## 1 Introduction  
Perforation can be defined as an artificial communication between the root canal space and the surrounding tooth tissue (periodontium), or oral environment [1]. Perforations can be pathological (caused by root resorption or caries) or iatrogenic (as a result of dental procedures during access cavity preparation, canal negotiation, or post space preparation) [1, 2]. Various factors, such as time elapsed before perforation repair, location or size of the perforation, the repair material used, and the experience of the operator, may all affect the treatment outcome [1, 3, 4, 5].  
The main goal of perforation management is to seal the defect in order to prevent bacterial contamination, inflammation, and loss of periodontal attachment and to prepare an optimal environment for tissue repair. In this context, the sealing ability and marginal adaptation of the repair material used are crucial in preventing the leakage of irritants and thus enhancing the chances of success [6, 7, 8]. A wide variety of materials including MTA, amalgam, glass ionomer cement, intermediate restorative material, and tricalcium phosphate have been tried in rural and root perforation repair [4, 7, 8, 9, 10, 11]. Notably, the use of biocompatible materials in perforation repair has been associated with a lower inflammatory response in the surrounding tissues [3; 9].  
The ability of different restorative materials to repair the perforation defect has been assessed by various in vitro experimental methods and reagents, including the bacterial leakage model [12], radioisotopes [13], dye penetration [11], and a fluid filtration method [14]. However, the reliability of these techniques has been questioned due to the fact that leakage may occur not only through the interface between the material and the dentine wall but also through other possible areas. This may be overlooked if the appropriate negative controls are not included. In addition, these methods do not provide information about the extent of bacterial penetration into the dentin. Significantly, while histological sections can be used to identify the presence and distribution of bacteria in the dentin tubules, they fail to evaluate the viability of the bacteria.  
These failings mean that alternative microscopic techniques are necessary in order to analyze more precisely the bacterial leakage and penetration depth into the dentinal tubules. Confocal laser scanning microscopy (CLSM) has been considered as an alternative microscopic technique that can provide quantitative and clinically relevant data about the presence or absence of bacteria and the extent to which the bacteria have colonized the sides of the dentinal tubules and root canal walls [15; 16; 17]. Thus, CLSM has an advantage over conventional in vitro experimental settings in comparing the bacterial colonization of perforation defects that have been repaired with various materials [18].  
In this study, we used CLSM to assess the colonization and proliferation of _Enterococcus faecalis_ in funcal perforations, repaired with MTA-Angelus, Endocem, or Biodentine. The null hypothesis was that bacterial penetration does not depend on the type of repair material.