We present the clinical applicability of fluorescence ratio reference standard (FRRS) to discriminate different stages of dental caries. Toward this, laser-induced autofluorescence emission spectra are recorded in vivo in the 400- to 800-nm spectral range on a miniature fiber optic spectrometer from 65 patients, with a 404-nm diode laser as the excitation source. Autofluorescence spectra of sound teeth consist of a broad emission at 500 nm that is typical of natural enamel, whereas in caries teeth additional peaks are seen at 635 and 680 nm due to emission from porphyrin compounds in oral bacteria. Scatter plots are developed to differentiate sound teeth from enamel caries, sound teeth from dentinal caries, and enamel caries from dentinal caries using the mean fluorescence intensity (FI) and ratios F500/F635 and F500/F680 measured from 25 sites of sound teeth and 65 sites of carious teeth. The sensitivity and specificity of both the FI and FRRS are determined. It is observed that a diagnostic algorithm based on FRRS scatter plots is able to discriminate enamel caries from sound teeth, dentinal caries from sound teeth, and enamel from dentinal caries with overall sensitivities of 85, 100, and 88% and specificities of 90, 100, and 77%, respectively.
Laser-induced autofluorescence (LIAF) is an emerging noninvasive technique in the biomedical field, especially for cancer detection. The goal of the study was to develop a spectral ratio reference standard (SRRS) to discriminate different grades of oral cancer.LIAF emission spectra from oral mucosa were recorded in the 420-720 nm spectral range on a miniature fiberoptic spectrometer from 14 anatomical sites of 35 healthy volunteers and 91 sites of 44 patients, with excitation at 404 nm from a diode laser.Histopathologic analysis of biopsy samples showed that oral mucosa of adjoining malignant sites in patients are not usually normal, but showed various degrees of epithelial dysplasia and hyperplasia. Therefore, instead of using LIAF data from apparently normal lesions of patients as control, spectral data values of the oral mucosa of healthy volunteers were used as control. The autofluorescence emission at 500 nm is characteristic of oral mucosa, whereas in malignant lesions a new peak is seen at 685 nm in addition to the previously reported peaks at 635 and 705 nm. Three spectral ratio reference standard (SRRS) scatterplots were created to differentiate the normal mucosa from hyperplasia, hyperplasia from dysplasia, and dysplasia from squamous cell carcinoma (SCC) using the mean fluorescence intensity ratios (F500/F635, F500/705 and F500/F685) measured from 40 sites in 20 patients and 11 sites in 35 healthy volunteers. During blind tests at 21 sites in 17 patients all 3 SRRS plots showed 100% sensitivity and specificity to discriminate hyperplasia from dysplastic and normal tissues, whereas only the F500/F685 SRRS showed the same sensitivity and specificity to differentiate dysplasia from SCC.An SRRS criteria based on scatterplots of autofluorescence spectral intensity ratios is described to discriminate oral mucosal variations and screen early stages of tissue progression toward malignancy.
Nitrogen laser-induced fluorescence (LIF) spectra of sound tooth consists of two broad bands centered at 440 and 490 nm, with two apparent side bands on either side. In order to locate the exact peak position of these bands and to effectively utilize the LIF spectral signatures for detection of tooth caries, the LIF spectra were curve-fitted using Gaussian spectral functions and the results were compared with those from diffuse reflectance spectral measurements.The excitation light at 337.1 nm was guided to the sound and caries-affected tooth samples through the central fiber of the fiber-optic probe of a laser-induced fluorescence reflectance spectroscopy (LIFRS) system. Six surrounding fibers of the probe collect tooth fluorescence or diffuse reflectance from the lesion and direct it to a miniature spectrometer. The in vitro spectra were obtained from healthy enamel, dentin, and pulp level tooth caries.As compared to sound tooth, the caries tooth showed lower fluorescence and reflectance intensities in the 350-700 nm region. The deconvoluted peaks in the LIF spectra of sound tooth were found centered at 403.80, 434.20, 486.88, and 522.45 nm, whereas in the case of pulp level caries, a new peak was observed at 636.78 nm. Curve-fitted parameters, such as peak center, Gaussian curve area, full width at half intensity maximum (FWHM), and their ratios, were also found to vary with the stage of tooth caries. The ratios involving the 435 nm band, such as F405/F435, F435/F490, and F435/F525 ratios derived from curve-fitted areas and amplitudes, were found to be sensitive to discriminate between sound, dentin, and pulp level caries. Among the various diffuse reflectance spectral intensity ratios, the R500/R700 was found to be most sensitive to distinguish between pulp and dentin level caries.Nitrogen laser-excited fluorescence spectral studies were found to be more suited for detection of caries lesions. The LIF measurement with spectral analysis, done by curve fitting, outscores the diffuse reflectance methodology and shows the potential to screen different levels of tooth decay in a clinical setting.
Diffuse reflectance (DR) spectroscopy is a simple, low-cost, and noninvasive modality with potential for distinguishing oral precancer. Recently, in an ex vivo study, the DR spectral ratio (R545/R575) of oxygenated hemoglobin bands at 545 and 575 nm was used for grading malignancy. This work presents the results of clinical trials conducted in 29 patients to detect oral precancer using this ratio. We use site-specific normal spectra from a group of 36 healthy volunteers for comparison with those of patients. Toward this, in vivo DR spectra from 14 anatomical sites of the oral cavity of healthy volunteers are recorded on a miniature fiber optic spectrometer with white light excitation. The R545/R575 ratio is lowest for healthy tissues and appears to increase with the grade of malignancy. As compared to scatter plots that use the mean DR ratio from all anatomical sites, those using site-specific data show improved sensitivity and specificity for early diagnosis and grading of oral cancer. In the case of buccal mucosa, using scatter plots of R545/R575 ratio, we obtain a sensitivity of 100% and specificity of 86% for discriminating precancer (dysplasia) from hyperplasia, and a sensitivity of 97% and specificity of 86% for discriminating hyperplasia from normal.
A low-cost, fast, and noninvasive method for early diagnosis of malignant lesions of oral mucosa based on diffuse reflectance spectral signatures is presented. In this technique, output of a tungsten halogen lamp is guided to the tissue through the central fiber of a reflection probe whose surrounding six fibers collects tissue reflectance. Ex vivo diffuse reflectance spectra in the 400 to 600-nm region is measured from surgically removed oral cavity lesions using a miniature fiber optic spectrometer connected to a computer. Reflectance spectral intensity is higher in malignant tissues and shows dips at 542 and 577 nm owing to absorption from oxygenated hemoglobin (HbO2). Measurements carried out, within an hour of surgical excision, on malignant lesion and adjoining uninvolved mucosa show that these absorption features are more prominent in neoplastic tissues owing to increased microvasculature and blood content. It is observed that reflectance intensity ratio of hemoglobin bands, R540/R575, from malignant sites are always lower than that from normal sites and vary according to the histological grade of malignancy. The diffuse reflectance intensity ratio R540/R575 of the hemoglobin bands appears to be a useful tool to discriminate between malignant lesions and normal mucosa of the oral cavity in a clinical setting.
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