Transverse Microradiography (TMR)
The “beauty” of microradiography is that mineral content is measured straightforwardly by the high x-ray absorption of enamel (and dentin) minerals as opposed to the organic and water content present in dental hard tissue. Transverse microradiography (TMR) is considered to be the “gold standard” for determination of the spatial distribution of mineral content across caries lesions from the surface into the deeper layers. Most commonly, 80–100μm thick sections are prepared perpendicularly to the tooth surface through the caries lesion.17 These sections are mounted in front of a high-resolution x-ray sensitive film or plate together with an aluminum step wedge for calibration. This set-up is irradiated with Cu K-α x-rays perpendicularly to the film and the cut surface, which is also referred to as transverse or transversal microradiography. The developed film is viewed under a microscope and can be used for qualitative assessment of the carious lesion (Fig. 3.12). By using optical densitometry of the film and correlation with gray values from the aluminum calibration step wedge, the mineral content at any given point of the specimen can be calculated, providing a quantitative method.18,19 Using backscatter-electron imaging of tooth sections with a scanning electron microscope (Fig. 3.13), qualitative information can be gained similarly to microradiography.
Fig. 3.13a, b Image of a cut through a carious lower molar tooth by backscatter SEM. Lighter gray levels indicate a higher mineral content.
a Early fissure caries with intact enamel surface. Note the unhomogeneous mineral loss within the enamel and the small amount of demineralization within the dentin.
b Deep dentin lesion with the enamel surface still intact. Despite severe demineralization within the enamel, its surface is still intact, most likely due to a lack of mechanical impact in this area. Dentin close to the enamel–dentin junction (EDJ) is severely demineralized. Closer to the pulp, brighter, well-mineralized zones have developed. The circular dark area in the lower third of the image is pulp that was exposed during sample preparation.
The nature of white spot caries lesions can be investigated using artificially created lesions. Typically, enamel specimens with a natural or a ground-and-polished surface are exposed to an acidic buffer solution or gel. Depending on the demineralization method used, a carious lesion forms within hours and up to several weeks or months.1,20,21 These lesions show most features of natural caries lesions (Fig. 3.12). From the outside toward the EDJ the mineral-rich surface layer is clearly visible as is the body of the lesion. From the area of the deepest demineralization on, the mineral content increases continuously toward deeper layers until it reaches the mineral content of sound enamel. The position of the dark and translucent zone visible in transmitted and polarized light microscopy cannot be identified separately in a microradiograph. In other words, the relative clear borders of these zones visible in transmitted and polarized light microscopy do not correspond to abrupt changes in mineral content. Whereas artificially created, carieslike lesions progress almost parallel to the enamel surface, natural enamel lesions develop in a more irregular pattern. This may be caused by the irregular distribution and properties of the cariogenic plaque covering the enamel surface and, in addition, changes of the plaque cariogenic potential with time.
A feature quite often observed with natural enamel caries is the formation of zones with varying mineral content, or bands of higher mineral content that extend through the body of the lesion. These bands run quasi-parallel to the surface, but may be curvy in shape, with the convex aspect toward the EDJ. In an artificial caries model with cyclic de-and remineralization, these bands may also appear. This supports the assumption that the mineral layering within a carious lesion is due to changes in cariogenicity of plaque covering the enamel. However, it also was suggested that layers of increased mineral content within the body of the lesion resemble the advancing front of the lesion at an earlier stage.22
Scanning Electron Microscopy (SEM)
Scanning electron microscopy of the enamel surface reveals the early stages of caries demineralization. Often, surface porosities can be observed in the imbrication lines of Pickerill along the perikymata (see Fig. 3.3a,b). A closer look reveals an enlargement of the interprismatic spaces that may extend from the surface into the depth of the lesion. Beneath areas of dissolution of the interprismatic space, dissolution of the prism core can be found (Fig. 3.14). In both cases, at the crystal level, the dissolution process starts from the intercrystal space, enlarging the intercrystal space and reducing crystal diameter and length. Also, dissolution from inside the crystals has been observed (see “TEM” section below). It has been reported that even in the body of the enamel lesion the outer area of the prisms sometimes is less prone to demineralization than the prism core. Reasons suggested for this are a change in crystal orientation at the outer area of the prisms23,24 as well as the higher organic content of the prism sheath. Also, remineralization (crystal growth) during the caries process25–27 can be assumed, because in the outer area of prisms of carious enamel, crystals of thicker diameter have been found (120–150nm diameter) as is the case in sound enamel. In this case the newly formed crystals most likely contain more fluoride and are less soluble than native enamel crystals (Chapter 2).
Overall, while caries lesions show an intact surface clinically even when the demineralization has reached the dentin, SEM reveals the presence of small cavitations at earlier stages of the disease.
Fig. 3.14 SEM image of a partially cavitated, interproximal white spot lesion. The black arrows indicate an enlarged interprismatic space. The white arrow points at dissolution and wear of a prism core.
Transmission Electron Microscopy (TEM)
Transmission electron microscopy is a technique for investigation of enamel at the ultrastructural level with a theoretical resolution limit of about 0.05nm. Using TEM it is possible to show specific features of carious enamel, particularly of the crystals. Individual crystals seem to dissolve either from the outside,28 starting with etching of the crystal surface, or from the inside along the lattice c-axis (longitudinal axis).28–30 It is not clear so far, under which circumstances either one is the preferred form of crystal dissolution. On the enamel surface of initial caries lesions lacunaelike defects can be found of about 5μm width and depth (see Fig. 3.4) that are filled with plaque, including bacteria. Although TEM reveals highly resolved images of ultrathin enamel sections, it shows differences between caries and sound dental enamel only from the body of the lesion, with a pore volume of about 10%–25%. Formation of narrow gaps (30–100nm width) around prisms of the body of the lesions can be seen in TEM images, but it is still not clear whether this is a sign of demineralization or an artifact from specimen preparation.28
NOTE
• A white spot lesion of enamel consists of a surface layer with mineral content lower than sound enamel, but higher than the underlying body of the lesion.
• Within a white spot lesion, prisms dissolve from the outside, starting with widening of the interprismatic