# Dental Microstructural Imaging: From Conventional Radiology to In Vivo Confocal Microscopy
## 3 In Vitro Imaging Techniques
### Other Microscopic Techniques  
Other than conventional microscopy, dental imaging histology can also be assessed with different techniques that can scan the sample's bulk or surface without the need for slicing or decalcification but offering details at microscopic resolution. The most applied techniques for dental imaging are reported below. _Confocal microscopy_ (CM) allows the imaging of horizontally oriented virtual slices of a sample by point-by-point illumination of various optical layers parallel to each others, whose microscopic image is instantly rebuilt by an integrated software to appreciate the microarchitecture with no need to physically cut, reduce, and slice the sample [40]. In CM devices, a nonharmful incident laser light at specific wavelengths stimulates the emission of refracted light from the dental tissue proportionally to the refractive indices specific to their organic and mineral compounds. Then, the refracted light emitted from the tooth is collected point by point by a detector, the signals are transduced into grayscale pixels, and integrated software then rebuilds en-face optical sections at microscopic resolution in a grayscale frame visible on a monitor [41]. Through this optical sectioning, it is possible to study the dental histology, its composition, and the disposition of each compound in the space with high resolution and contrast [42]. Furthermore, additional software offers the possibility of three-dimensional image reconstruction [43]. Images similar to those obtained with CM but cross-sectional oriented can be obtained with optical coherence tomography (OCT) that penetrates deeper than CM through the layers and utilizes near-infrared light to produce high-resolution images. OCT also avoids processing specimens, thus shortening and simplifying the imaging process. In detail, OCT uses incident infrared light, similar to CM, but detects the low-coherence interference of the light backscattered from tissues, similar to ultrasound-based technology: incident light diffuses through the tissues, which reflect it with different delay times according to their different compounds; then, signals are detected and converted to pixels on monitors, such as CM [41]. OCT is advantageously used in the studies of dental microscopic anatomy and to appreciate the marginal dental-restoration interface in studies on dental materials [44; 45]. _Scanning electron microscopy_ (SEM) scans the surface of a sample with a focused beam of electrons and offers striking micrographs with a considerable depth of field, thus presenting the characteristic three-dimensional appearance of the surface structure of the sample. In this case, SEM sends a focused flow of electrons over a surface; the electrons interact with the sample's atoms which emit secondary electrons that are attracted to a positively charged detector. Then, signals with different intensities are transmitted and integrated by software to display the surface's topography and composition [46]. In this way, SEM also offers thepossibility to visualize and measure microscopic details of dental defects and restorative materials in vitro [47] and, in some studies, also in vivo, on dental replicas [48]. Although the working principles are similar to SEM, in _transmission electron microscopy_ (TEM), electrons pass through ultrathin slices of biological tissues. Then, the information obtained on the microstructural details is recorded through changes in the momentum and phase of the electronic waves scattered by the tissues' thin slices, thus offering high-resolution images of dental defects and caries [49,50] and restorative compound interfaces [51]. In many cases, the limitations of each microscopic technique are balanced by complementary ones to offer notable and significant details of the analyzed enamel microstructures from various points of view. For example, TEM can be coupled with other imaging devices for synergic use, as reported in 2019 by [PERSON] et al.