-nm high-pass filter. The filter is necessary to capture only the wavelengths emitted by the fluorescence, and blocked all violet-blue light reflections of surface (QLF; Inspektor™ Research Systems, Amsterdam, the Netherlands). The image was captured, saved and processed.
The image was digitally stored on a computer for analyses. The difference between fluorescence intensity values gives three quantities; ∆F (average change in fluorescence, %), lesion area (mm2), and in later versions of the QLF software, ∆Q (area x ∆F), which gives a measure of the extent and severity of the lesion, but is not extensively used. The average loss of fluorescence, highlighted through color’s degree of yellow (high fluorescence loss), red, pink and purple (low fluorescence loss), was observed and dimensions calculated. The parameters ∆F and lesion size were obtained, first to objectively support/confirm the presence and the extent of the white-spot lesion. The image was stored, and the spot within the lesion with highest loss of fluorescence was used as a reference for the subsequent analyze.
Optical Coherence Tomography (OCT)
A commercially available OCT system was used (Spectral Radar SR-OCT: SR 930/Thorlabs, New Jersey, USA), operating in the spectral domain using a superluminescent diode (SLD) light source with central wavelength of 930 nm. This system consists of three main parts: a handheld scanning probe, a base unit that contains the SLD light source and a personal computer (PC) (Figure 1).
Figure 1: The commercial SR-OCT, OCP930SR, schematic diagram (adapted from Thorlabs New Jersey, USA).
The whole system is based on fiber optics couplers to direct the light from a broadband SLD source to the Michelson interferometer, which is located inside the handheld probe. After that, the light that travels back from sample and from the reference mirror, goes through the same fiber to the spectrometer and the image sensor located in the base unit. The base unit was connected to the PC, which was equipped with two high-performance data acquisition PC-cards.
OCT image acquisition
The system was configured to save images in automatic model, as stream mode, making possible to capture about 2.3 frames per second. Some other parameters were set under the following conditions: files saved as numeric array matrix; images composed by 2000 columns and 512 lines, providing a pixel resolution of 3µm x 2.88µm. Each cross-sectional image is a tomogram, known as the “B-scan”, with 6µm of transversal resolution and 4µm of axial resolution composed of several “A-scans”, along line produces information from a 'slice' of tooth tissue.
The handheld scanning probe from the OCT system (SR-OCT 930nm,Thorlabs) was firmly fixed into a stand perpendicular to the floor, and each sample was positioned in a micrometer translation stage controlled by a Motor Move system, about 0.5mm/s (Figure 2.a). Each sample had the surface scanned, through 4mm from mesial to distal on tooth surface, counting about 200 B-scans of 6mm cervical-incisal, totalizing an scanned area around 24mm2 captured in 8 seconds. All these B-scans were processed making possible to project data into a 2D map, named as C-scan, as schematically shown in Figure 2.b.
Figure 2: (a) The handheld scanning probe from the OCT system (Thorlabs) and the tooth in a micrometer translation stage controlled by a Motor Move system,
OCT Image Processing
A software was developed in Labview specifically to analyze/calculate the attenuation coefficient of all A-scan of each image. This procedure develops an attenuation coefficient bi-dimensional map of analyzed area, named C-Scan. Each new processed map gives information of particularly optical characteristic of the internal tissue structures evaluated. The whole sequence described can be explained by the block diagram below:
Figure 3: Diagram of the whole sequence processing.
Several B-Scan images were analyzed through each A-Scan, by the calculation of the attenuation signal that verify the distribution of the scattering of light that penetrated into each analyzed sample. The inherent curvature of the tooth surface was corrected graphically for each image, by aligning each A-Scan using the peak of the reflection of enamel/air interface as reference. The peak was excluded from attenuation coefficient calculation, as it represents only the abrupt change of refraction index between air and enamel. For the new C-Scan image, each B-scan represents one line and each A-Scan only one point that shows the value of attenuation coefficient, as an artbitrary number. The data which compose the C-Scan image was obtained by fitting its curve with a Beer-Lambert type function:
ܻ(ݔ) = ܣ݁ିஜ୶+ ܻ
where Y(x) is the OCT signal intensity, µ the attenuation coefficient, and x is the depth of light penetration (Cara et al., 2012;). The “x” number of points included on the exponential curvature line was 40 µm below reflect peak, and also the last 500µm was excluded as is a noise region. The points used to fit the exponential curvature line should be described as the value of coefficient can be modifiable. The C-Scan image was composed of the value of attenuation signal coefficient, of a total of 2000
A-scans (columns), from each B-scan. Through 6mm to be scanned, approximately 200 B-Scans were necessary, generating images that after process represented the new lines of the C-Scan image.
To better understand optical principles by OCT signal and images, it is important to consider that the system detector cannot identify huge difference between in homogenous material that has high absorption or high transmission. So a high attenuation coefficient signal can also represents loss of signal light by transmission. This fact enforces how important is to previously than OCT analyze, have samples optically characterized.
Stereomicroscope
As a complementary analysis simple images were captured by a stereomicroscope (magnification X10, Olympus), using the reflection light of the system, and also the transmitted extra light perpendicular to surface captured.These images added information about macroscopic effects of light properties of reflection and trans-illumination, between soud and caries regions.
Optical Polarized Microscope
After all techniques evaluation described, sections of each tooth were prepared by the Low Speed diamond Whellsaw, model 650, SBT inc., with water irrigation. The sections were obtained by cutting sample perpendicular to the buccal face. Selected sections were then ground using grinding stones until the required thickness of 200µm. The depth of caries lesions was observed under 50X of magnification using a transmitted Polarized Light Microscope (Olympus, USA) and through the measurements software it was possible to measure the real value of lesions depth, used as gold standard technique.
Results
The map reconstructed by the value of attenuation coefficient of OCT images permitted both quantitative and qualitative comparison with the QLF technique. As a representative guide, images by visible light were captured for better samples evaluation, as in figure 4. Comparing the lesion extension by both techniques, it was observed that OCT as a tomographic mode of capture showed better resolution and delimited contours.
Figure 4: a) Image obtained by reflection visible light; b) Image obtained by transilluminated visible light; c) Image from QLF software; d) The C- Scan image processed.
OCT processing model presented in figure 5 represents the calculation of light behaviour in each A-Scan. The value of attenuation coefficient excluded data of the peak first interference and it value consist an arbitrary number, although it is possible to compare the light behavior of the light on structure and shows difference between values for sound and carious points of the same sample.
(a)
Figure 5: Image reference and A-scan showing the decrease of light in evaluated by exponential decay, illustrating the alterations of attenuation coefficient
on carious signal. Fitting in decay signal curve of sound tooth (green line) and carious region (blue line).
Similar to QLF software, for each teeth structure analyzed, it was created a particular scale of colors through coefficient value with different intensity of colors between orange, yellow and blue. Considering the loss of fluorescence and the variation of the attenuation coefficient, it was possible to compare intensity of damages. Results from QLF were obtained by commercial software, and new parameters were established for OCT software, as presented in table 1.
Table 1: Data of fluorescence Intensity reduction (%) and attenuation coefficient increase (%) of each sample.
Sample Lesion Area (mm2) Fluorescence Intensity Reduction (%) Attenuation coeficiente increase (%) A 16.1 -28.3% 116% B 20.7 -34.2% 187% C 18.5 -27.2% 261% D 7.79 -19.7% 239% E 10.2 -13.3% 161% F 4.25 -11.9% 109%
QLF images identified caries lesions by the contrast of these areas that showed loss of fluorescence of 21.75% due to the increase of scattering and decrease of absorption. Although OCT images after data processing identified caries lesions by an increase of 178% on the value of attenuation coefficient µ. Cross sectional (B- scan) OCT images also shows that the higher attenuation of caries also represents an increase of reflectivity due to the increase of scattering in the first points of A-Scan signal, although the last points of the A-Scan signal shows that high attenuation of caries lesions, as observed when the enamel-dentine junction is near surface caries lesions doesn’t allow capture of this structure, as observed in figure 6.
Figure 6: (a) Enamel surface and dentine enamel junction (DEJ); (b) same region after artificial demineralization, not possible to see DEJ below carious surface.
Highlighting the optical principles, the tomographic technique based on backscattering, analyzed by B-Scan images (figure 7), allows measurements of how deep the light is still scattered by caries alterations on surface and subsurface, comparing sound and carious enamel. Measurements of caries lesion depths showed value about ~130micrometers.
Figure 7: (a) Polarized optical microscope image of 200um section; (b) Tomographic B-Scan image of the caries lesion.
Discussion
In this paper it was shown how optical methods offer advantages to observe and characterize the dental structure, mainly because of translucence, crystalline and regular structure of enamel, which has specifically interactions with radiation from UV to IR. Consequently, early alterations, as carious lesions, can be detected by alterations of reflected, back-scattered and absorbed light.
QLF and OCT optical fundamentals are interrelated because both are based at first on the increase of light scattering by caries lesion, which is much stronger than in sound enamel [Pine, ten Bosch, 1996]. In general, scattering causes the light path in the lesion to be much shorter than in sound enamel [Angmar Manson, 2001], although physics fundamentals of interactions between light and material depends on wavelength [Darling et al., 2006]. Used as a map guide of lesion, macroscopic images captured by visible light were just observed by contrast and brightness. And as expected, reflected visible light image shows the worst contrast and highest