Relationship between Changes in Chemical Composition of Enamel Subsurface Lesions and the Emitted Nonlinear Optical Signals: An in vitro Study

Abstract

The development of new diagnostic technologies based on the light scattering and autofluorescence properties of dental tissues is required to improve the diagnostic ability of initial caries lesions earlier than previously done and promoting the potential of treatment without surgical intervention. The aim of this study is to correlate fluorescence-based results provided by multiphoton microscopy (MPM) with confocal Raman microscopy records using phosphate level at 960 cm^–1 and the organic matrix at ∼2,931 cm^–1 in healthy and demineralized human enamel. Measurements on 14 teeth were made using two incident lights of different wavelengths, released by confocal Raman microscopy and MPM. Raman phosphate peak intensity at 960 cm^–1 along with organic to mineral ratio at (2,931/430 cm^–1) and nonlinear optical signals (second harmonic generation [SHG] and intrinsic two-photon excited fluorescence [I2PEF]) were recorded from the demineralized and healthy enamel sites. Raman spectral maps showed that the higher the organic/mineral ratio in the demineralized enamel, the lower the intensity of mineral component in the same zone. MPM revealed new optical indicators of carious lesion as shown by the presence of a red-shifted fluorescence peak in the 650- to 750-nm area of the fluorescence spectrum of demineralized enamel. Moreover, on sample regions with insignificant autofluorescence, the emergence of the SHG signal could be noted. By comparing I2PEF images with the structural motifs observed by the confocal Raman imaging system, the morphological similarity of the acquired images was quite evident. Any change in the I2PEF spectra reflects alterations in the chemical composition of enamel. These findings may provide an important basis for potentially valuable applications of photonic tools in the clinical diagnosis of tooth pathological conditions, besides exposing the fundamental role of organic matrix in enamel integrity and reparation.