Indiana University-Purdue University Indianapolis (IUPUI) / Background: Non-cavitated caries lesions form due to acid diffusion and demineralization of enamel subsurface with an intact surface layer (SL). Caries lesions progress when the outcome of demineralization and remineralization processes over time is net mineral loss. Lesions that continue to demineralize are called active, while those that display no evidence of further demineralization are called inactive. Micro-computed-tomography (µCT) analysis provides objective non-destructive measurements of the thickness of the surface layer (SL) and severity of caries lesions. Aims: 1) To investigate if visual/tactile suspected active non-cavitated early white spot lesions present a thinner surface layer than inactive ones; 2) To investigate if there is an association between the thickness of the surface layer (SLT) and caries activity, as determined by QLF during dehydration (△QD); 3) To determine lesion severity by comparing lesion volume and maximum depth correlation with △Q value at 15 s from QLF during dehydration. Materials and Methods: Thirty extracted human premolars exhibiting non-cavitated approximal white spot early lesions stored in 0.1.-percent thymol/4C and treated with 5.0-percent NaOCl/30 min were included in the study. Fifteen active and 15 inactive lesions were determined by visual/tactile examinations by consensus of two experienced examiners. Roughness measurements (Ra) were acquired using non-contact optical profilometry. Two-dimensional minimum (2D-min), maximum (2D-max), average (2D-avg) SL and three-dimensional (3D) analyses, volume and depth of lesions were determined from µCT image analysis. A series of fluorescence images were acquired at baseline (hydrated), at 1 s, at 5 s, at 10 s and at 15 s by QLF. During image acquisition, surfaces were dehydrated with continuous-compressed-air. △Q and △Q/s (△QD) were calculated. Data were analyzed using two-sample t-tests and Pearson correlation coefficients (p < 0.05). Results: Surface roughness of active and inactive lesions was not significantly different (p > 0.08). Overall lesion volume and depth in dentin were significantly larger in active lesions (p = 0.022, p = 0.009). SL thickness of active and inactive lesions was not significantly different (2D = 0.121, 3D = 0.080, 2D-avg = 0.446, 2D-min = 0.197, 2D-max = 0.122). △QD at 1s was significantly larger for active lesions (p = 0.046). ΔQ at 15 s of dehydration had a moderate positive association with lesion volume (r = 0.56). △QD had a weak negative association with SL thickness (2D-avg) and (2D-min). Conclusions: 1) Active and inactive non-cavitated lesions show no difference in SL thickness; 2) QLF during dehydration (△QD) does not correlate well with SL thickness; 3) ΔQ at 15 s of dehydration correlates moderately well with lesion volume and is consistent with caries activity assessed by visual/tactile examination.