## 4 Discussion  
The present study aimed to establish the feasibility of in vivo RCM to image the changes in enamel microscopic appearance after CPP-ACP treatments in vivo, on permanent teeth of children affected by EDs of various degree. As expected, CPP-ACP worked better on demineralized teeth than on hypoplastic ones because of the impossibility, to date, to re-create the enamel prisms where they lack. Indeed, the clinical responsiveness to treatment was higher in opacities than in hypoplastic EDs. The clinical results correlated with RCM imaging confirmed that CPP-ACP mineralization does not re-establish ex novo the normal prismatic enamel architecture where it was initially absent, because of the lack of prismatic formation during the normal amelogenesis. Therefore, according to the RCM in vivo images, the reported clinical improvement (and in some cases the disappearance) of the EDs after CPP-ACP treatment could be justified by the CPP-ACP compounds that stably fulfilled and deposited within the organic compounds of the demineralized enamel. These evidences are in line with what [PERSON] et al. reported in 2011 on the remineralizing effects of CPP-ACP mousese, revealing that the surfaces were covered with an amorphous deposit, completely obscuring the underlying prismatic structure [21]. Nevertheless, unlike [PERSON] et al., where the mineralizing CPP-ACP effects were evaluated ex vivo with Scanning Electron Microscopy (SEM) [21], in the present work the entire procedure was performed in vivo, on living teeth. In detail, in vivo RCM demonstrated that CPP-ACP complex mainly allows the organic proteins to adhere to the enamel layers, and to reduce the areas of lack of minerals, by recruiting minerals where initially absent, as previously proven and with similar microscopic pictures reported by further ex vivo studies with confocal laser scanning microscopy (CLSM) and SEM from the works of [PERSON] et al. [18] and [PERSON] et al. [39]. Moreover, in a case of severe hypoplasia, thanks to the total absence of enamel, dentinal tubules have been imaged in vivo for the first time, with their characteristic shape, similarly to what was depicted ex vivo by [PERSON] et al. [16], [PERSON] et al. [39] and [PERSON] et al. [40], thus overcoming the limited depth of penetration on healthy enamel, which impeded, in previous living studies [35; 36; 37], the imaging of the dentinal tissue. Although these results are merely qualitative and very preliminary, and a quantitative analysis of the improvements has been excluded due to the low number of subjects enrolled, they confirmed the RCM capability to highlight in vivo microscopic differences of EDs before, during and after remineralizing therapy. Furthermore, the imaging procedures revealed to be well tolerated by the children and the possibility to watch on the monitor the state of their \"teeth\" at microscopic resolution has intrigued and motivated children to be more compliant. In addition, the bad compliance of the patients as bias has been excluded by the feedback from their daily report of drink intake, which strongly motivated the patients to be collaborative. In conclusion, this novel approach to the study of enamel in vivo, could be applied to evaluate the responsiveness to remineralizing infiltrating agents, as well as to image in vivo the dental restoration interface, the margins of infiltrations, the duration and the deterioration of the compounds, and/or for longitudinal evaluation of non-invasive methods for white spot lesions arrestment, without the need for working on extracted and processed teeth, supporting and supported by the previous evidence of in vitro studies [18; 19; 20; 21]. Conceptualization, M.C., R.S. ([PERSON]) and A.L. ; data curation, M.C., F.d.V., D.L. and R.S. ([PERSON]); investigation, M.C. and R.S. ([PERSON]); methodology, M.C. ; project administration, R.S. ([PERSON]), R.S. ([PERSON]) and A.L. ; resources, R.S. ([PERSON]); supervision, R.S. ([PERSON]), R.S. ([PERSON]) and A.L. ; validation, F.d.V. and D.L. ; writing--original draft, M.C., D.D.S.