Bacterial biofilms are prevalent in the apical root canals of teeth with primary and post-treatment apical periodontitis. The rationale for endodontic treatments is to eradicate and prevent root canal infection. This goal may be accomplished by completing root canal treatment and adequate coronal restoration. The type of the root canal filling material may affect the invasion of bacterial penetration into the dentinal tubules, and the viability of these bacteria. Thus, a thorough understanding of the endodontic microbiota histopathology and the actual invasion routes of bacterial infections should be the basis for the success of endodontic treatments. The exact model of bacterial colonization in filled root end teeth is not fully understood. In Vitro studies attempted to evaluate leakage in the presence of root-end filling using different models such as the dye penetration model[27,28]. However, these studies were limited since they were using indirect laboratory models, incapable of evaluating the actual routes of bacterial penetration and colonization[26]. This thesis aimed to establish a novel and reliable experimental model that could enable to trace and quantify histologically the actual routes of bacteria and filling materials invasion into the dentinal tubules and in the total circumference of the root canal walls during various endodontic treatments, using Confocal Laser Scanning Microscopy (CLSM)-based model. CLSM was used together with live/dead bacterial staining techniques to find information regarding both the magnitude of dentin infection and the vitality of the proliferating bacteria within the infected tubuli in order to assess their viability.