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Stimulated Raman scattering microscopy (SRS) was deployed to quantify enamel demineralization in intact teeth. The surfaces of 15 bovine-enamel blocks were divided into four equal-areas, and chemically demineralized for 0, 8, 16, or 24 h, respectively. SRS images (spectral coverage from ∼850 to 1150 cm − 1) were obtained at 10-μm increments up to 90 μm from the surface to the dentin–enamel junction. SRS intensities of phosphate (peak: 959 cm − 1), carbonate (1070 cm − 1), and water (3250 cm − 1) were measured. The phosphate peak height was divided by the carbonate peak height to calculate the SRS-P/C-ratio, which was normalized relative to 90 μm (SRS-P/C-ratio-normalized). The water intensity against depth decay curve was fitted with exponential decay. A decay constant (SRS-water-content) was obtained. Knoop-hardness values were obtained before (SMHS) and after demineralization (SMHD). Surface microhardness-change (SMH-change) [ ( SMHD − SMHS ) / SMHS] was calculated. Depth and integrated mineral loss (ΔZ) were determined by transverse microradiography. Comparisons were made using repeated-measures of analysis of variance. For SRS-P/C-ratio-normalized, at 0-μm (surface), sound (0-h demineralization) was significantly higher than 8-h demineralization and 24-h demineralization; 16-h demineralization was significantly higher than 24-h demineralization. For SRS-water-content, 24-h demineralization was significantly higher than all other demineralization-groups; 8-h demineralization and 16-h demineralization were significantly higher than 0-h demineralization. SRS-water-content presented moderate-to-strong correlation with SMH-change and weak-to-moderate correlation with depth. These results collectively demonstrate the potential of using SRS microscopy for in-situ chemical analysis of dental caries.
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