## 3 Results  
Bacterial fluorescence was not detected in the negative control specimens, and fluorescence was detected in all the specimens of the positive control group. The extents of the stained areas (bacteria and filling) were larger in the lingual and buccal areas compared to the distal and mesial areas. However, the differences between these areas was not statistically significant between the different retrograde filling material groups (\(p=0.094\)) (Figure 2a,f). Looking at all groups, the filling material and bacterial penetration depths within the tubuli were variable, with minimal and maximal filling penetration depths of 11 and 957 \(\upmu\)m, respectively (mean of \(130\pm 158\)\(\upmu\)m), and minimal and maximal bacterial penetration depths of 9 and 1480 \(\upmu\)m, respectively (mean of \(167\pm 317\)\(\upmu\)m) (Table 1). In addition, a negative correlation was found between the depth of filling material penetration and the bacterial penetration, where greater penetration depth of the filling material into the tubuli was associated with significantly shallower bacterial penetration depth (live and dead bacteria combined) (\(p=0.015\)). Figure 2: (**a**–**f**): Demonstrating confocal laser scanning microscopy (CLSM) images (magnification scale; a: 0–750 \(\upmu\)m, b–f: 0–100 \(\upmu\)m), from the positive control group) of the _E. faecalis_ bacterial invasion into the dentinal tubules. Live bacteria are presented in green (**e**), dead bacteria are presented in red (**d**), and the retrograde filling is presented in blue (**c**). The bacteria and the filling materials can be seen at high (**b**–**e**) and low (**a,f**) magnification. The minimal, maximal, and mean depths of bacterial and filling penetration within the tubuli were assessed by defining the canal wall as the beginning point (**f**). When comparing the filling and bacterial penetration depths between the filling groups, in the Biodentine group, bacteria penetrated deeper within the tubuli, while the filling penetration depths were shallower in comparison to the other materials (IRM, MTA) (\(p=0.004\)) (Table 1). Generally, more dead bacteria were detected compared to live bacteria in all the experimental groups (\(p=0.003\)), without significant differences between the filling materials (\(p=0.087\)) (Figure 3). However, a significant negative correlation was found between the depth of filling material penetration and the penetration depth of live bacteria, where deeper filling penetration was associated with shallower penetration depths of live bacteria (\(p=0.024\)) (Figures 4 and 5). \begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline  & \multicolumn{2}{c}{**Filling Material Penetration**} & \multicolumn{4}{c}{**Bacterial Penetration**} & \multirow{2}{*}{**Group**} \\
**SD** & **Median** & **Max** & **Min** & **SD** & **Median** & **Max** & **Min** & \\ \hline
198 & 183 & 644 & 58 & 158 & 146 & 500 & 33 & MTA \\
158 & 311 & 957 & 11 & 342 & 129 & 996 & 9 & IRM \\
155 & 93 \({}^{\text{a}}\) & 535 \({}^{\text{a}}\) & 32 & 451 & 663 \({}^{\text{b}}\) & 1480 & 160 & Biodentine \\ - & - & - & - & 189 & 1200 \({}^{\text{c}}\) & 1610 & 890 \({}^{\text{c}}\) & Control \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Presents the penetration depths (in \(\upmu\)m) of the retrograde filling materials and bacteria within the tubuli. The minimal and maximal bacterial penetration depths into the dentinal tubules were 1 and 1480 \(\upmu\)m, respectively, with a mean of 167 \(\upmu\)m. The minimal and maximal filling penetration depths into the dentinal tubules were 0 and 957 \(\upmu\)m, respectively, with a mean of 130 \(\upmu\)m. In the Biodentine group, in comparison to the other materials (mineral trioxide aggregate (MTA), intermediate restorative material (IRM)), bacteria penetrated deeper into the dentin (\({}^{\text{b}}\): one-way ANOVA, \(p=0.0021\)), while the filling depth was lower (\({}^{\text{a}}\): one-way ANOVA, \(p=0.04\)).