# Dental Microstructural Imaging: From Conventional Radiology to In Vivo Confocal Microscopy
## 4 In Vivo Dental Imaging
### In Vivo Confocal Microscopy  
Recently, a strong interest in the potential clinical role of in vivo _reflectance confocal microscopy_ (RCM) in atomatology has arisen [85]. Conventionally, the in vivo RCM system functions using a diode laser at 830 nm combined with a water immersion objective lens. The laser power provides a variable power setup ranging between 5 and 10 mW, based on the depth of the tissue to be examined and the nature of the tissue itself; the powers emitted do not cause any tissue damage or heating [86]. As the ex vivo counterpart, in vivo RCM allows optical scanning on horizontal virtual planes, layer by layer, with the ability to deeply penetrate both ex vivo samples and in vivo tissues. In the case of in vivo dental imaging, the penetration depth is strongly limited by the reflectance of the hard mineralized tissues that allow the imaging within the first layers of the enamel, up to 300 \(\upmu\)m from the dental surface [87]. The first in vivo dental observations under RCM were executed by [PERSON] et al. in 1992 [88] and by [PERSON] et al. in 1996 [89], achieving promising results despite the prototypes' technical limitations. In 2014, [PERSON] et al. endorsed the feasibility of an RCM device (commercially available for skin imaging) for the in vivo microscopic imaging of dental surfaces [87]. The authors collected en-face dental images from 15 healthy volunteers, obtaining scanned horizontal slices of 500 \(\times\) 500 \(\upmu\)m, 5\(\upmu\)m thick, and with a lateral resolution of 0.5-1 \(\upmu\)m, similar to the histological standards. Despite producing very high microscopic resolution images, the device did not reach the most posterior sites of the oral cavity due to the rigidity of the probe. Nonetheless, the images obtained in their study show the regular prismatic and interprismatic structures of the healthy dental elements and identify the cracked enamel surfaces' fracture lines of the affected teeth. In addition, the device produced images capable of distinguishing the deep enamel structure from resin composite restorations. One year later, [PERSON] and colleagues conducted a similar in vivo exploration study to investigate the microanatomic differences between deciduous and permanent enamels. For this purpose, they imaged permanent and deciduous teeth in adults and children, respectively, and reported the differential features observed during in vivo RCM scanning [90]. Lower mineralization percentage results signified lower mechanical strengths; such aspects of the deciduous teeth explained the irregular appearance of dark areas in which the architecture appeared optically empty. Later, the same research group also applied in vivo RCM imaging to preliminarily identify and distinguish various kinds and degrees of enamel defects, such as hypoplasia and hypomineralization [91], as well as the remineralizing effect in vivo of amorphous calcium phosphate agents in a series of children with enamel defects. This study reported encouraging results on RCM's capability to monitor treatment and the patient's compliance [92]. Examples images of the hard and soft tissues of the oral cavity as they appear under RCM are shown in Figure 1.  
Figure 1: Examples of in vivo RCM images of the hard and soft oral tissues. (**a**) The clinical picture shows the points corresponding to the RCM frames: B, gingivitis, reported in b1-3; C, healthy gingiva, corresponding to frames c1-3; D, enamel defect, reported in d1-3; E, healthy enamel, shown in e1-3. (**b1**) Gingival epithelial surface, showing a massive presence of bacterial plaque (white arrows) covering the mucosal surface. (**b2**) Inflammation persists up to the basal epithelial layers with blood cells within the subepithelial and basal epithelial layers. (**b3**) Higher magnification of the bright roundish dental plaque bacteria from the picture in the red square of b1. (**c1**) Healthy mucosa at the intermedium epithelial layers. (**c2**) Healthy mucosa at the basal layer.