## 4 Discussion
In another study that used the calcium-related marker Fluo-3 to investigate the penetration depth of bioceramic sealers, it has been shown that the sealer penetration is lower compared to the values that can be expected from studies using Rhodamine B [27; 28], which also corroborates the findings of the present study. Few studies describe the need to introduce Fluo-3 instead of Rhodamine B for the evaluation of tubular penetration. Studies such as those by [PERSON] et al. [8] and [PERSON] et al. [29] detailed that the use of Rhodamine B for the assessment of tubular penetration is not the most appropriate technique to visualize this penetration, due to the high affinity of Rhodamine B to humidity, causing the obtainment of results that appear to be higher under CLSM. It is important to indicate the fact that Fluo-3 is an indicator with an unquestionably higher price than Rhodamine B and this increases the financial plan of the studies to a notorious degree. Even so, in the present study, we can observe that the use of Fluo-3 for the evaluation of the tubular penetration of bioceramic sealers should be the best choice to avoid variations that may alter the results. For the study sample, single-rooted premolars with a single canal were chosen. The ignorance of the age of teeth used and their function in the oral cavity, could have an influence on the tubular obliteration. Thus, an explanation can be given for the existence of extreme cases in the studied groups. Therefore, researchers should consider investigating new methods for standardizing the type of dentin in subsequent studies. It should be noted that the illumination power of the laser light source in the confocal microscope significantly increases the image intensity [30]. An interesting finding in this study was the statistically lower percentage of sealer penetration of Fluo-3 in medial slices compared to its control tooth at this level (\(p<0.05\)). If Fluo-3 increases its fluorescence in the presence of calcium ions dissociated from the sealer, it was surprising that the tubular penetration ability was statistically higher in the control tooth. The explanation given for this finding was the presence of a higher permeability of the dental tubules and a possible overexposure of the sample. Furthermore, it should be noted that more teeth should have been used in the control group to allow for more statistical variability. The method of analyzing the tubular penetration capacity and the maximum penetration of the sealer into the dental tubules was based on the methodology of [PERSON] et al. [12] and [PERSON] et al. [13], among others. This method is a way of analyzing penetration that has a certain subjectivity [31]. The main problem with any image-based methodology is its extrapolation to numerical values. Even so, two trained researchers carried out the measurements to try to minimize this fact. Using the intraclass correlation coefficient (ICC), agreement values of around 0.90 were obtained, concluding a high level of affinity between the examiners. Further investigations on the tubular penetration of sealers and methodological approaches by the direct detection of sealers in tubules below the surface of a specimen are needed. Furthermore, it seems likely that the penetration of the sealer into the dentine tubule does not contribute relevantly to canal sealing [21]. Likewise, penetration into the tubule must be considered in combination with other factors, such as sealability and bond strength. Following the conditions of this study and independent of the fluorescent stain used, there is less tubular penetration in the apical sections than in the medial sections of the dental root when a calcium silicate-based sealer is used. Rhodamine B achieves a higher tubular penetration than the fluorescent stain Fluo-3, but there are only significant differences between groups when comparing the maximum penetration at the apical level.