Laser Induced Fluorescence of Carious Lesion Porphyrins
Danijela Matoević (1), Zrinka Tarle (1), Sneana Miljanić (2), Zlatko Meić (2), Lana Pichler (3), Goran Pichler (3)
1 - School of Dental Medicine, University of Zagreb
2 - Department of Chemistry, Faculty od Science, University of Zagreb
3 - Laboratory for Femtosecond Laser Spectroscopy, Institute of Physics, Zagreb
Address for correspondence:
Received: March 11, 2010
Accepted: June 8, 2010
Available online: June 15, 2010
Acta Stomatol Croat. 2010;44(2):82-89.
Original scientific article
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Objectives: This paper reports the preliminary results of the measurements of laser induced fluorescence in several porphyrin solutions. Coproporphyrin and uroporphyrin are common constituents of carious lesions. Their property to exhibit fluorescence when irradiated with a light of certain wavelength could be used as a means to detect carious lesions. Materials and methods: Absorption coefficient measurements of coproporphyrin I dihydrochloride and uroporphyrin I dihydrochloride solutions were performed under different pH conditions in order to identify spectral regions for effective laser excitation. Lasers with discrete wavelengths at 420 nm, 473 nm and 532 nm were used for the induction of the fluorescence. Results: At all laser wavelengths interesting fluorescence bands peaking at 591 nm, 619 nm and 652 nm for coproporphyrin and at 617 nm and 680 nm for uroporphyrin were observed. Conclusions: When combined together all bands should correspond to the spectral band structures found in real carious lesions.
Key Words: Porphyrins; Coproporphyrins; Uroporphyrins; Dental Caries; Spectrometry, Fluorescence
Standard dental diagnostic tools for caries detection, such as clinical examination and radiographic methods, that have been the golden standard for the last half of century, are being replaced by modern diagnostic means. The reason might be that the traditional methods are more appropriate for the detection of advanced stages of caries (1). Also, the widely spread usage of fluoride supplements as caries preventive agents, has changed the clinical image of caries. Depending on the rate of the superficial remineralization of enamel, it may become harder and slow or arrest the progression of caries, but it may also contribute to the higher rate of undetected caries which has passed through the dentino-enamel junction. The current advances in the preventive and therapeutic agents for arresting and reversing the carious process, like topical fluoridation, calcium phosphate application, antimicrobial therapy, alteration of diet or ozone treatment, are significantly progressed in the last years. To be able to use them, it is of essential importance to have an adequate diagnostic system which could detect the early stages of caries development during which the remineralization treatment is the most efficient. Bearing all this in mind, it makes no surprise that studies searching for more accurate methods of caries detection can frequently be found in the current literature. Changes in optical properties of light reflected from carious tooth seem to interest many scientists. The initial diagnostic trials by means of laser induced fluorescence exhibited at least partial improvements. A few new approaches have contributed to caries lesions detection, such as quantitative light-induced fluorescence (QLF) (1) and diode laser-based fluorescence detection (DIAGNODent (DD), KaVo, Biberach, Germany) (2). Number of researchers studied the fluorescence of tooth and caries induced by light. Enamel fluorescence was first described by Benedict as early as in 1928. (3) and subsequently proposed as a means to detect dental caries, as the fluorescence seemed to be absent in the areas overtaken by caries (4). Foreman has investigated the excitation and emission spectra of fluorescent component of dentine (5). Teeth naturally fluoresce upon irradiation with ultraviolet and visible light (6). Considering caries detection, Alfano et al. (7) and Bjelkhagen et al. (8) demonstrated that laser induced fluorescence of endogenous fluorophores in human teeth could be used as a basis for discrimination between carious and sound tissue. Upon illumination with near-UV and visible light and imaging the emitted fluorescence in the range 600 to 700 nm, carious or demineralized surfaces appear dark. An origin of endogenous fluorescence in teeth in this particular wavelength range has not been established (6). König et al. (9) found fluorescence in carious lesions that conformed to metal-free porphyrin monomers, supposedly a product of bacterial metabolism. According to these authors, porphyrins in carious lesions fluoresce only in red spectral area (9). Furthermore, Hibst and Paulus (10) have identified fluorescing compounds of caries as being uroporphyrin (UP) (Figure 1), coproporphyrin (CP) (Figure 2) and protoporphyrin IX. Porphyrins are organic compounds composed of four pyrrole rings linked by C=H bridges. Various porphyrins have the same aromatic macrocycle but differ in the substituents on the pyrrole unities. Hence, uroporphyrin have four acetic and four propionic acid side chains attached to the pyrrole rings (Figure 1), while coproporphyrin contains four propionic acid side chains and four methyl groups (Figure 2). Protoporphyrin IX is the porphyrin of heme and it caries methyl, ethenyl and propionic acid moieties. Due to highly conjugated structure porphyrins have highly stabilized electronic excited states and intensively absorb visible light (11). The detailed spectroscopic studies of their absorption and emission spectra are necessary for improving caries diagnostic tools. Previous fluorescence studies of the caries lesions have been concentrated on the spectral composition of all constituents of the caries, but none of them aimed to distinguish the contribution of each individual component. Due to the diversity of caries lesions, their composition may strongly vary in quantity and quality of the chromophores. Therefore it is of essential importance to resolve the laser induced fluorescence (LIF) of each chromophore and eventually combine their fluorescence into possible complex spectra that should be compared with real cases. The primary goal of our investigation was to further explore the origin of the fluorescence in decayed teeth excited by laser light. We want to establish what is the contribution of UP and CP to the fluorescence of caries lesions in various pH conditions (12). The second goal is to find out which laser wavelength would be the most appropriate for the excitation of the porphyrin monomers in order to facilitate the precise fluorescence diagnostics of caries.
Materials and methods
Uroporphyrin I dihydrochloride (UP) and coproporphyrin I dihydrochloride (CP) were purchased from Sigma-Aldrich Co. (St. Louis, Missouri, USA). Stock solutions of the porphyrins were prepared according to Komagoe et al. (12) by dissolving 3.6 mg of CP and 1.0 mg of UP in 10 mM NaOH followed by neutralization with 100 mM HCl and dilution with distilled water. The concentration of the resulting stock solutions were 5.0×10-4 M and 1.1×10-4 M for CP and UP, respectively. Solutions of different pH values were prepared by dilution of the stock solutions with an appropriate acid, base or buffer solution (10 mM NaOH, 10 mM Na2HPO4/NaH2PO4 (pH 7.0), 10 mM NaH2PO4/H3PO4 (for pH 4.0 and 5.0), 1 M H3PO4, 1 M HCl). The concentration of the working solutions was 1×10-5 M.
Absorption spectra were taken on a Varian spectrophotometer (model CARY 3, Varian Inc., Palo Alto, USA). Conventional quartz cells (10 mm×10 mm) were used throughout. Spectra were always recorded immediately after the preparation of the working solution. For pH measurements, a Mettler Toledo pH meter (model MP 220, Mettler Toledo Inc., Greifensee, Switzerland) with a Mettler Toledo InLab®413 combined glass-calomel electrode was used. The pH meter was calibrated with standard aqueous buffer solutions of pH 7.00 and 4.01. The pH values of the working solutions were measured after the absorption spectra were recorded.
Laser induced fluorescence (LIF) measurements
Three laser diodes i.e. at 420 nm (TOPTICA LD 100, TOPTICA Photonics AG, Gräfelfing, Germany), 473 nm (CNI laser, Changchun New Industries Optoelectronics Tech. Co., Ltd., Changchun, China) and 532 nm (HC Photonics Corp., Hsinchu, Taiwan) were used. They all have been continuous wave lasers of nominally 15, 20 and 5 mW power, respectively. 473 nm and 532 nm lasers were actually handy laser pointers, whereas the laser at 420 nm was highly monochromatic external cavity enhanced laser diode. HR4000CG-UV-NIR high resolution digital spectrometer (OceanOptics, Dunedin, FL, USA) covers the spectral range 200 - 1100 nm, has a resolution of about 1 nm, and was used in continuous wave mode of operation.
In Figure 3 and Figure 4 we present the absorption spectra of CP and UP for several values of pH. Distinctive absorption spectra were observed for both porphyrins at different pH values as a result of the pH dependent aggregation and protonation of the molecules (12-17). In an alkaline medium of pH 12.0 an absorption maximum at 390 nm was assigned to the monomeric species of CP (13, 14) (Figure 3). In the spectrum of the neutral solution, however, an additional maximum appeared at a lower wavelength due to formation of CP dimers. Dimers with face-to-face stacking clearly dominated in the solution of pH 5.0 absorbing at 371 nm (13-15). Increasing acidity to pH 3.9 a broad absorption band was noted and assigned to a highly aggregated form (13, 16, 17). In highly acidic media of pH 1.7 and 0.8 sharp absorption maxima at 400 nm and 401 nm, respectively, indicated presence of monomers protonated at the inner nitrogen atoms of the molecules (N-protonated monomer) (15). A similar behavior was observed for the UP molecules, although with the less degree of aggregation. According to the absorption spectra, the same monomeric species were present in the highly alkaline solution and in the neutral solution contributing to the absorption maximum at 396 nm (14, 17) (Figure 4). In the spectra of UP solutions of pH 5.2 and 4.3 a shoulder at around 385 nm corresponded to the aggregated molecules (12), whereas a sharp absorption maximum at 405 nm indicated that protonation of monomeric molecules occurred already under the mild acidic conditions (15). N-protonated molecules were dominant species in highly acidic solutions, pH 1.6 and 0.8, showing an intense absorption at 405 nm (15). In Figure 5 and Figure 6 we present laser induced spectra of CP and UP for different laser excitations. In Figure 5 all three laser lines may be seen and we may readily deduce that the 532 nm laser line was the most effective in inducing fluorescence. It is interesting to note that the maxima of the LIF spectra almost coincide for all three laser excitation wavelengths. In Figure 6 the UP LIF spectra are very similar for all three laser lines. Only 420 nm laser line is visible, whereas the other two laser lines were subtracted.
In this study we investigated the origin of fluorescence in carious lesions. The results are potentially of considerable importance for many caries detection devices. The absorption spectra of two important ingredients of carious tissue, CP and UP, have been taken under different pH conditions. Depending on the hydrogen ion activity the porphyrins exist in solution in several forms: as monomers, N-protonated monomers, dimers, and highly aggregated species. Each form is characterized by an appropriate absorption spectrum. The goal of pH dependent measurement of porphyrin absorption was to determine adequate laser wavelength for the excitation of fluorescence and thus to find the most efficient diagnostic means. The results indicate that absorption maxima for CP are in the range 370-401 nm and for UP from 390 up to 405 nm. Therefore, of all the lasers used in this study, the laser with discrete wavelength at 420 nm should be the most appropriate. It is interesting to note that for the different laser wavelengths we obtained essentially the same fluorescence spectrum in both porphyrin studied. The laser wavelengths at 420, 473 and 532 nm are relatively well separated, however the LIF spectrum appears almost the same. This is the consequence of the cascading from the upper excited states in both UP and CP molecules to the common lowest emitting state. The maxima in LIF spectra for CP are around 590, 620 and 650 nm and for UP are at 620 and 680 nm. These results closely correlate with studies of caries fluorescence on teeth, but similar study was not conducted on porphyrin solutions. Buchalla (18) has found distinct fluorescence bands at 624, 650 and 690 nm in both light spot lesions, which represent early stage in the development of caries, and dark spot lesions in advanced caries process, whereas white spot lesions also show a single peak at 635 nm. The research has been carried out on freshly extracted human teeth with pulsed xenon discharge lamp at excitation wavelengths from 360 to 580 nm, in steps of 20 nm. However, Zezell et al. (19) report fluorescence bands around 590, 625 and 635 nm on natural carious teeth excited by a 405 nm blue diode laser, but they suggest that all observed bands occur in both natural and carious enamel. The position of bands is unaltered by a lesion, only its intensity changed. Fluorescence on in vitro caries model was studied by Borisova et al. (20). Common caries forming bacteria were introduced in artificially demineralized teeth and irradiated with a 337 nm nitrogen laser. The fluorescence peaking was found in the 590-650 nm spectral range, which is in accordance with previous studies. Small differences in maxima in LIF spectra among studies which used extracted carious human teeth and the present study can be explained by complex conditions in oral environment, especially in dynamic carious lesions. Under these terms, it is possible that those other fluorescing compounds exist in carious tissue (such as protoporphyrin IX) and that there is a certain interaction among them. Namely, the emission of electromagnetic radiation from one fluorescing molecule can excite another fluorescing compound which absorbs a particular wavelength and a summarized emission of all of them might be the reason of the slight shift in LIF spectra. Commercial devices for detection of caries, QLF and DD, operate on different principles. The QLF determines the relative loss of fluorescence within a carious lesion due to increased scattering, whereas the DD measures the level of fluorescence from the carious lesion due to the presence of bacterial by-products such as porphyrins (21). DD device uses diode laser at the wavelength of 655 nm for the excitation of porphyrins and long-pass filter to detect the fluorescence in the near-infrared spectral region (2). Hibst and Gall reported that fluorescence intensity is lower using red light (635 or 655 nm) for excitation of carious teeth compared to excitation wavelengths in violet to green spectral region (406 or 488 nm). The decrease is more pronounced for sound compared to carious enamel or dentine, so it is proposed that caries is detected by fluorescence intensity rather than by analyzing spectral differences (22). The pH values in oral cavity are subject to various internal and external influences. One of the most interesting variables is acidity of caries lesions. As porphyrins are constituents of carious lesions, we were interested if there is a difference in their absorption spectra under the different pH condition. Komagoe et al. (12) have investigated the influence of various pH on aggregation of porphyrins and the efficiency of photogeneration of hydrogen peroxide, important for photodynamic therapy of tumors. Comparing the results of the present study and previous studies which studied the fluorescence of caries lesions, we can conclude that CP and UP have a significant contribution in the caries fluorescence. To complete the spectral image of caries, it is necessary to include the third porphyrin, protoporphyrin IX, which is considered to be the main important porphyrin of carious lesions (10, 20). Therefore, our future work is aimed to establish absorption spectra of protoporphyrin IX under different pH values, as well as combining these porphyrins in order to get basic caries model for any diagnostic means based on fluorescence.
The carious tissue fluorescence might be contributed to coproporphyrine and uroporphyrine. The results of our study are confirmed by numerous studies, whereas this study distincted the origin of the specific emission spectra of each of these chemicals. Our main intention was to study in vitro laser induced fluorescence of uropophyrine and coproporphyrin solutions of different pH values. We presented the relevant absorption spectra and LIF spectra produced by three types of lasers at 420, 473 and 532 nm. We found that all three lasers induce almost the same LIF spectrum of UP and CP solutions. Characteristics of the LIF spectra CP and UP reflect the actual conditions of dental caries. This basic research of porphyrine fluorescence in carious lesion is important for many devices for early caries detection.
We gratefully acknowledge the support of the Ministry of Science, Education and Sport of Republic of Croatia (Projects 035-0352851-2857, 065-0352851-0410 and 119-1191342-2959) and Alexander von Humboldt Foundation (Germany).
1. Stookey GK. Quantitative light fluorescence: a technology for early monitoring of the caries process. Dent Clin North Am. 2005 Oct;49(4):753-70. :. ABSTRACT :.
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