Despite of currently having multiple methods to measure caries lesions in vitro, little is known of their validity and how they compare to each other. Objective: To compare in vitro methods currently used to measure incipient caries lesions in vitro. Methods: Human enamel specimens were demineralized for 14, 40, or 96 h in a demineralizing solution, and then, analyzed using Vickers surface microhardness (MHN). Next, a 100-120-µm thin section was obtained from each specimen and analyzed for lesion size [area (a) and/or depth (d)] and severity [signal intensity (i)] using stereomicroscopy (SM), transmitted bright-light microscopy (TLM), polarized-light microscopy (PLM), confocal microscopy (CLSM), fluorescent microscopy (FM), transversal microradiography (TMR), and SEM in low (LV-BSE) and high-vacuum conditions (HV-BSE, HV-SE). Data obtained from the different methods were correlated (Pearson's). Additionally, the demineralization groups were compared for each method (one-way ANOVA). Results: Correlations (r) between PLM (d), TLM (d), TMR (d, ΔZ), FM (a, d), CLSM (a, d), SM (a, d) were very high (r: >0.85). When these data sets were compared to MHN and SEM (d), correlations were slightly lower (r: 0.71-0.78, 0.53-0.82, respectively). While most methods were able to show statistically significant differences (p<0.05) among all demineralization times, some of the data sets collected using SEM (HV-BSE and LV-BSE), CLSM (i), FM (i) and SM (i) were not able to do so; they also showed lower correlations with the other methods (r: 0.2-0.8). Conclusion: All methods showed high correlations and discrimination determining lesion size and could be considered for determining caries lesion size in vitro. However, data collected for signal intensity (i) and SEM backscattered electrons (BSE) showed lower correlations values and lower discrimination determining lesion size and should not be the first choice for measuring caries lesion size. Partially supported by NIH (#P01 DE13540).

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