## 2 Materials and Methods  
This article represents a narrative review regarding recent applications of Raman Microspectroscopy on sound and pathological hard dental tissues. It was performed in accordance with the Scale for the Assessment of Narrative Review Articles [50; 51]. An electronic literature search was performed on PubMed, Scopus, and Web of Science from January 2016 to October 2021; the scientific publications earlier than 2016 were cited only when they were essential for advancing the discussion. A combination of the following keywords was used: \"Raman\" OR \"ATR-FTIR\" AND \"dental lesions\" OR \"human teeth application\" OR \"tooth structure\". Related articles cited in the reference list of relevant studies were manually searched. Articles were selected in two phases: title and abstract screening and then full-text screening; if the content of the abstract was unclear, the article was selected for full-text review. All scientific articles were evaluated according to specific predetermined inclusion and exclusion criteria. The inclusion criteria were: (1) articles published from January 2016 to October 2021, (2) dental lesions and dental structure analyzed by Raman Microspectroscopy or ATR-FTIR spectroscopy, (3) analysis of human teeth, (4) English language, and (5) full-text publication. The exclusion criteria were: (1) not human teeth, (2) duplicate literature and duplicate data, (3) full-text not accessible, and (4) not available in English. There were no disagreements during the article selection process. the study of both inorganic and organic components. Since the spectral profile and the peaks' position can be influenced by composition and structure [60], thus making their interpretation challenging, the detailed analysis of the spectral bands related to the chemical composition of the different dental components is mandatory. In Figure 1, the main IR and Raman bands representative of enamel, dentin, and cement in sound teeth are reported. Regarding enamel, the main Raman bands are assigned to phosphates (PO4\({}^{3-}\), 960 cm\({}^{-1}\)), and A- (CO3\({}^{2-}\), 1102 cm\({}^{-1}\)) and B-type (CO3\({}^{2-}\), 1070 cm\({}^{-1}\)) carbonate groups in HA [61], with the latter representing the \(\
u_{1}\) symmetric stretching mode of CO3\({}^{2-}\) ions replacing PO4\({}^{3-}\) in the apatite lattice [62]. Carbonate substitution affects HA in terms of solubility and reactivity [63]. Significant changes in the intensities of these bands were found in carious lesions with respect to healthy enamel and were mainly attributed to demineralization-induced alterations of enamel crystallite morphology and/or orientation [13, 64, 65]. It is well known that crystallinity increases when crystals are larger and/or more perfect, and when there is less substitution [66]. Moreover, in Raman enamel spectra, a weak band at 1660 cm\({}^{-1}\), attributable to the amide I band and hence to the organic matrix, can be detected. The Raman spectrum of dentin is characterized by bands at 1660 cm\({}^{-1}\), 1450 cm\({}^{-1}\), and 1242 cm\({}^{-1}\) (attributable to collagen) and 1070 cm\({}^{-1}\) (CO3\({}^{2-}\)) and 960 cm\({}^{-1}\) (PO4\({}^{3-}\) of hydroxyapatite). Finally, cementum shows Raman bands at 960 cm\({}^{-1}\), and 1452 cm\({}^{-1}\) and 1660 cm\({}^{-1}\), the first assigned to the mineral component and the other two to the organic matrix [13]. By the analysis of Raman spectra, some parameters, such as the full width at half maximum of a single band (FWHM) and band ratios obtained using the areas (A) or the intensities (I) of the most significant bands, can be calculated and used as markers to provide accurate diagnostic information related to diseases affecting dental tissues (Figure 2). Among all, the main analyzed parameters are mineral/matrix (A\({}_{960}\)/A\({}_{1660}\)), crystallinity (inversely proportional to FWHM\({}_{960}\)), P (I\({}_{960}\)), and carbonate-to-phosphate ratio (C/P) or gradient in mineral content (GMC) (I\({}_{1070}\)/I\({}_{960}\)) [48].