Lasers in Surgery and Medicine 39:422–427 (2007)
Nondestructive Measurement of the Inhibition
of Demineralization on Smooth Surfaces Using
Polarization-Sensitive Optical
Coherence Tomography
Sherri L. Chong, DDS, MS, Cynthia L. Darling, PhD, and Daniel Fried, PhD*
School of Dentistry, University of California, San Francisco, California 94143
Background and Objective: The objective of this study
was to test the hypothesis that the inhibition of deminer-
alization in an in vitro simulated caries model by different
fluoride agents could be monitored nondestructively using
polarization-sensitive optical coherence tomography (PS-
OCT) on smooth enamel surfaces peripheral to orthodontic
brackets.
Materials and Methods: Sixty human molar samples
used for this in vitro study were divided into four groups of
fifteen. The groups consisted of a control, a fluoride
releasing glass ionomer cement, an adhesive fluoride
sealant, and fluoride in solution (2-ppm fluoride). The
reflectivity from tooth surfaces was monitored using a PS-
OCT system at different time points after exposure to a pH
cycling demin/remin model at 3-day intervals for 15 days.
Polarized light microscopy (PLM) was used to examine
lesion depth after day 15.
Results: PS-OCT was effective at measuring significant
differences in the integrated reflectivity in depth between
the control and fluoride groups (P<0.001). The fluoride
sealant demonstrated a greater protective effect than the
fluoride in solution and the glass ionomer cement.
Conclusions: These results suggest that PS-OCT is well
suited for the nondestructive assessment of caries inhibi-
tion by anti-caries agents. Lasers Surg. Med. 39:422–427,
2007. ß 2007 Wiley-Liss, Inc.
Key words: optical tomography; PS-OCT; dental caries;
dental enamel; fluoride; demineralization
INTRODUCTION
Optical coherence tomography (OCT) is a noninvasive
technique for creating cross-sectional images of internal
biological structure [1–4]. The intensity of reflected and
backscattered light is measured as a function of its axial
position in the tissue. Low coherence interferometry is used
to selectively remove or gate out the component of back-
scattered signal that has undergone multiple scattering
events, resulting in high resolution images of reflectivity
versus depth (<15 mm). Lateral scanning of the probe beam
across the biological tissue is then used to generate a two-
dimensional intensity plot, similar to ultrasound images,
called a b-scan. OCT has been used to acquire images of
both oral soft and hard tissues [5–8]. Polarization resolved
ß 2007 Wiley-Liss, Inc.
OCT or polarization-sensitive optical coherence tomogra-
phy (PS-OCT) is advantageous for imaging dental caries
[9–12] since the strong surface reflection is removed from
the image taken in the polarization state perpendicular to
the incident polarization and the reflectivity can be
integrated with depth to provide a measure of the lesion
severity.
Orthodontic appliances, on the smooth surfaces can
become ‘‘plaque traps’’ increasing the risk of decalcification
around bracket bases. Maintaining oral hygiene and
following the diet regimen are challenging during ortho-
dontic treatment. Noncompliance can lead to a higher
prevalence of demineralization seen when removing ortho-
dontic braces. The development of caries during orthodon-
tic fixed appliance therapy is a significant concern. Early
decalcification, demineralization, or ‘‘white spot lesions’’ as
they are often referred to, may develop and progress very
rapidly around brackets and bands. White spot lesions have
been shown to develop within 1 month around brackets and
bands [13]. Such areas of decalcification are called white
spot lesions due to the loss of translucency of the enamel in
that area. Typically there is no cavitation, but it may
present as a rough surface. The noncavitated surface layer
is about 40–50-mm thick, exhibits less than 20% deminer-
alization, and subsurface layers can be up to 70%
demineralized. The initial appearance is chalky white,
but if mineral loss continues, subsurface lesions will result.
There is a possibility that white spot lesions may regress or
disappear due to surface abrasion, but may still pose an
esthetic problem even 5 years post-treatment [14]. The
incidence of this demineralization after full fixed appliance
treatment can be as high as 50% [15] or as noted
elsewhere between 2% and 96% of patients [14]. If these
initial lesions are detected early, they can be arrested, or
even reversed, by a variety of noninvasive methods
Contract grant sponsor: NIDCR; Contract grant number: R01
DE017869.
*Correspondence to: Daniel Fried, PhD, Department of Pre-
ventive and Restorative Dental Sciences, University of California,
707 Parnassus Ave., San Francisco, CA 94143.
E-mail: daniel.fried@ucsf.edu
Accepted 21 February 2007
Published online 18 June 2007 in Wiley InterScience
(www.interscience.wiley.com).
DOI 10.1002/lsm.20506
 PS-OCT OF ARTIFICIAL DEMINERALIZATION
including fluoride administration, anti-bacterial rinses,
dietary changes, or low-intensity laser irradiation.
Fluoride inhibits dissolution of calcium and phosphate in
enamel, by dissolving during an acid challenge, preventing
the tooth structure from being dissolved [16]. Another role of
fluoride is to bring calcium and phosphate ions together
forming a more acid-resistant surface that makes decay less
likely to progress [16]. Daily fluoride should be applied to the
surface of the tooth to have the most effect. Oral hygiene
maintenance and a fluoride rinse are usually recommended
to orthodontic patients to prevent decalcifications from
developing. It has been shown that regular fluoride tooth-
paste use alone is unable to inhibit the development of lesions
around brackets [13,17]. The use of a neutral 0.05% sodium
fluoride rinse along with a fluoride toothpaste can signifi-
cantly reduce the appearance of white spot lesions, depend-
ing on patient compliance [18,19]. The use of a fluoride-
releasing adhesive for bonding reduced lesion development
around brackets compared to the use of a nonfluoride-
releasing adhesive [13,15,20]. Resin-modified glass ionomer
cement releases fluoride but also has greater adhesive
strength than conventional glass ionomer [15]. Most of the
fluoride is released during the first day after bonding, but
there is continued release over a few months [14]. The
fluoride from the adhesive acts locally, so the caries-
inhibiting effect is not due to an increase in the fluoride level
in saliva [21]. In a high concentration fluoride solution, glass
ionomer cements can actually absorb the fluoride from
solution and be a continuous source of fluoride over time
[14]. The amount of fluoride release increases as the sodium
fluoride content in the adhesive increases [22]. Topical
fluoride application following initial acid etching of the tooth
has been shown to be an additional source of fluoride without
altering the bond strength of the adhesive [23].
The purpose of this study was to test the hypothesis that PS-
OCT can nondestructively measure significant differences in
the rate of development of simulated caries lesions in vitro due
to inhibition by fluoride. A glass ionomer adhesive, a fluoride
sealant, and fluoride in solution were investigated.
MATERIALS AND METHODS
Sample Preparation
Teeth extracted from patients in the San Francisco Bay
area were collected, cleaned and sterilized with Gamma
radiation. Following root resection, human molars are
sectioned into buccal and lingual halves with an Isomet
2000 precision saw (Buehler, LakeBluff, IL). The 60 tooth
hemi-sections were embedded in acrylic blocks of similar
dimension, with the buccal (or lingual) side exposed from
the acrylic (see Fig. 1). Each exposed surface was polished
with aluminum oxide to ensure a clean surface for imaging
and bonding before application of the brackets. Teeth are
stored in a moist environment to maintain tissue hydration,
and 0.1% thymol was added to prevent bacterial growth.
PS-OCT Imaging
An all fiber-based Optical Coherence Domain Reflecto-
metry (OCDR) system with polarization maintaining (pm)
423
Fig. 1. Sample tooth hemi-section mounted in Resin block.
Area scanned by the PS-OCT system is shown by arrow. Other
areas were covered by red acid—resistant varnish. [Figure can
be viewed in color online via www.interscience.wiley.com.]
optical fiber, high speed piezoelectric fiber-stretchers and
two balanced InGaAs receivers that was designed and
fabricated by Optiphase, Inc., Van Nuys, CA, was inte-
grated with a broadband high power superluminescent
diode (SLD) (SLED-59, COVEGA, Jessup, MD) and a high-
speed XY-scanning system (ESP 300 controller & 850G-HS
stages, National Instruments, Austin, TX) and used for
in vitro optical tomography. This system is based on a
polarization-sensitive Michelson white light interferom-
eter. The polarized SLD source operating at a center
wavelength of 1310 nm with a spectral bandwidth FWHM
of 50 nm was aligned using a polarization controller to
deliver 20-mW into the slow axis of the polarization
maintaining (pm) fiber of the source arm of the inter-
ferometer. This light was split into the reference and
sample arms of the Michelson interferometer by a 50/50
pm-fiber coupler.
The sample arm was coupled to an AR coated fiber-
collimator to produce a 6-mm diameter collimated beam.
That beam was focused onto the sample surface using a 20-
mm focal length AR coated plano-convex lens. This
configuration provided axial and lateral resolution of
approximately 20 mm with a signal to noise ratio of greater
than 40–50 dB. Both orthogonal polarization states of the
light scattered from the tissue are coupled into the slow and
fast axes of the pm-fiber of the sample arm. A quarter wave
plate set at 22.58 to horizontal in the reference arm rotates
the polarization of the light by 458 upon reflection. After
being reflected from the reference mirror, the sample and
reference beams are recombined by the pm fiber-coupler. A
polarizing cube splits the recombined beam into its
horizontal and vertical polarization components or ‘‘slow’’
and ‘‘fast’’ axis components, which are coupled by single
mode fiber optics into two detectors. The light from the
reference arm is polarized at 458 and therefore split evenly
between the two detectors. Readings of the electronically
demodulated signal from each receiver channel represent
the intensity for each orthogonal polarization of the back-
scattered light. Neutral density filters were added to the
424 CHONG ET AL.
reference arm to reduce the intensity noise for shot limited
detection. The all-fiber OCDR system is described in more
detail in reference [24]. The PS-OCT system is completely
controlled using LabviewTM software (National Instru-
ments). Acquired scans are compiled into b-scan files.
Image processing was carried out using Igor ProTM, data
analysis software (Wavemetrics, Inc., Lake Oswego, OR).
In our PS-OCT system, linearly polarized light is incident
on the tooth and reflected light is collected by a polarization
preserving fiber in both polarization states (k- and \-axes)
and delivered to separate detectors for each polarization
state resulting in two images for each scan called the (k-
axis) and (\-axis) images, respectively. The k-axis image
includes all of the reflected light from the tooth, including
surface reflections, created by the air–enamel interface
[25]. This can mask surface or subsurface scattering and
create artifacts (spikes) in the image [25]. The (\-axis)
image arises from light depolarized by strong light scatter-
ing such as that which occurs in caries lesions. It has been
shown that PS-OCT can successfully detect artificial lesion
progression on occlusal surfaces and under sealants and
composite restorations [26]. Defects between the sealant/
composite and the tooth surface can create strong reflec-
tions. The surface reflection is not strong in the perpendi-
cular axis because the light is not depolarized with
reflection at 08 incidence. For sound enamel, the perpendi-
cular axis signal in the enamel is weak due to weak
scattering and depolarization in the sound enamel. With
demineralization, the perpendicular axis signal is high,
similar to the parallel axis, due to the intense depolariza-
tion by light scattering. This occurs with the increase in
light scattering of 2–3 orders of magnitude. Therefore, the
strong increase of the signal in the perpendicular axis
serves to represent the severity of demineralization.
The samples were randomly assigned to one of four
groups, each containing 15 human molar halves (n ¼ 60)
bonded with metal orthodontic brackets. All bonding
agents were used according to manufacturers specifica-
tions.
The four groups are:
(1) Control—nonfluoride-containing adhesive-bonded
brackets, EnlightTM (Ormco, Orange, CA)
(2) Fluoride—fluoride in solution (2-ppm F
in solution)
with nonfluoride-containing adhesive-bonded brack-
ets (EnlightTM adhesive)
(3) Sealant—topical fluoride sealant, ProsealTM (Reliance
Orthodontic Products, Itasca, IL) with nonfluoride-
containing adhesive-bonded brackets (EnlightTM
adhesive)
(4) Glass Ionomer—glass ionomer adhesive-bonded
brackets, Fuji Ortho LC 2-part adhesive (GC America,
Alsip, IL).
All groups were subjected to cycles of demineralization
and remineralization (pH changes similar to in vivo) for a
total of 15 days. The demineralization-remineralization
cycling is explained in detail in Featherstone et al. [27]. The
samples were exposed to 6 hours of demineralization and
17 hours of remineralization at 3-day intervals. The
demineralization solution consisted of 40 ml samples
containing 2.0 mmol/L calcium, 2.0 mmol/L phosphate,
and 0.075 mol/L acetate at a pH of 4.5 and a temperature of
378C. The remineralization solution consisted of 20 ml
samples containing 1.5 mmol/L calcium, 0.9 mmol/L
phosphate, 150 mmol/L KCl, and 20 mmol/L cacodylate
buffer at a pH of 7.0 and a temperature of 378C. Progressive
scans at periods of 0, 3, 6 9, 12, and 15 days were taken
during lesion development. A line profile (one point on the
b-scan where the backscattering reading was taken) was
extracted from each b-scan on one side adjacent to the
bracket base (see Fig. 3). Comparison of all the line profiles
was used to determine the effectiveness of the PS-OCT
system in monitoring the rate of lesion development, and
the efficacy of each mechanism of fluoride delivery. Each
line-profile from the PS-OCT (\-axis) b-scan scans was
integrated over a distance from the tooth surface to a depth
of 200-mm to yield the integrated reflectivity which we call
DR and it has units of reflectivity (decibels (dB)depth
(mm)). The background reflectivity (25 dB) was subtracted
before integration. This unit is analogous to the DZ value
used for transverse microradiography to quantify lesion
severity, namely the product of volume % mineral loss and
depth (Vol.%mm).
Polarized Light Microscopy (PLM)
After PS-OCT scanning tooth hemi-sections were serially
sectioned into sections of 200 mm thickness for polarized
light examination. Thin sections were imbibed in water and
examined at up to 500 with a polarizing microscope
interfaced to a high-resolution digital camera. Samples
were examined in the brightfield mode with crossed
polarizers and a red I plate with 500-nm retardation.
Demineralization due to strong scattering causes loss of
birefringence in the lesion and it appears dark. Measure-
ments of lesion depth were made with calibrated image
analysis software that is capable of direct length and area
measurements.
RESULTS
PS-OCT (\-axis) b-scan scans are shown in Figure 2 for
representative samples from all four groups acquired
before demineralization (day 0) and after 15 days of
demineralization. For the control sample on day 0 there is
a very weak signal that is barely visible from the tooth
surface, however an area of strong scattering representing
an area of demineralization almost 200-mm deep is visible
after 15 days of pH cycling. The samples with fluoride in
solution show similar behavior to the control samples
however, the lesion is not as deep after 15 days. The b-scans
for the sealant group show a thin transparent layer on the
tooth surface due to the transparent sealant. The reflection
from the surface of the sealant is visible while scattering
from the sealant itself is not visible. The signal from the
underlying enamel does not appear to increase markedly
after 15 days. The images for the last group, the glass
ionomer cement show the position of the bracket and the
 PS-OCT OF ARTIFICIAL DEMINERALIZATION
Fig. 2. Sample PS-OCT (\-axis) b-scans taken at day 0 and
day 15 for all four groups. Half of each b-scan is shown from the
edge of the bracket to the edge of the tooth for the control,
fluoride in solution and sealant groups. The b-scan for the glass
ionomer shows the position of the bracket as indicated. A red-
white-blue color intensity scale was used with high reflectivity
in red, moderate reflectivity in white and low reflectivity in
blue. [Figure can be viewed in color online via www.
interscience.wiley.com.]
images after 15 days of pH cycling are not markedly
different from day 0.
Figure 3 shows line profiles taken from the control
sample shown in Figure 2 for day 0 and day 15. The
reflectivity increases by more than two orders of magnitude
(20 dB) in the area of the lesion created after 15 days of pH
cycling. The line profiles were integrated to yield DR
(dBdepth (mm)) values for each sample for each time
Fig. 3. The two line-profiles or a-scans taken from two points
on the day-0 (red) and day-15 (blue) b-scans shown in Figure 2
taken from one of the samples in the control group. The
reflectivity is plotted in decibels (dB) showing a 2- to 3-order of
magnitude increase in reflectivity of the enamel after demi-
neralization. The front surface of the tooth occurs at position
(A) and the lesion extends to position (B). The lesion depth is
calculated by dividing the depth indicated in the a-scan
(distance between pts. A and B by the refractive index of
enamel (n ¼ 1.63). The depth of this lesion is more than 200-mm
deep. [Figure can be viewed in color online via www.
interscience.wiley.com.]
425
period. The integrated reflectivity measurements for all
four groups are summarized in Table 1.
Originally it was our intent to take the PS-OCT line-
profiles used for comparison of the four groups next to the
bracket base. However, even though care was taken to
avoid leakage of remnant cement from beneath the bracket,
in many of the samples the cement penetrated beyond the
bracket base confounding measurement close to the base.
Therefore, we decided to take line profiles halfway between
the left side of the bracket base and the left edge of the tooth.
Along a smooth surface the demineralization should be
relatively uniform, so the data taken from the middle point
should be the same as that taken from the side of
the bracket base if all excess cement was removed. It can
be argued that there may have been differences between
these two points due to differences in the thickness and
quality of the enamel at different areas of the tooth, but
these differences would be expected to even out over all the
samples in the group.
The mean integrated reflectivity (DR (dBmm)) mea-
sured with PS-OCT between the control group and the
three groups treated with fluoride was significantly
different (P<0.001) after the 15th day of pH cycling.
Figure 4 shows (DR (dBmm)) for each of the four groups
at day 15. The mean and standard deviations for each group
were as follows: control (350  65), fluoride in solution
(194  66), fluoride sealant (110  33), and glass ionomer
(170  63). A one-way analysis of variance (ANOVA)
followed by the Tukey–Kramer post-hoc multiple compar-
isons test was used to compare each group. The control
group had a significantly greater integrated reflectivity
than the other three groups (P<0.001). The fluoride sealant
group showed significantly less reflectivity than the other
two fluoride groups, but there was no significant difference
between the fluoride in solution and glass ionomer groups.
Each group was also analyzed at each of the six time
points. Repeated-measures analysis of variance (ANOVA)
followed by the Tukey–Kramer post-hoc multiple compar-
isons test was used. Table 1 lists the mean DR (dBmm) and
the standard deviations at days 0, 3, 6, 9, 12, and 15 for the
four groups when the scans were taken halfway between
the side of the bracket base and the edge of the tooth. The
greatest increase in reflectivity for the control group
occurred during the first cycling period, day 0–3. The rate
of increase in reflectivity slowed after 3 days and there was
only a slight increase between day 12 and day 15. There was
a significant (P<0.001) difference between day 0 and each of
the other five time points, and between day 3 and day 15.
There was also a significant difference (P<0.01) between
day 3 and day 9.
The fluoride in solution group showed a large increase in
reflectivity between day 0 and day 3 with little change after
the first period. There was a significant (P<0.001) differ-
ence between day 0 and day 3, 6, 9, and 15, and between day
3 and day 12. There was also a significant difference
(P<0.05) between day 0 and day 12.
There was a larger delay before a significant increase in
the integrated reflectivity was observed for the sealant
group, and the greatest reflectivity increase occurred
426 CHONG ET AL.

TABLE 1. Integrated Reflectivity After Each 3-Day Period of Demineralization Mean (s.d.)

Day 0 Day 3 Day 6 Day 9 Day 12 Day 15

Control 58 (36)a 247 (66)b 306 (57)b,c 322 (47)c 277 (81)b,c 350 (65)c
Sealant 53 (44)a 55 (29)a 110 (39)b 115 (50)b 136 (42)b 110 (33)b
Fluoride 88 (59)a 236 (68)b 202 (68)b,c 195 (69)b,c 149 (44)c 194 (66)b,c
Glass Ionomer 55 (46)a 115 (47)b 164 (71)b,c 176 (78)c 206 (91)c 169 (63)c
a,b,c
Same letter in the same row or group are not significantly different.

There was a significant (P<0.001) difference between day 0

Fig. 4. Comparison of the integrated mean reflectivity after 15
days of pH cycling between the control, fluoride, sealant, and
glass ionomer groups. The error bars represent standard
deviations. The bars with the same shading pattern are not
significantly different.
between day 3 and day 6. There was a significant (P<0.001)
difference between day 0 and day 9, between day 0 and day
12, between day 3 and day 9, and between day 3 and day 12.
There was also a significant difference (P<0.01) between
day 0 and day 6 and 15, and between day 3 and 6 and day 3
and 15.
The reflectivity progressively increased in each dissolu-
tion time period up to day 12 for the glass ionomer group.
Fig. 5. Comparison of the mean lesion depth after 15 days of
pH cycling between the control, fluoride, sealant, and glass
ionomer groups measured using polarized light microscopy of
thin sections. The error bars represent standard deviations.
and days 6, 9, 12, and 15, and between day 3 and day 12.
There was also a significant difference (P<0.05) between
day 0 and 3, and between day 3 and 15.
PLM data is shown in Figure 5. The lesion depth for each
successfully sectioned sample was measured in micro-
meters. The mean lesion depth  s.d. for each group were:
control (177  52 mm), fluoride in solution (119  42 mm),
fluoride sealant (87  25 mm), and glass ionomer (112  46
mm). The control group had the deepest lesions, followed by
the fluoride in solution group, the glass ionomer group, and
the shallowest lesions were seen with the fluoride sealant
group. The control group exhibited significantly deeper
lesions than the fluoride in solution (P<0.01), fluoride
sealant (P<0.001), and glass ionomer (P<0.05) groups.
These results are in agreement with the PS-OCT data.
DISCUSSION
All of the fluoride modalities were expected to provide
some protection to tooth structure, therefore significantly
less reflectivity was anticipated for the PS-OCT measure-
ments with all the fluoride groups in comparison with the
control group. All three (fluoride in solution, fluoride
sealant, and glass ionomer cement) did indeed show
significantly less reflectivity compared to the control group.
It was also expected that the reflectivity from the control
group samples would progressively increase from day 0 to
day 15 as demineralization progressed. The control group
showed a large initial increase in demineralization from
day 0 to day 3, then a continual increase until day 15, which
shows that the absence of fluoride allowed demineraliza-
tion to progress rapidly. The greatest increase in the
integrated reflectivity occurred in the first 3 days of
exposure to pH cycling which is consistent with the
expected rate of demineralization of a diffusion controlled
reaction [28]. All the fluoride groups did experience less
demineralization than the control group. Significant
demineralization was also noted in all groups, signifying
that fluoride does not completely protect teeth from
demineralization but does significantly reduce the severity
of the lesions.
The fluoride in solution group demonstrated a large
initial increase in demineralization from day 0 to day 3 in a
similar manner as the control group, followed by a
progressive decrease in the demineralization as the fluoride
appeared to be more effective. The demineralization/
remineralization solutions were changed before day 9 for
 PS-OCT OF ARTIFICIAL DEMINERALIZATION
all four groups, therefore the fluoride in solution group was
the only fluoride group to receive two applications of
fluoride in 15 days. However, the fluoride in solution was
not the most effective fluoride modality, and may have been
less effective with only one application.
The glass ionomer group showed a similar time progres-
sion of lesion development as the control group. This may
have occurred because the line profiles were taken too far
away from the side of the bracket and the glass ionomer
cement is most effective in the immediate area around the
bracket base. Glass ionomer has been shown to release
fluoride in the immediate vicinity and be a continual source
of fluoride over time [13–15]. This could explain why the
reflectivity, even though similar to controls, was still less
for the glass ionomer group than the control group.
The fluoride sealant group showed the least deminer-
alization overall. Moreover, the sealant group did not show
the initial increase in demineralization seen with the
control and glass ionomer groups. There was a marked
delay in rise of reflectivity that could be anticipated, since it
takes time for the acid to penetrate the sealant. Although,
the lesions produced under the sealant group were the least
severe, it was surprising that there was measurable
demineralization under the sealant. Reapplication of the
fluoride sealant may have provided even greater protection
from the acid attack.
In summary, this study showed that PS-OCT system can
be used to nondestructively measure the inhibition of
artificial demineralization on smooth surfaces around
bracket bases by fluoride. PS-OCT was successful in
tracking the development of white spot lesions on the
smooth surfaces around orthodontic brackets, and to test
the efficacy of a fluoride-releasing adhesive (glass ionomer),
a fluoride sealant, and fluoride in solution in inhibiting
decay around brackets.
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