## 2 Materials and Methods  
Thirty teeth with single round-shaped root canals extracted for periodontal or orthodontic reasons were selected. The crowns were removed with a 0.3 mm low-speed diamond saw ([PERSON], Buehler, Lake Bluff, IL, USA), and the root canal length was standardized to 12 mm. Root canal preparation was performed using the Wave One Gold ([PERSON], Tulsa Dental Specialties, Johnson City, TN, USA) reciprocating system in the working length up to the Wave Gold Medium instrument (35.08). A K-File 40 was used to ensure and standardize the apical diameter after shaping. After instrumentation, 2.5 mL NaCl S.25% (Niclor, Ogna, Muggio, Italy) was used as irrigant. The canals were subsequent irrigated with 17% EDTA (Ogna), which was allowed to act for 5 min, and subsequently by 2 mL of 5% NaCl (Ogna) for one minute and sterile water for another minute. Canals were then dried with paper points, and the dentin walls were coated with Topseal sealer (Dentsply, De Trey, Constance, Germany). For this purpose, a paper point was moved 40.06 mm to 1 mm short of the WL in a pumping motion for 5 s. The sealer was previously labeled with a 0.1 wt% rhodamine B dye ([PERSON], Arese, Italy) for confocal laser scanning microscopy (CLSM). Samples were divided into three groups characterized by the different obturation techniques.  
* Group 1 (SB--10 teeth): System B (EIE Analytic Technology, Orange, CA, USA) and Calamus (Dentsply, Maillefer).
* Group 2 (T--10 teeth): Thermafil obturators 40 (Dentsply, Tulsa Dental Specialties, Johnson City, TN, USA).
* Group 3 (ST--10 teeth): Thermafil obturators 40 (Dentsply. Tulsa Dental Specialties, Johnson City, TN, USA) with sonication performed by SONICflex quick 2008/L Air Scaler (KaVo Dental Ltd., Corinium Industrial Estate, Amersham Bucks, UK), specifically adapted for Thermafil obturators (Figure 1). Vibration time: 10 s at a frequency of 6.5 Hz.  
The teeth were stored at 100% humidity in a dark environment and at 4 \({}^{\circ}\)C. After seven days, teeth were cut transversely with a 0.3 mm Isomet saw (Isomet, Buehler, IL, USA) 3 and 6 mm from the apex to obtain sections approximately 1 mm thick. The surfaces were then polished with superfine Sof-Lex disks (3M) on a low-speed handpiece to remove any dentin debris that may have been generated during cutting. Specimens were examined at 10x magnification under CLSM 510 (Zeiss, [PERSON], MD, USA). The ImageJ program was used to measure the depth, area, and percentage of penetration of the sealer penetration into the denutinal tubules. The emission wavelengths were 540/590 nm. The sealer penetration depth was determined as the mean depth of eight different measurements. In addition, the maximum penetration was measured from the canal wall to the end of the maximum depth of sealer infiltration. Instead, the area was calculated by subtracting the space of the root canal from the areas infiltrated by the sealer [26].  
Statistically significant differences in sealer penetration between groups at 3- and 6-mm depths were investigated through a non-parametric test (Kruskal-Wallis test with Dunn's procedure). The normality was checked through the Shapiro-Wilk test. Statistical significance was set at 5% (\(p<0.05\)). Power post hoc was calculated. Statistical analysis was performed using STATA16 (StataCorp, College Station, TX, USA).