Optical approach to the salivary pellicle

Abstract

The salivary pellicle plays an important role in oral physiology, yet noninvasive in situ characterization and mapping of this layer remains elusive. The goal of this study is to develop an optical approach for the real-time, noninvasive mapping and characterization of salivary pellicles using optical coherence tomography (OCT) and optical coherence microscopy (OCM). The long-term goals are to improve diagnostic capabilities in the oral cavity, gain a better understanding of physiological and pathological processes related to the oral hard tissues, and monitor treatment responses. A salivary pellicle is incubated on small enamel cubes using human whole saliva. OCT and OCM imaging occurs at 0, 10, 30, 60 min, and 24 h. For some imaging, spherical gold nanoparticles (15 nm) are added to determine whether this would increase the optical signal from the pellicle. Multiphoton microscopy (MPM) provides the baseline information. In the saliva-incubated samples, a surface signal from the developing pellicle is visible in OCT images. Pellicle "islands" form, which increase in complexity over time until they merge to form a continuous layer over the enamel surface. Noninvasive, in situ time-based pellicle formation on the enamel surface is visualized and characterized using optical imaging.

Figures

Fig. 1

Sample preparation. Teeth set in silicone mold for OCT imaging. Two oil ink markers indicate the area for imaging.

Fig. 2

Saliva preparation. Clarified saliva after centrifugation. In this study, 1000-g ×20-min centrifugation was performed and the resulting clear supernatant (red arrow) was collected using careful pipetting to prevent disturbance in the precipitate (yellow arrow). (Color online only.)

Fig. 3

Imaging dish specifically designed for salivary pellicle imaging. To protect against dehydration and mechanical irritation of the developing salivary pellicle, the imaging dish border was sealed using silicone (top), and the window border was protected by 100-μm-thick plastic film. The gap between the imaging glass and the salivary pellicle assures an intact and unchanged salivary pellicle throughout imaging.

Fig. 4

Time-based 2-D OCT images extracted from 3-D reconstructed images. (a) Top view of the enamel surface incubated in distilled water for 10, 30, and 60 min showing no change over time. (b) Top view of the enamel surface incubated in saliva for 10, 30, and 60 min showing development of the salivary pellicle.

Fig. 5

OCM images of salivary pellicle. OCM images (green signal) were superimposed with fluorescent (red signal) and secondharmonic (blue signal) microscopic images. (a) Top view of 3-D reconstructed OCM image of distilled water-incubated sample. After 120-min incubation, there is no distinctive layer between teeth and water. The light-pink-colored signal is related to fluorescence from the imaging glass (black arrows). (b) Optical section of 3-D image, providing a lateral view of (a). (c) Top view of 3-D reconstructed OCM image of saliva-incubated sample. A distinctive pellicle layer between tooth and saliva is clearly visible (white arrows). (d) Optical section of 3-D image, providing a lateral view of (c). The pellicle with an average thickness of 20 μm is clearly visible on the tooth surface (white arrows). (Color online only.)

Fig. 6

MPM images of salivary pellicle. (a) Tooth incubated in distilled water for 120 min. Enamel rods are clearly visible in this 3-D reconstructed top view image. (b) Optical section of 3-D image, providing a lateral view of (a) and confirming the absence of pellicle. (c) Isoline plot of (b). The outermost zone of enamel shows high signal intensity. (d) Tooth incubated in saliva for 120 min. Due to the development of an overlying pellicle layer, the enamel rods are mostly obstructed in this top view image. (e) Optical section of 3-D image, providing a lateral view of (d). The pellicle layer covers the outer tooth surface. (f) Isoline plot of (e). The new signal on the outside tooth surface, which is not seen in the samples incubated in water (c) implies the existence of a salivary pellicle.

Fig. 7

Growth and development of pellicle. OCM images after 120-min incubation in saliva. Globular growth of pellicle is clearly visible. (a) Top view of 3-D reconstructed image. Fluorescein in dentinal tubule (white arrows) and pellicle (yellow arrows) shows red signal. (b) Optically sectioned lateral view of 3-D reconstructed image. Pellicle islands are also visible in later view (yellow circle). (Color online only.)

Fig. 8

Progressive growth and development of pellicle (white arrows) on one tooth sample. Top view of 3-D reconstructed MPM images at progressive saliva incubation time points. (a) 10-min incubation. (b) 30-min incubation. (c) 60-min incubation. (d) 24-h incubation. Blue signal originates from tooth and saliva; pink and red signals originate from the salivary pellicle. Over time, the number and diameter of pellicle islands gradually increased. (Color online only.)