2.1.

Sample Preparation

Fifteen human teeth with suspected lesions on the occlusal surface were collected with the approval of the Committee of Human Research and sterilized with gamma radiation. Teeth were visually examined and those that scored ICDAS 1 or 2 were selected. Teeth were mounted in black orthodontic acrylic blocks. Samples were stored in a moist environment of 0.1% thymol to maintain tissue hydration and prevent bacterial growth. The outlines of $5\times 5\text{-}\mathrm{mm}$ windows $\sim 50\text{-}\mu \mathrm{m}$ deep were cut on the occlusal surface of each tooth using a ${\mathrm{CO}}_2$ laser (Impact 2500, GSI Lumonics, Rugby, United Kingdom) around the suspected lesion area. The channels cut by the laser serve as reference points for imaging and serial sectioning and are sufficiently narrow that they do not interfere with calculations of the image contrast.

A clear sealant (without an optical opacifier), DELTON® FS+ pit and fissure sealant (Dentsply, York, Pennsylvania) was applied to the suspect fissure area according to the manufacturer’s instructions. NIR and PS-OCT images of the occlusal surface were acquired before and after the sealant was applied. A diamond suspension (3000 mesh, 6 μm) from CrystaLite Inc. (Lewis Center, Ohio) was sprayed onto the tooth surface to show the surface of the sealant for PS-OCT imaging in order to determine the sealant thickness.

2.2.

Composite Attenuation Measurements

Delton clear sealant was cast into rods using clear straws and light cured for the recommended duration. Discs were cut using an IsoMet 2000 wet saw from Buehler (Lakebluff, Illinois) to produce samples 1.0, 1.5, 2.0, 2.5, and 3.0 mm in thickness ($n=3$ for each thickness). Sample disks were serially polished on both sides with 12, 9, 5, 3, and 0.3 μm Buehler fibermet aluminum oxide polishing discs and measured with a digital caliper 500 series from Mitutoyo (Aurora, Illinois). Light from an Ocean Optics (Dunedin, Florida) fiber-coupled tungsten-halogen lamp, Model HL-2000-FHSA, with a filter wheel with bandpass filters, FB series [full-width half-maximum (FWHM) 12-nm] from Thorlabs (Newton, New Jersey) centered at 50-nm intervals from 1300 to 1650 nm was used to illuminate specific areas of the sample with a spot size of $\sim 100\mu \mathrm{m}$. The spot size was confirmed through knife-edge beam profiling techniques. Phase sensitive detection was employed using a lock-in amplifier (Model SR 850) and an optical chopper from Stanford Research Systems (Stanford, California). A large-area Ge photoreceiver Model 2033 from New Focus (Santa Clara, California) equipped with a variable aperture was used for detection. This procedure ensured that the position of the incident collimated light beam was fixed while cycling through the filters. Three measurements of collimated transmission were recorded at various sections within each sample and averaged. The collimated signal ($I$) at the detector was compared with the initial intensity of the beam ($I_{\mathrm{o}}$). Using this ratio and the thickness of the samples ($d$), the attenuation coefficient (${\mu }_t$) was calculated using Beer–Lambert plots:

The aperture in the front of the detector was sufficiently small to ensure that light scattered at small angles did not contribute significantly to the collimated transmission.

2.3.

Visible-Light Depth Composition Tooth Images

Images of the tooth occlusal surfaces were examined using a digital microscopy system, the VHX-1000 from Keyence (Elmwood, New Jersey) with the VH-Z25 lens with a magnification from 25 to $175\times$. Images were acquired by scanning the image plane of the microscope and reconstructing a depth composition image with all points at optimum focus displayed in a two-dimensional image. Figure 1 shows depth composition images of a tooth before and after placement of the transparent sealant. The sealant does change the appearance of the occlusal surface, increasing the contrast of the edges of the pits and the fissures. The sealant itself is not visible and one cannot estimate the sealant thickness and coverage by visual examination alone.

Fig. 1

Digital microscopy images of one of the samples before and after application of the transparent sealant. These are depth composition images with all surfaces in focus.

2.4.

Near-IR Cross Polarization Reflectance Images

In order to acquire reflected light images, NIR light was directed toward the occlusal surface through a broadband-fused silica beam splitter (1200 to 1600 nm) Model BSW12 (Thorlabs, Newton, New Jersey), and the reflected light from the tooth was transmitted by the beam splitter to the imaging camera (Fig. 2). Crossed polarizers were placed after the light source and before the detector and used to remove specular reflection (glare) that interferes with measurements of the lesion contrast. The NIR reflectance images were captured using a $320\times 240$ element InGaAs area camera Model SU320-KTSX from Sensors Unlimited (Princeton, New Jersey) with a 25-μm pixel pitch. Reflectance measurements were taken for three spectral bands using two bandpass filters and a longpass filter. The bandpass filters were Model #'s BP1300-90 and BP1460-85 from Spectrogon (Parsippany, New Jersey). The 1500-nm-longpass filter Model # the FEL 1500 from Thorlabs (Newton, New Jersey), which resulted in a spectral range of 1500 to 1700 nm due to the sensitivity of the InGaAs camera.

Fig. 2

(a) Occlusal transillumination setup with A. line lights, B. prism, and C. InGaAs camera with 1300-nm filter. (b) Reflectance setup with A. polarized light source, B. beam splitter, and C. InGaAs camera camera with filters and polarizer.

2.5.

Near-IR Transillumination Images

A 150-W fiber-optic illuminator, Model FOI-1 from the E Licht Company (Denver, Colorado) with a low profile fiber optic with dual-line lights, Model P39-987 (Edmund Scientific, Barrington, New Jersey), was used with each light line directed at the cement-to-enamel junction beneath the crown on the buccal and lingual sides of each tooth (Fig. 2). Light leaving the occlusal surface was directed by a right angle prism to the SU320 InGaAs camera equipped with a Navitar (Rochester, New York) SWIR-35 lens, a 75-mm plano-convex lens LA1608-C Thorlabs (Newton, New Jersey) and a 90-nm-wide bandpass filter centered at 1300 nm, BP1300-90 Spectrogon, (Parsippany, New Jersey).

2.6.

Polarization Sensitive—Optical Coherence Tomography

An all-fiber-based optical coherence domain reflectometry system (time-domain) with polarization maintaining optical fiber, high-speed piezoelectric fiber-stretchers, and two balanced InGaAs receivers that was designed and fabricated by Optiphase, Inc., Van Nuys, California, was used. This two-channel system was integrated with a broadband superluminescent diode Denselight (Jessup, Maryland) and a high-speed $XY$-scanning system (ESP 300 controller and 850G-HS stages, National Instruments, Austin, Texas) for in vitro time-domain OCT. The system had a lateral resolution of $\sim 20\mu \mathrm{m}$ and an axial resolution of 10 μm in air. The PS-OCT system is completely controlled using Labview software (National Instruments, Austin, Texas). The system is described in detail in Refs. 21 and 22. Acquired scans are compiled into b-scan files. Image processing was carried out using Igor Pro data analysis software (Wavemetrics Inc., Lake Oswego, Oregon).

The CP-OCT scans were assessed to evaluate the integrated reflectivity ($\mathrm{\Delta }R$) over the depth of demineralization in units of ($\mathrm{dB}\times \mu \mathrm{m}$). A program written in Labview as described in Ref 23 was used to automatically calculate the lesion depth using an edge detection approach and then integrate the reflectivity over that depth.

2.7.

Image Analysis and Statistics

Line profiles were extracted across the same position in the lesions within the $5\times 5\text{-}\mathrm{mm}$ boxes for each spectral band, and the image contrast was calculated using the equation $(I_{\mathrm{L}}-I_{\mathrm{S}})/I_{\mathrm{L}}$; where $I_{\mathrm{S}}$ is the mean intensity of the sound enamel outside the window area, and $I_{\mathrm{L}}$ is the mean intensity of the lesion inside the window for reflectance. Transillumination $(I_{\mathrm{S}}-I_{\mathrm{L}})/I_{\mathrm{S}}$ has the reverse contrast, i.e., the intensity in lesion areas is lower than the sound enamel. The image contrast varies from 0 to 1 with 1 being maximum contrast and 0 no contrast. All image analysis was carried out using Igor Pro software (Wavemetrics, Lake Oswego, Oregon). Repeated measures of one-way analysis of variance followed by the Tukey–Kramer posthoc multiple comparison test was used to compare groups for each type of lesion employing Prism software (GraphPad, San Diego, California) before and after application of the sealant.

3.1.

Attenuation Measurements

The optical attenuation coefficients as a function of wavelength are plotted in Fig. 3 from 1300 to 1650 nm. The coefficients lie between 5 and $10\mathrm{c}{\mathrm{m}}^{-1}$ and are consistent with the varying absorption of water with the lowest attenuation at 1300 nm and the highest attenuation at $1450\mathrm{c}{\mathrm{m}}^{-1}$ matching the water absorption band.

Fig. 3

Plot of the attenuation coefficient of the Delton clear sealant versus wavelength.

3.2.

PS-OCT Measurements

PS-OCT b-scans of both polarization states of the occlusal surface of one of the teeth before and after application of the sealant are shown in Fig. 4. The laser marks 5-mm apart are visible on the left and right sides of the sample near each cusp and they appear as small depressions along with very strong surface reflection; they also strongly attenuate light and there is a loss of signal intensity under them. They are marked by the stars in the copolarization image in Fig. 4(a). A small shallow lesion is located at the base of the central fissure and the dentinal-enamel junction (DEJ) is clearly visible in the initial b-scans for both polarizations [Fig. 4(a)]. The lesion is located in the center of the white circle. After application of the sealant, the underlying DEJ is no longer visible [Fig. 4(b)] in either polarization. It is also very difficult to resolve the surface of the sealant in both polarization images [Fig. 4(b)]. In addition, the reflectivity from the lesion at the base of the fissure is greatly reduced in the copolarization image, whereas it is not reduced in the cross-polarization image.

Fig. 4

Polarization sensitive optical coherence tomography (PS-OCT) scans of an occlusal fissure before application of the sealant (a) after application of the sealant (b) and with the diamond suspension added (c) to view the sealant surface. The upper images are the copolarization images, whereas the lower images are the cross-polarization ($\perp$) images. In (A) the stars mark the position of the laser incisions and the white circle marks the position of the lesion in the fissure. The images are red-white-blue false color with the intensity in decibels.

A 6-μm grit diamond powder suspension was sprayed onto the tooth surface, which rendered the surface of the sealant visible without greatly reducing the OCT imaging depth [Fig. 4(c)]. This facilitated measurement of the sealant thickness. The $\mathrm{\Delta }R$ values were calculated from images without the diamond powder.

The $\mathrm{\Delta }R$ values were measured and calculated for the lesions before and after application of the sealant. The mean (s.d.) of $\mathrm{\Delta }R$ for all the samples for the copolarization and cross-polarization ($\perp$) images, pre and postsealant, is plotted in Fig. 5. Bars of the same color are statistically similar ($P>0.05$). Only the copolarization image manifested a significant reduction ($P<0.05$) in $\mathrm{\Delta }R$ after application of the sealant.

Fig. 5

The mean (sd) of the integrated reflectivity with lesion depth, $\mathrm{\Delta }R$ ($\mathrm{dB}\times \mu \mathrm{m}$) calculated from the PS-OCT images before and after application of the sealant. Bars of the same color are statistically similar $P>0.05$.

3.3.

Near-IR Reflectance

Figure 6 shows NIR reflectance images of a tooth before and after sealant placement for three wavelength regions 1300, 1450, and 1500 to 1700 nm. There is shallow demineralization in the central fissure, which appears whiter than the surrounding enamel, and it shows up with the highest contrast in Figs. 6(c) and 6(e). The fissure is located in the center of the $5\times 5\mathrm{mm}$ reference box cut into the tooth. In Fig. 6(a) (1300 nm) it appears that either cracks or the premolar anatomy interfered with light transmission in the tooth causing segmentation of the light; one section of the tooth appears much brighter than the rest of the tooth. After application of the sealant, the reflected light is more uniform [Fig. 6(b)]. It is possible that the sealant filled in the cracks allowing better transmission of light through the tooth. It is very difficult to resolve the increased light reflectivity from the lesion in the fissure in Fig. 6(a), and it is not at all visible after application of the sealant [Fig. 6(b)]. The narrow zone of demineralization in the fissure appears with high contrast in Figs. 6(c) and 6(e) (1450 and 1500 to 1700 nm) and the surrounding sound enamel appears very dark. After application of the sealant the demineralization appears less continuous in the fissure and has lower contrast [Figs. 6(d) and 6(f)].

Fig. 6

Near-IR reflectance images before application of the sealant at 1300, 1450, and 1500 to 1700-nm (a, c, e) and after application of the sealant at 1300, 1450, and 1500 to 1700-nm (b, d, f). The demineralization in the fissure is located at the position indicated by the white arrows in (c) and (e).

The mean (s.d.) of the lesion contrast in reflectance is plotted in Fig. 7 for the three NIR spectral regions before and after application of the sealant. The lesion contrast was significantly higher ($P<0.05$) at 1460 and 1500 to 1700 nm versus 1300 nm before application of the sealant. Application of the sealant significantly reduced ($P<0.05$) the lesion contrast at 1460 and 1500 to 1700 nm. The lowest mean contrast was at 1300 nm (0.08) and it dropped by 37% after adding the sealant. The highest contrast was at 1500 to 1700 nm (0.26) and it decreased by 49% after adding the sealant. The greatest change in contrast occurred at 1460 nm where the initial contrast (0.19) dropped by 80%. After application of the sealant, the mean lesion contrast at 1500 to 1700 nm was significantly higher than that for both 1300 and 1460 nm ($P>0.05$).

Fig. 7

The mean (sd) of the lesion contrast calculated from the near-IR reflectance images before and after application of the sealant at each wavelength range investigated. The dark bars (left) are before application of the sealant and the light bars (right) are after application of the sealant.

3.4.

Near-IR Transillumination

Figure 8 shows NIR transillumination images at 1300 nm of a tooth before and after placement of the sealant. Note that the contrast is inverted for transillumination and the lesion appears darker instead of whiter than the surrounding sound tissues. The previous studies have shown that 1300 nm performs best for transillumination of the occlusal surfaces. The other wavelengths, 1450 nm and 1500 to 1700 nm, failed to yield high contrast due to the high water absorption of the sealant and the underlying dentin. Moreover, NIR transillumination yields poor contrast for shallow lesions on tooth surfaces.²⁴

Fig. 8

Near-IR transillumination images acquired at 1300 nm before (a) and after (b) application of the sealant. The demineralization in the fissure is located at the position indicated by the white arrows.