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The purpose of this study was to investigate the depths of penetration of a calcium silicate–based sealer in dentinal tubules by using 3 different obturation methods.One hundred extracted human permanent anterior teeth were endodontically prepared and divided equally into 3 experimental groups and 1 control group as follows: CPoint single cone (CPSC), gutta-percha single cone (GPSC), gutta-percha vertical condensation (GPVC), all with a calcium silicate–based sealer and calcium indicator Fluo-3, and CPoint single cone with a calcium indicator Fluo-3 (CPF3) without sealer as the control. The roots of the teeth in each group were axially cross-sectioned, and the surfaces were examined under confocal laser scanning microscopy at ×10 and ×20 magnifications. The sealer penetration depths were measured at their maximum depths and at 4 circumferential depths (12, 3, 6, and 9 o'clock) by using fluorescence.Statistical analyses by using one-way analysis of variance and repeated measures analysis with linear mixed models showed no statistically significant difference among the mean maximum depth measurements (CPSC, 283.83 μm; GPSC, 318.66 μm; and GPVC, 313.03 μm; P = .7553) and among the average depths across all points (CPSC, 111.24 μm; GPSC, 135.38 μm; and GPVC, 126.62 μm; P = .5304) for the 3 experimental groups.The pressure derived from hygroscopic expansion of CPoint or warm vertical condensation did not enhance penetration depths of the calcium silicate–based sealer. Sealer penetration into the dentinal tubules occurred independent of the obturation technique.This project study demonstrated that calcium silicate–based root canal sealers penetrated into the dentinal tubules by using different obturation techniques.This project reveals that the fluorescent dye (Fluo-3) can be an appropriate marker for tracing the penetration of the calcium silicate–based root canal sealers in the dentinal tubules.