QUANTITATIVE EVALUATION OF SIMULATED ENAMEL
DEMINERALIZATION AND REMINERALIZATION USING
PHOTOTHERMAL RADIOMETRY AND MODULATED
LUMINESCENCE
by
Adam Hellen
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Graduate Department of Dentistry
University of Toronto
© Copyright by Adam Hellen, 2010
II
Quantitative Evaluation of Simulated Enamel Demineralization
and Remineralization Using Photothermal Radiometry and
Modulated Luminescence
Adam Hellen
Master of Science
Graduate Department of Dentistry
University of Toronto
2010
Abstract
Detection modalities that can evaluate the early stages of dental caries are indispensable. The
purpose of this thesis is to evaluate the efficacy of photothermal radiometry and modulated
luminescence (PTR-LUM) to non-destructively detect and quantify simulated enamel caries.
Two experiments were performed based on the PTR-LUM detection mode: back-propagation or
transmission-mode. Artificial demineralized lesions were created in human molars and a subset
was further exposed to an artificial remineralizing solution. PTR-LUM frequency scans were
performed periodically during de/re-mineralization treatments. PTR data was fitted to a
theoretical model based on optical and thermal fluxes in enamel to extract opto-thermophysical
parameters. Lesion validation was performed using transverse microradiography (TMR). Optical
and thermal properties changed with the development and repair of the caries lesions while
theory-derived thicknesses paralleled those determined microradiographically. These trends
coupled with the uniqueness-of-fit of the generated parameters illustrate the efficacy of PTR-
LUM to non-destructively detect and quantify de/re-mineralized lesions.
III
Acknowledgements
I would like to thank all the lab members at the Center for Advanced Diffusion-Wave
Technologies (CADIFT) and Quantum Dental Technologies (QDT) for their insight and support.
Specifically, I would like to acknowledge Dr. Raymond Jeon, Dr. Anna Matvienko and Dr.
Koneswaran Sivagurunathan for their invaluable assistance, lengthy discussions, suggestions and
guidance over the years. Thank you to Dr. Stephen Abrams for his endless motivation and
clinical insight into the application and development of caries detection aids. His clinical
expertise and knowledge of dental economics are truly invaluable to the dental profession as he
continually tries to promote a shift from the traditional ―drill, fill and bill‖ approach to dentistry.
I would like to express my sincere gratitude toward my supervisors, Dr. Andreas Mandelis and
Dr. Yoav Finer for their endless support, leadership and expertise. Thank you to my advisory
committee member, Dr. Paul Santerre, for his support and discussions. Thank you to Prof.
Mandelis, whose scientific knowledge, perspicacity and motivation made this research project
possible and inspired me to meticulously explore the interminable world of quantitative, non-
destructive science. I would also like to thank Dr. Bennett Amaechi at the University of Texas
Health Science Center at San Antonio for transverse microradiography analysis and his
invaluable discussions relating to the experimental protocol and principles of cariology.
I would like to acknowledge the following funding agencies for financial support: the Ministry of
Research and Innovation (MRI), the Ontario Premier‘s Discovery Award, the Ontario Research
Fund from the Canadian Foundation for Innovation (CFI-ORF) and lastly the Natural Sciences
and Engineering Research Council of Canada (NSERC).
IV
Table of Contents Abstract ........................................................................................................................................... II
Acknowledgements ....................................................................................................................... III
Table of Contents .......................................................................................................................... IV
List of Tables ................................................................................................................................ VI
List of Figures ............................................................................................................................... VI
1 Literature Review................................................................................................................ 1
1.1 Tooth structure ........................................................................................................ 1
1.2 Dental Caries ........................................................................................................... 3
1.3 The demineralization process ................................................................................. 5
1.4 Histopathology of early caries lesions .................................................................... 7
1.5 The remineralization process .................................................................................. 8
1.6 Demineralization and remineralization evaluation techniques ............................. 11
1.6.1 Physical principles underlying light – tooth interactions .......................... 11
1.6.2 Microradiography ...................................................................................... 16
1.6.3 Optical fluorescence techniques ................................................................ 16
1.6.4 Optical Coherence Tomography ............................................................... 19
1.6.5 Photothermal Radiometry and Modulated Luminescence ........................ 20
2 Rationale ........................................................................................................................... 24
3 PTR-LUM Backscatter Mode: Materials and Methods .................................................... 26
3.1 Sample Collection and Sterilization ...................................................................... 26
3.2 Sample Preparation ............................................................................................... 26
3.3 Demineralization and Remineralization Treatments ............................................ 27
3.3.1 Demineralization ....................................................................................... 27
3.3.2 Remineralization ....................................................................................... 28
3.4 PTR-LUM Experimental Setup ............................................................................ 28
3.5 PTR-LUM frequency scans .................................................................................. 30
3.6 Theoretical Model ................................................................................................. 31
3.6.1 Optical Field .............................................................................................. 32
3.6.2 Thermal Wave Field .................................................................................. 36
3.7 Multiparameter Fitting of Experimental Curves ................................................... 37
3.8 Transverse microradiography (TMR) and image analysis.................................... 42
3.9 Statistical analysis ................................................................................................. 43
4 Results ............................................................................................................................... 44
4.1 Sound enamel ........................................................................................................ 44
4.1.1 Microradiographic Analysis and PTR-LUM signals ................................ 44
4.1.2 Theoretical analysis of untreated enamel samples .................................... 45
4.2 Demineralization Group........................................................................................ 46
4.2.1 Microradiographic Analysis and PTR-LUM Signals ................................ 46
4.2.2 Theoretical analysis of demineralized enamel samples ............................ 49
4.3 Remineralization Treatment Groups ..................................................................... 53
V
4.3.1 Microradiographic analysis and visual appearance ................................... 53
4.3.2 Fluoride-free remineralization group ........................................................ 54
4.3.3 Low fluoride (1 ppm) remineralization group........................................... 59
4.3.4 High fluoride (1000 ppm) remineralization group .................................... 63
5 Discussion ......................................................................................................................... 67
5.1 PTR-LUM signals and multiparameter fits of sound enamel ............................... 67
5.2 PTR-LUM signals during short and long-term demineralization ......................... 70
5.2.1 Multiparameter fits of PTR signals during demineralization .................... 71
5.2.2 LUM signal generation during demineralization ...................................... 79
5.3 PTR-LUM signals during short and long-term remineralization .......................... 82
5.4 Errors and Limitations in the extraction of opto-thermophysical properties ........ 89
5.5 Comparison of irradiation wavelengths and future directions .............................. 91
5.6 Summary ............................................................................................................... 92
CHAPTER 2: Transmission mode PTR – LUM........................................................................... 94
6 Rationale ........................................................................................................................... 94
7 Materials and Methods ...................................................................................................... 95
7.1 Sample Preparation ............................................................................................... 95
7.2 PTR-LUM frequency scans .................................................................................. 96
7.3 Demineralization and Remineralization Treatments ............................................ 97
7.4 PTR-LUM Experimental Setup ............................................................................ 97
7.5 Transverse microradiography (TMR) and image analysis.................................... 98
8 Results ............................................................................................................................... 98
8.1 Time-series demineralization experiments ........................................................... 98
8.2 Time-series remineralization experiments .......................................................... 100
9 Discussion ....................................................................................................................... 102
9.1 PTR-LUM signals and time-series demineralization .......................................... 102
9.2 PTR-LUM signals and time-series remineralization .......................................... 106
9.3 Errors and limitations of transmission PTR-LUM measurements ...................... 107
9.4 Future directions ................................................................................................. 108
10 Significance..................................................................................................................... 109
11 Summary ......................................................................................................................... 113
12 Conclusions ..................................................................................................................... 113
13 Appendices ...................................................................................................................... 115
13.1 Appendix 1 .......................................................................................................... 115
13.2 Appendix 2 .......................................................................................................... 119
13.3 Appendix 3 .......................................................................................................... 123
13.4 Appendix 4 .......................................................................................................... 128
13.5 Appendix 5 .......................................................................................................... 131
14 References ....................................................................................................................... 136
VI
List of Tables
Chapter 1
Table 1. Published optical properties of sound and carious enamel at relevant wavelengths. ..... 14
Table 2. Published set of thermal properties of sound and carious enamel. ................................. 15
Table 3. Laser parameters for backscatter PTR-LUM measurements. ......................................... 29
Table 4. Treatment groups for backscatter PTR-LUM ................................................................. 27
Table 5. Composition of the remineralizing solution. .................................................................. 28
Table 6. The list of parameters fitted from the theoretical analysis. ............................................. 32
Table 7. Fixed upper and lower limits of the fundamental parameters defined for the
multiparameter fitting of untreated enamel. .......................................................................... 40
Table 8. Mean (± s.d.) set of derived optical and thermal parameters of intact enamel layers .... 46
Table 9. General trends in the main physical parameters following short and long term
demineralization.. .................................................................................................................. 53
Table 10. Average mineral loss and lesion depth of remineralization and demineralized treatment
groups.. .................................................................................................................................. 54
Table 11. General trends in the main physical parameters following demineralization and short
vs. long term remineralization. .............................................................................................. 67
Chapter 2
Table 2.1. Treatment groups for transmission-mode PTR-LUM study. ....................................... 97
List of Figures
Chapter 1
Figure 1. A human molar in situ showing the primary tissue components and surrounding tooth
structures. ................................................................................................................................. 1
Figure 2. A scanning electron microscopic view of (A) the three primary tissues of teeth, and the
spatial organization of enamel and relationship between rods (R) and inter-rod (IR) spaces
viewed longitudinally (B) or in cross section (C). (D) Mature, permanent, human enamel
showing superficial aprismatic enamel overlying prismatic enamel... .................................... 2
Figure 3. The dynamic demineralization-remineralization equilibrium at the plaque-enamel
interface. Saliva acts as a source of mineral and fluoride ions promoting lesion
remineralization. ...................................................................................................................... 4
Figure 4. The pH dependence on enamel caries formation and remineralization. ......................... 5
VII
Figure 5. (A) Classical enamel caries lesion with surface layer and demineralized lesion body
clearly evident under polarized light microscopy. A clear distinction of carious zones is
evident in the image (A) and microdensitometric profile of the lesion (B). The mineral
volume percent is plotted vs. depth (d) from the surface. ....................................................... 8
Figure 6. Light- tissue interaction. ................................................................................................ 12
Figure 7. Wavelength dependence of (A) the water absorption coefficient and (B) the infrared
transmission properties of the primary absorbers in dental enamel. ..................................... 13
Figure 8. Photothermal and luminescence effects upon excitation with an intensity modulated
laser beam. ............................................................................................................................. 22
Figure 9. Experimental setup for backscatter-mode PTR-LUM study ......................................... 30
Figure 10. The 3-layer geometrical representation used for theoretical analysis and associated
optical and thermal parameters of each layer. ....................................................................... 33
Figure 11. Schematic geometry of effective layers for multiparameter fittings of sound enamel.38
Figure 12. Schematic structure of effective layers used for fits of demineralized and
remineralized enamel. ............................................................................................................ 40
Figure 13. Schematic mineral content profile for the theoretical determination of layer
thicknesses. ............................................................................................................................ 41
Figure 14. Visual appearance of (a) sound enamel and (b) white-spot appearance after 10-days of
acid treatment. ....................................................................................................................... 44
Figure 15. PTR-LUM amplitudes and phase curves for a representative sound enamel sample
under 660-nm laser excitation. The densitometric tracing (top right) and microradiographic
image (bottom right) are presented in the adjacent figures.. ................................................. 45
Figure 16. PTR-LUM amplitudes and phase curves for a 10 day demineralized sample under
660nm laser excitation. Error bars, when not visible, are of the size of the symbols. The
densitometric tracing (top right) and microradiographic image (bottom right) of the lesion
are presented in the adjacent figures. ..................................................................................... 48
Figure 17. PTR-LUM signals for the 40 day demineralized lesion under 660-nm. Error bars,
when not visible, are of the size of the symbols. The densitometric tracing (top right) and
microradiographic image (bottom right) of the lesion are presented in the adjacent figures. 49
Figure 18. The change in optical absorption (a) and scattering (b) coefficients and thermal
conductivity (c) and diffusivity (d) parameters as a function of time, over the 10 day
demineralization period.. ....................................................................................................... 50
VIII
Figure 19. Changes in the thickness of layer 1 and layer 2 as a function of time for the 10 day (a)
and 40 day (b) demineralized samples. The inset in (b) shows the details of layer 1 thickness
over time on an expanded scale.. ........................................................................................... 51
Figure 20. The change in optical absorption (a) and scattering (b) coefficients and thermal
conductivity (c) and diffusivity (d) parameters as a function of time over the 40 day
demineralization period.. ....................................................................................................... 52
Figure 21. Visual appearance of representative samples from each remineralization treatment
group. (a) Remineralized in the absence of fluoride; (b) remineralized in the presence of low
fluoride; (c) remineralized in the presence of high fluoride levels. ....................................... 53
Figure 22. Microradiographic image and mineral volume profile for an exemplary sample from
the fluoride-free treatment group. .......................................................................................... 55
Figure 23. PTR-LUM amplitude ratios and phases differences with respect to the final
demineralization state for a sample in the fluoride-free treatment group, under 660nm laser
excitation.. ............................................................................................................................. 56
Figure 24. Change in optical absorption (A) and scattering coefficients (B) over treatment time
for a sample in the fluoride-free treatment group.. ................................................................ 57
Figure 25. Change in thermal conductivity (A) and diffusivity (B) over treatment time for a
sample in the fluoride-free treatment group.. ........................................................................ 58
Figure 26. Change in layer thicknesses over treatment time for a sample in the fluoride-free
treatment group. ..................................................................................................................... 58
Figure 27. Microradiographic image and mineral volume profile for an exemplary sample from
the low fluoride treatment group. .......................................................................................... 59
Figure 28. PTR-LUM amplitude ratios and phases differences with respect to the final
demineralization state for a sample in the low fluoride treatment group, under 660nm laser
excitation. .............................................................................................................................. 60
Figure 29. Change in optical absorption (A) and scattering coefficients (B) over treatment time
for a sample in the low fluoride treatment group.. ................................................................ 61
Figure 30. Change in thermal conductivity (A) and diffusivity (B) over treatment time for the
low fluoride sample. ..................................................................................................................
............................................................................................................................................... 62
Figure 31. Change in layer thicknesses over treatment time for a sample in the low fluoride
treatment group. ..................................................................................................................... 62
Figure 32. Microradiographic image and mineral volume profile for an exemplary sample from
the high fluoride treatment group. ......................................................................................... 63
IX
Figure 33. PTR-LUM amplitude ratios and phase differences with respect to the final
demineralization state for a sample in the high fluoride treatment group, under 660nm laser
excitation.. ............................................................................................................................. 64
Figure 34. Change in optical absorption (A) and scattering coefficients (B) over treatment time
for a sample in the high fluoride treatment group.. ............................................................... 65
Figure 35. Change in thermal conductivity (A) and diffusivity (B) over treatment time for a
sample in the high fluoride treatment group. ......................................................................... 66
Figure 36. Change in layer thicknesses over treatment time for a sample in the high fluoride
treatment group. ..................................................................................................................... 66
Chapter 2
Figure 2.1. Experimental apparatus for transmission experiments. .............................................. 96
Figure 2.2. Experimental setup for transmission-mode PTR-LUM. ............................................ 98
Figure 2.3. Exemplary microradiograph (a), densitometric tracing (b), and visible light
transmission image (c) of a demineralized enamel section. .................................................. 99
Figure 2.4. Time-series transmission-mode PTR-LUM amplitude and phase signals at 1Hz (PTR)
and 89Hz (LUM) for a sample demineralized for 15 days. ................................................... 99
Figure 2.5. Time-series transmission PTR-LUM amplitude and phase signals at 1Hz (PTR) and
89Hz (LUM). Vertical dashed lines divide de-and remineralization treatments. The visible
light transmission image (top right) and microradiographic image (bottom right) are
presented in the adjacent figures. ........................................................................................ 101
Figure 2.6. Microradiograph and mineral content profile of a de- and remineralized sample. The
corresponding PTR-LUM signals are presented in Fig. 2.7.. .............................................. 101
Figure 2.7. Time-series transmission PTR signals at 1 Hz (A) and transmission LUM at 89Hz
(B). (C) Time-series LUM signals at 89 Hz viewed in backscatter mode.. ......................... 102
1
1 Literature Review
1.1 Tooth structure
Human teeth are composed of three primary tissues and moving from the surface exposed to the
oral environment inward they are: enamel, dentin, and pulp (Fig. 1). Enamel is the outer
mineralized tissue exposed to the oral environment, forming a protective layer at the anatomical
crown of the teeth. Dentin occupies the largest portion of the overall tooth structure and encloses
the pulp chamber.
Figure 1. A human molar in situ showing the primary tooth tissue components and surrounding
tooth structures [Adapted from Nanci 2003].
Enamel is the complex, non-vital, secretory product of specialized epithelial cells called
ameloblasts. The process of enamel formation can be broadly categorized into two stages. The
first stage, called the secretory stage, involves the organized secretion of a protein-rich and
acellular matrix, packed with thin, ribbon-like crystals of hydroxyapatite (OHAp) into prisms (or
rods) (Smith 1998). The second stage, referred to as the maturation stage, involves the growth of
crystals at the expense of protein and enamel fluid, which are absent in mature enamel (ten Cate
1998). In mature enamel, OHAp crystals are approximately 30 – 40 nm in diameter and can be
up to 10 µm long. The OHAp crystals pack together forming the enamel prisms, which typically
have an overall cross-section of 4 – 6 µm (Fig. 2). The end result of enamel formation is the
creation of an acellular tissue which is composed of approximately 95 wt% (≈85 vol%) of an
impure mineral OHAp with the remainder made up of non-collagenous protein and water
2
(Dowker et al. 1999; Featherstone 2000). The spaces created at the interface between prisms is
occupied by ≈12 % by volume water and ≈3 % by volume organic material, thus creating about
≈15 % by volume of available diffusible space for acids and inorganic mineral ions (Kidd and
Joyston-Bechal 1997; Featherstone 1999)(Fig. 2).
Figure 2. A scanning electron microscopic view of (A) the three primary tissues of teeth, and the
spatial organization of enamel and relationship between rods (R) and inter-rod (IR) spaces
viewed longitudinally (B) or in cross section (C) [Adapted from Nanci 2003]. (D) Mature,
permanent, human enamel showing superficial aprismatic enamel overlying prismatic enamel.
ES = enamel surface. Bar = 50 µm. [Adapted from Kodaka et al. 1991].
At the outermost surface of enamel, a region of aprismatic enamel has been described distinct
from underlying prismatic structure (Gwinnett 1967; Kodaka 2003) (Fig. 2D). The term
―aprismatic‖ does not imply the layer is altogether structure-less, but rather refers to the absence
of characteristic prism markings with the parallel arrangement of needle-shaped crystallites. The
aprismatic enamel layer is most likely formed as a result of the reduced functional activity of
ameloblasts during the terminal stages of amelogenesis (Ripa et al. 1966). It is prevalent in both
unerupted and erupted permanent and deciduous dentition, although more likely to be observed
in unerupted and deciduous teeth, with a range of thicknesses from a few microns to
approximately 60 µm in erupted teeth (Kakaboura et al. 2005). It is present in about 70% of
permanent erupted human molars, with its prevalence most likely linked to its wear over time as
a result of abrasive and masticatory forces and post-eruptive maturation processes (Gwinnett
1967; Ripa et al. 1966). As permeability and caries susceptibility of newly erupted enamel is
very high, the natural post-eruptive maturation process occurs to create a more stable and less
soluble enamel surface through the precipitation of fluoridated-OHAp phases.
The impure nature of enamel mineral is reflected in its propensity to accommodate numerous
ionic substitutions and vacancies within its crystal lattice without losing its apatitic structure
A B C D
3
(Aoba 2004). Extraneous ions most commonly found within the enamel apatite include cations,
magnesium and sodium and anions, carbonate and fluoride (Robinson et al. 2000). As a
consequence of these impurity atoms, defects are introduced within the OHAp crystal lattice
thereby altering the solubility product of the mineral (ten Cate and Featherstone 1991).
Composition of the impurity elements varies with depth, with higher carbonate and magnesium
concentrations in areas of lower crystallinity closer to the dentin-enamel junction (DEJ) and
higher fluoride levels at the enamel surface (Weatherell et al. 1974).
As enamel is highly mineralized and brittle, in order to avoid fracture under regular masticatory
forces it requires additional support, which is provided by the more resilient dentin (Nanci 2003).
In contrast to enamel, it is a vital tissue made up of closely-packed micrometer-sized cylinders
with a higher mineralized shell, called dentinal tubules, which contain dentinal fluid and the
cytoplasmic extensions of the odontoblasts, the dentin-forming cells. Dentin, as in bone, is
formed with OHAp crystals organized about an abundant organic matrix. It is composed of about
47 vol.% carbonated-rich and calcium deficient OHAp mineral, about 33 vol.% organic material,
mainly Type-I collagen, and the remaining ≈20 vol.% water (Marshall et al. 1997; Curzon and
Featherstone 1983). The lower crystallinity and higher organic component of dentin makes it a
porous and more acid soluble tissue. The pulp chamber, enclosed by the dentin, contains soft
connective tissue and is innervated by nerves. It serves several functions including the formation
of dentin, provides nutrients to avascular dentin and can repair dentin when required (Nanci
2003).
1.2 Dental Caries
Dental caries is an infectious, ubiquitous and multifactorial disease affecting nearly all mankind.
The chronic, slowly progressing caries process involves the localized destruction of dental hard
tissue, initially in enamel followed by dentin, as a result of acids produced from bacterial
fermentation of dietary carbohydrates by the cariogenic microflora of dental plaque (Selwitz et
al. 2007). Caries encompasses a continuum of disease states where an intimate balance between
demineralization and remineralization can be found sub-clinically localized initially to the outer
enamel, progressed into dentin or advanced in root tissue (Fig. 3).
4
Figure 3. The dynamic demineralization-remineralization equilibrium at the plaque-enamel
interface. Saliva acts as a source of mineral and fluoride ions promoting lesion remineralization
[Adapted from Winston and Bhaskar 1998].
Demineralization refers to the process by which bacterially-derived plaque acids generated under
mildly acidic conditions diffuse into the tooth mineral structure (Fig. 2), causing destruction of
enamel and/or dentin crystals. As enamel destruction continues and the lesion progresses into the
underlying dentin, bacterial invasion will occur with the concomitant diffusion of acids. As a
result, death of the pulp and spread of infection to periapical tissues may occur (Kidd and
Joyston-Bechal 1997). Under strongly acidic conditions, such as those generated by gastric acids
and acidic beverages, an erosive challenge can result in the often irreversible loss of tooth
structure (Fig. 4). The repair process, remineralization, occurs under near-neutral physiological
pH conditions whereby calcium and phosphate mineral ions are re-deposited within the caries
lesion from saliva and plaque fluid (Fig. 3). Although the processes of demineralization and
remineralization are described separately, it is their intimate balance, modulated by host factors
such as diet, salivary flow and composition, carbohydrate frequency and duration and the
maintenance of oral hygiene, that ultimately determines an individual‘s overall caries risk
(Selwitz et al. 2007; Aoba 2004). As a result, over time, early lesions left uncared for may
cavitate, while if diagnosis at the incipient stage timely therapies can be instituted to repair the
lesions to restore form and functionality.
5
Figure 4. The pH dependence on enamel caries formation and remineralization (Adapted from
Pretty 2006, modified from Mount and Hume 2005).
1.3 The demineralization process
Demineralization refers to the destruction of OHAp by organic acids (predominately lactic acid)
in their unionized form, generating a concentration gradient for the dissolution of calcium and
phosphate and diffusion from the bulk tissue (Featherstone et al. 1979) (Fig. 3). The process of
demineralization involves active mineral loss at the advancing front of the lesion, at a depth
below the enamel surface, with the transport of acid ions from the plaque to the advancing front
and mineral ions from the advancing front toward the plaque. Thus over time, the advancing
lesion front moves deeper into the enamel at the expense of underlying sound enamel, while an
intact surface layer above the lesion modulates the reaction between the internal demineralizing
structure and external solution (Fig. 5). During the initial caries attack, direct surface enamel
dissolution causes an enlargement of the intercrystalline spaces over the enamel surface thereby
facilitating the movement of acids and mineral ions into and out of the porous enamel structure.
This may indicate preferential loss of more acid-soluble phases of enamel mineral, carbonate and
magnesium-rich phases, as well as facilitated diffusion of acids through the enamel
microstructure due to lower crystal density at these particular sites (Robinson et al. 2000).
Thermodynamically, appropriate physicochemical conditions at the tooth-solution interface are
needed in order to favour either demineralization or remineralization. These conditions refer to
the saturation level of the solution in direct contact with enamel. The pH at which the solution in
direct contact with the tooth is saturated with respect to enamel mineral is referred to as the
‗critical pH‘ value and is typically around 5.5 (Dawes 2003). Below the critical pH value
dissolution will be favoured, whereas above mineral precipitation reactions are favoured (Fig. 4).
The rate at which mineral is lost from enamel and the depth to which the lesion progresses is a
HA is hydroxyapatite FA is fluorapatite
6
function of the rate-limiting process. Historically, the dissolution process in enamel was
described by a diffusion-control mechanism, where the rate-determining step was the transport of
mineral ions to-and-from the dissolution site at the advancing front of the lesion (Gray 1962;
Featherstone et al. 1979; Christoffersen and Arends 1982; Poole et al. 1981). More recent
evidence has suggested a surface-controlled mechanism of enamel demineralization, where the
rate of reaction was determined by the crystalline-level chemical dissolution processes at the
advancing front of the developing lesion (Josselin de Jong et al. 1987; Margolis and Moreno
1992; Gao et al. 1993b; Margolis et al. 1999; Anderson et al. 1998). It is likely however, that
dissolution processes in vitro and in vivo, entail a continuum of surface and diffusion controlled
mechanisms (Elliott et al. 2008), as enamel porosity, the presence of dissolution inhibitors from
saliva, mineral solubility and exposed surface area for dissolution may all influence the
governing reaction control mechanism (Anderson et al. 1998).
In order to gain insight into de- and remineralization mechanisms in a more controlled state,
artificial demineralizing systems have been developed to reproducibly create carious lesions
indistinguishable from natural caries lesions. A requirement for chemical demineralizing systems
is the incorporation of a surface inhibitor, partial saturation conditions and/or the modification of
the viscosity of the demineralizing medium. Surface inhibitors have taken form as one or a
combination of the following: incorporation of fluoride ions, diphosphonates, polyacrylic acid or
hydroxyethyl cellulose/carboxymethyl cellulose. The observation that an essentially intact outer
layer is maintained or forms superficial to a demineralized lesion body is remarkable. Over the
years, several theories have been proposed as to explain the origin of this layer. Inhibitors of
surface dissolution have been implicated in surface layer formation by adsorbing to enamel
surfaces rendering it less soluble and as a result acid must diffuse to deeper depth to find more
soluble mineral phases. Surface layer formation has also been related to the intrinsic structural
and chemical gradients in enamel, which significantly impact thermodynamic solubilities.
However, as early studies demonstrated that subsurface lesions could be created in OHAp pellets
and other permeable solids in the absence of surface inhibitors, fluoride, or chemical and
structural gradients, these influences alone could not be responsible for subsurface lesion
formation in enamel and other mechanisms were sought to explain surface layer formation (Aoba
et al. 1978; Anderson and Elliott 1985). These include the kinetic and thermodynamic model
7
based on the calcium phosphate solubility diagrams (Margolis and Moreno 1985), the coupled-
diffusion mechanism, where the chemical potential of one component influences the diffusion
properties of another (Anderson and Elliott 1987; Anderson et al. 2004) and gradients and
transport processes occurring during the dissolution process, where the out-diffusion of mineral
ions from subsurface layers to the bulk solution will reprecipitate within the surface zone
(Moreno and Zahradnik 1974). In summary, it is likely that the aforementioned mechanisms
operate in concert in order to produce subsurface demineralized lesion in enamel. The selection
of an appropriate demineralizing agent is critical in studies investigating efficacies of
remineralizing agents. It is important to note that parameters of a developed artificial lesion, such
as mineral loss, lesion depth and the mineral content of the surface layer, have a marked impact
on the outcome of subsequent remineralization (Lynch et al. 2007).
1.4 Histopathology of early caries lesions
The earliest macroscopic clinical evidence of caries in enamel is the presence of a ‗white-spot
lesion‘. The chalky-white discoloration of a white spot lesion as compared to sound enamel is the
result of a site-specific increase in porosity, which alters the light scattering properties of enamel
(Kidd and Joyston-Bechal 1997). White spot lesions are characterized by subsurface
demineralization below an apparently intact, mineralized surface layer (Fig. 5). The histological
appearance of a white spot lesion manifests itself as a crude 3-layered geometrical profile (Fig.
5). This profile consists of a relatively intact and unaffected surface layer at the most superficial
aspect of the lesion and overlying a demineralized lesion body, which makes up the largest
portion of a carious lesion, where bulk mineral loss occurs. Deep to the demineralized lesion
body is the presence of unaffected, sound enamel. The surface zone is essential in controlling the
rate and extent of both demineralization and remineralization processes and its destruction and
collapse will lead to cavitation (Robinson et al. 2000; Kidd and Joyston-Bechal 1997).
8
Figure 5. (A) Classical enamel caries lesion with surface layer and demineralized lesion body
clearly evident under polarized light microscopy (Adapted from Featherstone 2008). A clear
distinction of carious zones is evident in the image (A) and microdensitometric profile of the
lesion (B). The mineral volume percent is plotted vs. depth (d) from the surface. dsl denotes the
approximate thickness of the surface layer (SL) covering the lesion body (L); df refers to the
position of the lesion front (adapted from Arends and Christoffersen 1986).
1.5 The remineralization process
At neutral pH without acid challenge, the main driving force for remineralization is the passive
transport of salivary or plaque calcium and phosphate ions down their concentration gradient into
the lesion body (Chow and Vogel 2001) (Fig. 3). Soluble calcium phosphate phases are
transformed to a solid and less acid soluble phase through the precipitation of fluorapatite (FAP)
or fluoridated hydroxyapatite (F-OHAp) on existing demineralized crystallites or through the
nucleation of new crystallites. This remineralization process is a natural chemically inorganic
process that does not require soft-tissue or cellular biological processes (Featherstone 2009), as
in bone and dentin remodelling mechanisms. As the remineralization process in vivo takes
considerable amount of time, a multitude of studies have investigated ways to enhance this
process, the most well-documented being fluoride incorporation. Fluoride has been known for
decades to inhibit lesion demineralization and promote consolidation by crystal growth as
fluoride enhances the driving force for mineral precipitation (ten Cate and Featherstone 1991).
Due to its anionic character, fluoride has a high affinity for positive ions such as calcium (CDC
2001). Fluoride ions can substitute completely, or partially, for the OH- ions in the OHAp lattice
to give rise to the formation of the mineral FAP or F-OHAp, respectively. The displacement of
OH- ions with F
- ions in the OHAp lattice results in increased stability, and reduction in the
volume of the unit cell, creating a less soluble apatitic phase (Aoba 1997). Plaque contains a
large amount of fluoride in many different forms, free, loose, bound or firmly bound and each
(A) (B)
9
affect relative de- and remineralization processes in different ways (ten Cate 1983). In its tooth-
bound state, fluoride incorporated during enamel maturation is stable and does not play a
significant role in the remineralization process, whereas ambient free fluoride levels have been
implicated as the major determinants in promoting remineralization of enamel lesions (Faller
1995; Chow and Vogel 2001; Wong et al. 1987). As a result, the post-eruptive effect of a
topically applied fluoride, through varnishes, dentifrices or mouthwashes, is essential in
modulating the dynamic equilibrium at the tooth‘s surface promoting remineralization of
demineralized lesions (Fejerskov et al. 1994, Aoba 1997).
The mechanism of fluoride incorporation as FAP concomitant with its strong affinity for calcium
ions has been the basis for numerous in vivo and in vitro studies demonstrating enhanced
remineralization, particularly in the presence of low fluoride concentrations in solution
(Featherstone and Zero 1992; ten Cate 1990; ten Cate 1997). Varughese and Moreno (1981)
found an increase in the rate of crystal growth when fluoride was added to a calcifying solution
at concentrations as low as 0.05 ppm. Silverstone et al. (1981) found a substantial reduction
(72%) in lesion area when fluoride at 1 ppm was added to a calcifying solution compared to the
same solution in the absence of fluoride (22%). In the same study and in others, no additional
effect was observed in solutions containing 10 ppm fluoride, illustrating that only low levels of
fluoride are required to induce remineralization throughout the body of the lesions (Zahradnik
1979; Silverstone et al. 1981). A similar significant uptake of mineral was observed in other
studies implementing a 1 ppm fluoride solution (ten Cate and Arends 1977; Zahradnik 1979;
Koulourides et al. 1974; ten Cate 2001; Thuy et al. 2008; Nancollas 1979; Lammers et al. 1991;
Amjad and Nancollas 1979; Iijima et al. 1999; Zimmerman et al. 1978). Crystal diameters that
decreased substantially within the demineralized zones were found to increase significantly in
size following remineralization toward the sound enamel level and in some cases produced
diameters 2 - 3x the size of sound enamel crystals (Silverstone 1977; Silverstone et al. 1981;
Silverstone 1983; ten Cate and Arends 1977; Arends and Jongebloed 1979). Employing high
fluoride concentrations, at the levels contained in mouthrinses, varnishes and dentifrices, calcium
fluoride (CaF2) or a calcium-fluoride-like material is formed, and has been suggested to act as a
pH-dependent fluoride reservoir which would release fluoride under subsequent acidic
challenges (ten Cate 1997).
10
Although intuitively mineral reintroduction into porous demineralized enamel from saliva seems
simple, this process does not proceed without its challenges. One challenge relates to the
limitation of remineralization by the diffusion of ions from the external solution (ten Cate 1990).
With excessive fluoride concentrations and/or large enamel supersaturation conditions rapid
mineral deposition may occur preferentially within the enamel surface layer and obstruct surface
enamel porosities leading to a disconnect between the external environment (saliva and plaque
fluid) and the internal environment (subsurface lesion body). Thus, the deposition of mineral at a
particular depth within a caries lesion depends on both the local availability of partially
demineralized crystallites, which act as mineral scaffolds, as well as the local supersaturation,
fluoride levels and pH (ten Cate 1990). The aforementioned mechanism of enhanced surface
layer mineral deposition has been proposed to explain the fact that remineralization is never
complete (Larsen and Fejerskov 1989; Pearce et al. 1995; Chow and Vogel 2001; Arends and ten
Cate 1981; Lagerweij and ten Cate 2006) and points to the accelerated effects of fluoride-
induced remineralization during the initial stages whereas a plateau effect is often observed at
later periods (Al-Khateeb et al. 2000). However, additional factors such as the presence of
salivary proteins in vivo also play a vital role (Zahradnik 1979; Fujikawa et al. 2008). A clinical
lesion judged as not progressing (or inactive) and where surface enamel is restored chemically
and mechanically above a subsurface lesion is termed an arrested lesion. In contrast to active
lesions, arrested lesions are inherently stable and do not respond to remineralization therapies.
Thus, the clinical activity of a lesion is vital in determining susceptibility to remineralization
therapies and preventing overtreatment of lesions that are chemically stable.
An unfavourable trade-off of fluoride ingestion to reduce caries is dental fluorosis. This results
from excessive intake of fluoride during enamel development (Aoba and Fejerskov 2002).
Fluorotic enamel appears histologically as hypomineralization of the subsurface enamel, covered
by a well-mineralized outer surface layer (Fejerskov et al. 1994). The severity of dental fluorosis
is due to the cumulative effect of fluoride over time and therefore can act as a direct indication of
the degree of past fluoride exposure. However, even low levels of fluoride ingestion
(approximately 0.03 mg/kg body weight) can result in weak fluorotic manifestations in the
enamel (Aoba and Fejerskov 2002). More severe cases of dental fluorosis are characterized by
extensive hypomineralization of the porous subsurface layer with exceptionally brittle
11
mineralized surface enamel that may be susceptible to the formation of defects under regular
forces during mastication (Fejerskov et al. 1994). Moderate-to-severe fluorosis is observed as
pits and grooves on the enamel surface or a mottled appearance (Limeback 1994).
1.6 Demineralization and remineralization evaluation techniques
1.6.1 Physical principles underlying light – tooth interactions
When light interacts with teeth, a fraction is reflected, scattered or transmitted from the tissue
and a part may be absorbed within the tissue (Fig. 6). The absorbed energy can be converted
non-radiatively or radiatively as heat or fluorescence, respectively. Scattering refers to the
process whereby a photon changes its path without losing its energy. This typically occurs
following the interaction of photons with small particles or inhomogeneities within the tissue
volume. Following absorption events, further scattering or transmission events can take place.
Fundamental processes underlying light-tissue interactions involve optical phenomena within the
tissue volume. Parameters used to characterize tissue optical properties include, photon
scattering (μs) and absorption coefficients (μa), which refer to the average number of absorption
and scattering events per unit length of a photon propagating though the medium (Minet et al.
2006). Together they amount to the total attenuation coefficient, given as: μt = μa + μs and is an
important optical parameter defining the total optical penetration depth in a tissue at a given
excitation wavelength (Minet et al. 2006). As noted earlier, enamel and dentin are composite
materials with chemical and structural gradients varying as a function of depth. As a result, light
propagation in enamel and dentin, as in other biological media, can be described as a highly
scattering random medium, i.e. turbid tissue. Scattering in tissues is a result of structural
inhomogeneities and a function of the wavelength of light. There is a direct relationship between
scattering and the wavelength of light, where longer wavelengths scatter less than shorter
wavelengths and consequently offer deeper penetration into tissues. However, deeper penetration
comes at the expense of a poorer resolution (Hall and Girkin 2004). In addition to μs, the
scattering phase function (g), also referred to as the anisotropy factor or cosine of the scattering
angle, describes the directional nature of scattering events and is highly dependent on the nature
of the scattering medium. Biological tissues are highly forward scattering (g ≈ 1) in the visible,
meaning light paths are long with high absorption probabilities, with typical g values between
12
0.79 - 0.98 (Cheong et al. 1990). By measuring the aforementioned optical constants, light
transport process within a tissue, such as tooth enamel, can be almost completely described.
Figure 6. Light- tissue interaction. See text for description. [Adapted from Hall and Girkin
2004].
For the detection of early caries the selection of the correct wavelength of light is vital. The
wavelength must be chosen such that sound enamel is transparent, meaning light absorption is
minimal. This typically occurs at the longer wavelength end of the visible spectrum (visibly red)
and into the infrared, as light scattering and absorption processes are much lower and therefore
transmission is optimal (Featherstone and Fried 2001). However, farther into the infrared
absorption coefficients markedly increase due to the strong absorption peak of water (Fig. 7A,
B). Wavelengths in the range ≈2 µm < λ > ≈650-nm ensure optimal light penetration within
biological tissues (Gupta et al. 2007).
13
Figure 7. Wavelength dependence of (A) the water absorption coefficient (Modified from Gupta
et al. 2007) and (B) the infrared transmission properties of the primary absorbers in dental
enamel (Modified from Featherstone and Fried 2001).
The presence of defects in enamel (caries lesion) will alter optical scattering and absorption
properties which subsequently influence converted energy signals, such as thermal emissions or
fluorescence intensity (Hall and Girkin 2004). A detailed list of optical absorption and scattering
coefficients for sound and carious enamel are presented in Table 1. These optical properties play
a vital role in determining the energy distribution within the tissue volume. Larger values for
these coefficients indicate that incident photons are absorbed rapidly in near-surface regions,
resulting in smaller penetration depths and poorer resolution of deeper underlying structures. The
origin of light scattering in enamel has been ascribed to both the crystallites and prisms
components of enamel. Typical absorption and scattering properties for dentin, not included
within Table 1, are much larger than the corresponding values for enamel. A larger absorption
coefficient of approximately 300 – 400 m-1
is typical of dentin and may be related to the larger
organic makeup of dentin compared to enamel. Although not considered in the present
investigations, the scattering properties of dentin were found to be highly dependent on the
density of tubules rather than mineral content and varied from 3000 – 20 000 cm-1
over the
visible light spectral range (400 – 700 nm) (Zijp and ten Bosch 1991).
Teeth, similar to other biological tissues, are complex multi-layered structures with absorption
and scattering properties changing as a function of depth. As a result, attempts to derive
complete analytical solutions to model light propagation within the tissue volume are both
impractical and virtually impossible. Rather approximations are implemented. Historically,
(A) (B)
14
optical properties of enamel and dentin have been determined by measurements of the
reflectance and transmission through thin sections followed by the application of a theoretical
formalism to the experimental data to extract optical absorption and scattering coefficients.
Theoretical models typically used include Kubelka-Munk theory (Ko et al. 2000; Zijp 2001) or
the Monte Carlo approach (Fried et al. 1993; Fried et al. 1995; Mujat et al. 2003). The
determination of the aforementioned optical parameters (μa, μs and g) is a difficult task requiring
the use of complex numerical models, assumptions and approximations which explains why
there have been relatively few reports on optical evaluation of dental tissues. Furthermore, the
use of thin sections, mostly of known thickness, is a requirement for transmission measurements.
These preparative samples add additional variability that does not reflect the conditions in the
oral cavity, thus making it difficult to relate in vitro determined optical properties of prepared
sections to intact substrates in vivo (Chebotareva et al. 1993).
Table 1. Published set of optical properties of sound and carious enamel at relevant wavelengths.
* Bovine enamel sections
ffi Zijp (2001), calculated from Spitzer and ten Bosch (1975)
† From diffuse reflectance and transmittance by a modified CCD camera method
Optical Properties of Enamel
Wavelength (nm)
Absorption coefficient μa, [m
-1] Scattering coefficient
μs, [m-1]
References
Sound Enamel
543 <100 10 500 Fried et al. (1995)
600 7 000* Groenhuis et al. (1981)
600 3 300 Groenhuis et al. (1981)
600 <100 ≈6400 Mobley and Vo-Dinh (2003)
632 <100 6 000 ± 1 800 Fried et al. (1995)
632 97‡ 2 300‡ Spitzer and Ten Bosch (1975)
633 6 600 Zijp et al. (1995)
633 97 110 Zijp (2001) and references therein
633 40 1 000‡ Spitzer and Ten Bosch (1975)
700 <100 ≈5000 Mobley and Vo-Dinh (2003)
700 5 500* Groenhuis et al. (1981)
700 2 700 Groenhuis et al. (1981)
800 <100 ≈3300 Mobley and Vo-Dinh (2003)
1000 <100 ≈1600 Mobley and Vo-Dinh (2003)
1053 <100 1 500 Fried et al. (1995)
1 730 – 5 140†* Ko et al. (2000)
Carious Enamel
600 55 000 Groenhuis et al. (1981); Zijp (2001)
633 157 000 Spitzer and ten Bosch (1977)
633 42‡ 32 000‡ Spitzer and ten Bosch (1975)
5 300 – 14 280†* Ko et al. (2000)
15
As most laser-tissue interactions are thermal in nature, the optical-to-thermal energy conversion
reactions following photon absorption and the subsequent non-radiative heat conversion,
propagation/decay and tissue responses are important physical parameters to consider. Thermal
parameters include thermal diffusivity (α) and thermal conductivity (κ), which refer to the rate
and amount of heat diffusion through a medium, respectively. Thermal diffusivity is an
important thermophysical parameter given by:
C
, (1)
where α is the thermal diffusivity, κ is the thermal conductivity, ρ is the density and C is the heat
capacity. Investigations reporting thermal properties of sound and carious enamel and dentin are
scarce. Furthermore, of those investigations, majority, if not all, are concerned with the
properties of sound enamel. A list of parameters derived from published literature is presented in
Table 2. Direct measurements of thermal conductivity and diffusivity are difficult to assess in
intact samples as well as thin slabs. Measurements are often carried out using thin enamel
sections and thermocouples. Considering that enamel and dentin sectioning can induce cracks,
defects or other anomalies which all significantly affect optical and thermal properties, being
able to extract optical and thermal parameters from whole teeth non-destructively would be
invaluable.
Table 2. Published set of thermal properties of sound and carious enamel.
* Carious sample
ffi Compressed hydroxyapatite powder
Thermal Properties of Enamel
Thermal Conductivity, κ *W/m·K+
Thermal Diffusivity, α *m2/s]
References
0.65 Soyenkoff et al. (1958)
0.87 Saitoh et al. (2000)
0.88- 1.07 Craig and Peyton (1961)
0.90/0.72* El-Brolossy et al. (2005)
0.93 4.7 x 10-7 Brown et al. (1970)
0.77 3.22 x 10-7 Minesaki (1990)
0.88 4.69 x 10-7 Braden (1985)
0.93 4.1 x 10-7 Barker et al. (1972)
0.92 4.2 x 10-7 Braden (1964)
4.09 x 10-7 O’Brien (1997); Panas et al. (2003)
2.27 x 10-7 Panas et al. (2003)
4.0 x 10-7‡ Rodriguez et al. (2001)
16
1.6.2 Microradiography
The most common microradiographic method is transverse microradiography (TMR) (Arends
and ten Bosch 1992). TMR is a destructive analytical technique that yields the most detailed
quantitative information to date (Damen et al. 1997). It provides direct measurements of the
mineral content of the examined dental tissues. In TMR, samples are cut into thin slices and
oriented perpendicularly to the anatomical tooth surface. The sections are placed on a piece of
high-resolution radiographic film and together with an aluminum step-wedge are irradiated with
monochromatic x-rays. X-ray absorbance shown by the optical density of the developed film can
be used to calculate two different parameters, mineral loss (vol%.μm) and lesion depth (μm)
(Arends and ten Bosch 1992). As enamel or dentin is treated with demineralizing solutions lesion
depth and mineral loss values increase, whereas both parameters are reduced upon
remineralization. The main setback of TMR is its destructiveness and tedious procedure of
sample sectioning and preparation. A recent advancement in the TMR software allows for the
accurate determination of mineral content in curved tooth surfaces by an algorithm that
mathematically flattens the section (de Josselin de Jong and van der Veen 2007). This is
completed through several scans of the microradiographed section and avoids additional sources
of error in mineral loss and lesion depth calculations and their overestimation due to the natural
curvature of tooth surfaces. Damen et al. (1997) judged TMR as the current ―gold standard‖ and
an appropriate tool for quantifying minor changes in mineral density over time.
1.6.3 Optical fluorescence techniques
Recently evolving methods of caries detection, utilize light sources as a tool to indirectly assess
the state of a tooth‘s health, or level of mineral loss. Considering the ordered structure of enamel
prisms and dentinal tubules, light, in the visible and near infrared part of the electromagnetic
spectrum, is able to propagate fairly well through teeth (Hall and Girkin 2004). As a result,
disruptions in the regular pattern of a tooth, i.e. a carious lesion or any other anomaly, will create
discontinuities within the medium, leading to enhanced light scattering and an alteration of the
optical path lengths. In addition to scattering, absorption and fluorescence properties may
likewise be altered by the presence of defects. Fluorescence is the phenomenon whereupon
electrons are excited from a ground or lower energy state to a higher energy state, and upon de-
excitation results in the emission of energy as longer wavelength light (Tranæus et al. 2005).
17
Although, some of the inherent fluorescence, or autofluorescence, of teeth can be attributed to
chromophores, the material responsible for the fluorescent properties remains uncertain (Stookey
2005). Thus, the baseline fluorescence of sound teeth may be a result of the combination of
inorganic matrix with absorbing organic molecules (Hibst and Paulus 1999).
Two non-destructive optical techniques that take advantage of fluorescence emission are
Quantitative Light-induced Fluorescence (QLF) and DIAGNOdentTM
.
1.6.3.1 Visible light Fluorescence- QLF
QLF is an optical fluorescence technique that distinguishes carious from non-carious regions
based on fluorescence radiance, which is lower in demineralized lesions (Angmar-Månsson and
ten Bosch 2001). Instruments using optical fluorescence, like QLF imaging, capture images of a
tooth by illuminating with blue-violet light from an arc lamp, which emits, with a wavelength of
370nm, white light filtered through a blue-transmitting filter based on xenon gas excitation and
emission technology. The basis of the technology is that demineralized areas will fluoresce less
than sound enamel and this difference is quantified as the fluorescence loss parameter, Q. The
cause of fluorescence loss was proposed to be due to 2 sources. The first is the presence of
chromophores, where in a demineralized area protein chromophores will be removed, resulting
in a loss in autofluorescence. The second source is due to the light scattering and absorption
properties of a healthy versus demineralized area (Stookey 2005). Due to the enhanced light
scattering properties found in demineralized lesions, the path of the light traveling within the
enamel is much shorter, compared to healthy enamel, and as a result the smaller amount of
absorption per volume yields lower fluorescence values. Furthermore, the scattering within
demineralized lesions creates an obstruction which prevents light travel from the fluorescing
dentin to the surface and excitation light from the surface to the dentin (Tranæus et al. 2005;
Angmar-Månsson and ten Bosch 2001). QLF has been demonstrated both in vitro and in vivo to
be sensitive to smooth-surface caries (Shi et al. 2001), occlusal caries (ten Cate et al. 2000),
secondary caries (Ando et al. 2004) and root caries (Gonzalez-Cabezas et al. 2001), while
attaining values of sensitivity and specificity from the aforementioned studies of no lower than
0.76 and well above 0.78, respectively. QLF, is, however, limited by its penetration depth to ≈
400 μm (Tranæus et al. 2005). Amaechi and Higham (2002) demonstrated the efficacy of QLF as
18
a tool to detect and longitudinally monitor the progression of artificial caries-like lesions and
their subsequent remineralization. Potential disadvantages of the QLF system are derived from
the fact that dehydration can significantly affect lesion fluorescence, putting into question the
clinical interpretation of the fluorescence values (Al-Khateeb et al. 2002, Pinelli et al. 2002).
Furthermore, repositioning the QLF probe at the same measurement point for longitudinal
monitoring of lesions was found to be challenging, especially when lesion parameters change
over time. Lastly, QLF is unable to accurately detect lesions interproximally (Pretty et al. 2002).
1.6.3.2 Laser fluorescence—DIAGNOdentTM
The type of fluorescence emission depends on the wavelength of the source light. Blue
fluorescence and yellow-orange fluorescence are emitted from incident light sources in the near-
ultraviolet region and in the blue-green region, respectively. The third type of fluorescence is red
fluorescence, where incident light is in the red or near-infrared region (Tranæus et al. 2005). A
device called DIAGNOdentTM
(DD) takes advantage of the red fluorescence by emitting light in
the visible-red region (λ = 655 nm) to induce red and near-infrared fluorescence, which is
collected by a photodiode combined with a long pass filter (transmission > 680 nm) as the
detector. The DD device involves point measurements given on a linear scale and presented on
the display screen as an integer between 0 and 99 (Pretty 2006). The nature of red fluorescence
has been attributed to different protoporphyrins that are present as bacterial breakdown products
and metabolites for other oral bacteria. The exact mechanism by which DD monitors incipient
caries has not been fully elucidated. However, due to the nature of red fluorescence, the DD
readings appear to be based on bacterial porphyrins rather than enamel mineral dissolution
(Pretty and Maupomé 2004; Astvaldsdottir et al. 2010). While DD had demonstrated greater
sensitivity compared to traditional diagnostic methods (visual inspection, radiography), a greater
prospect of encountering false-positive diagnoses has limited its clinical usefulness. For
example, DD readings have been found to be complicated by factors such as stains, plaque and
tooth hydration levels and over-scored due to disturbances in tooth mineralization as a result of
developmental hypomineralization (Ferreira Zandoná and Zero 2006). Disadvantages in the DD
system are related to the aforementioned high incidence of false-positive diagnoses. In
accordance with all other optical techniques, the presence of stains will highly confound the
19
technique (Hall and Girkin 2004). In addition to stains, calculus, plaque, some composite resin
filling materials, remnants of polishing pastes and developmental anomalies can all affect the
device readings leading to false-positive diagnoses (Pinelli et al. 2002, Lussi et al. 1999, 2005,
Shi et al. 2001). As a result, DD readings, should not solely be the basis for primary clinical
diagnosis, but rather should be used as adjuncts to current diagnostic tools.
1.6.4 Optical Coherence Tomography
Optical coherence tomography (OCT) is a non-destructive optical imaging technique based upon
light interference within semi-transparent materials, such as teeth. This methodology has been
developed and used in the field of opthamology for over a decade in addition to applications in
imaging of skin and gastrointestinal tissues (Hall and Girkin 2004). The OCT system illuminates
the teeth at 850 nm or 1310 nm wavelengths, which result in an optical imaging depth of 0.6 – to
2.0 mm, respectively. Through near-IR illumination of the tooth, a combination of high spatial
resolution (≈10-20 μm) and real-time 2-D depth visualization can be acquired (Amaechi 2009).
An OCT system contains a Michelson interferometer that splits the incident light beam into 2
coherent beams of light, the sample beam and the reference beam. The sample beam will enter
the tissue and scatter both in the forward and back direction, where the scattering properties are
dependent on anomalous structures and changes in refractive index within the tissue. The back
scattered sample beam is recombined with the reference beam and the generation of interference
patterns are observed by a photodetector. The intensity of the generated interference pattern is a
function of the scattering and caused by changes within the tooth structure (Hall and Girkin
2004). Generating a depth profile at a single point along the laser trajectory depth from a single
point on the tooth surface is referred to as an A-scan (Choo-Smith et al. 2008). Taking several A-
scans as a function of position along a line produces information from the assembled adjacent A-
scans in order to optically slice the tooth tissue, producing a 2-dimensional depth image referred
to as a B-scan (Hall and Girkin 2004). As conventional OCT systems were found to be highly
susceptible to the strong surface reflection from the high refractive index enamel, a modification
of the system to produce polarization-sensitive OCT (PS-OCT) was developed and uses linearly
polarized light which does not depolarize from surface reflection (Jones and Fried 2006). The
PS-OCT system had been successfully implemented to image artificial and natural caries lesions
and assessed severity in terms lesion depth in enamel (Fried et al. 2002) and the severity of
20
demineralization on dentin surfaces (Manesh et al. 2008). Furthermore, additional studies have
demonstrated that PS-OCT can image fluoride-enhanced surface layer remineralization in
enamel (Jones and Fried 2006) and remineralization of dentin (Manesh et al. 2009). OCT is still
too sensitive to natural crystal defects in enamel and produces a non-zero reflection baseline
which compromises its sensitivity to detect caries.
1.6.5 Photothermal Radiometry and Modulated Luminescence
Following initial absorption events within a medium, atoms or molecules are raised to excited
electronic states. Common modes of de-excitation processes involve a series of either radiative
transitions/decays, resulting in the production of longer wavelength light, fluorescence, and non-
radiative transitions which result in the production of heat. The basis of frequency-domain
photothermal radiometry (PTR) relies on the conversion of absorbed optical energy into thermal
energy, and the subsequent observation of modulated mid-infrared emission (Fig. 8). A periodic
heat source, such as an intensity modulated laser beam, results in the generation of a periodic
temperature distribution within a material. This oscillatory temperature field arising within each
light-absorbing layer of a material launches temperature waves known as ‗thermal waves‘ which
rapidly decay over the sample depth. Thermal waves are very heavily damped with decay
constants equal to the thermal diffusion length (Almond and Patel 1996). The thermal diffusion
length is a quantity given by equation (2):
ff
(2)
where α is the thermal diffusivity (given in equation 1) and f is the modulation frequency. The
thermal diffusion length is indicative of the depth penetrability of a thermal wave technique
analogous to the optical absorption depth of electromagnetic waves. It is clear from the formula
of the thermal diffusion length, that there is a strong dependence of the thermal diffusion length
on the thermal properties of the medium, more specifically thermal diffusivity (α), and laser
modulation frequency. Thus, a high diffusivity material, or low modulation frequency will
effectively enhance propagation and capture of thermal waves deeper into the medium. In
metals, optical absorption depths are limited to the nanometer scale resulting in heat generation
at the surface of the metal diffusing into the bulk. Therefore, conductive heat transfer dominates
21
and thermal diffusion lengths are much larger than optical penetration depths. This underscores
the sensitivity of photothermal techniques to the inspection of optically opaque materials well
beyond the range of optical imaging devices. In non-metallic polycrystalline materials, such as
teeth, the case is much different. Longer optical absorption depths mean that absorption
processes are not limited to the surface and as a result photothermal effects cannot be considered
as a simple surface heat source. As optical absorption depths are longer, elemental heat sources
can arise within the bulk of the medium. Thus, thermal wave generation within the bulk of an
optically-absorbing semi-infinite translucent material will depend on both optical absorption
processes, in addition to the thermal diffusion length. The resultant PTR signal is derived from
optical absorption and thermal wave generation, which creates a modulation in the temperature
of the sample surface by integrating all contributions over the depth of the sample (Almond and
Patel 1996).
Thermal contributions to the overall PTR signal are generated in 2 distinct modes, conductively
and radiatively. The conductive component dominates in the near-surface regions of a sample, as
described above, and is a function of the thermal properties of the sample and the laser beam
modulation frequency. The radiative component, a function of the longer penetration depth of the
diffusively scattered optical field, will also influence IR signal generation. An enhanced radiative
component, providing information from deep subsurface structures, originates in optically
absorbing and thermally-emitting subsurface features like incipient carious lesions (Jeon et al.
2004a). From a single PTR scan, the generated amplitude and phase signals and the improved
signal-to-noise ratio (SNR) due to lock-in detection (Mandelis 1994) increases the amount of
information gathered, compared to time-domain methods, which is the prominent feature of
frequency-domain techniques.
22
Figure 8. Photothermal and luminescence effects upon excitation with an intensity modulated
laser beam.
As mentioned above, PTR involves the monitoring of IR emissions using mid-IR detectors.
However, where the IR signal is collected depends on individual experimental methods. The
observation point on the sample can occur in 2 modes. Light excitation and IR emissions
collected from the same surface are referred to as ‗backscattered PTR‘ whereas excitation and
collection of IR emissions on opposite surfaces is referred to as ‗transmission-mode PTR‘.
As a complementary signal channel, modulated luminescence (LUM) monitors the optical-to-
radiative energy conversion, where photon absorption and excitation to a higher-energy state is
followed by de-excitation to a lower energy state and emission of longer wavelength photons
(Fig. 8). As a purely optical technique the high scattering coefficients of sound and carious
enamel and dentin significantly limit optical penetration depths. Investigation of the modulated
luminescence signal from enamel samples revealed the existence of a long (≈ ms) and short (≈
μs) relaxation lifetimes, the longer of which was present in all teeth and is insensitive to the
overall health of a tooth. In contrast, the shorter lifetime established a degree of sensitivity to the
quality of enamel (Nicolaides et al. 2002).
The combination of PTR and LUM amplitude and phase signals provides four sensitive channels
to assess the physical condition and the state of health of teeth. Changes in enamel and/or dentin
mineral density and porosity, characteristic of de-and re-mineralization processes, will alter the
optical and thermal properties of the tissue and the resultant mid-IR emissions. PTR-LUM has
demonstrated the ability to explore up to 5 mm below the enamel surface and provide
information regarding minor subsurface perturbations (Jeon et al. 2004a). PTR-LUM has been
23
shown to have the potential to detect and longitudinally monitor early interproximal
demineralization (Jeon et al. 2007), pit and fissure caries (Jeon et al. 2004), and artificial
demineralized and remineralized carious lesions on roots and enamel of human teeth (Jeon et al.
2008). The continual development and enhancement of the PTR-LUM system as a laboratory
investigation device has been investigated for nearly a decade at the Centre for Advanced
Diffusion-Wave Technologies, at the University of Toronto. A clinical prototype based on PTR-
LUM phenomena is currently in development by Quantum Dental Technologies, marketed as
The Canary Dental Caries Detection System.
Thanks to the development of PTR to detect thermal waves along with its strong, comprehensive
theoretical underpinning, describing the generated thermal signal inside a sample, the extraction
of optical and thermophysical material properties have been made possible. PTR measurements
have been used previously for depth analysis and non-destructive extraction of optical and
thermal properties from opaque materials (Tam 1985). Furthermore, in layered materials, PTR
has been used to investigate changes in the thermal properties of specific subsurface layers
(Balageas et al. 1986). In terms of teeth, PTR in its pulsed-mode has been investigated toward
the evaluation of optical absorption coefficients of enamel (Zuerlein et al. 1999; Zuerlein et al.
1998) and dentin (Chebotareva et al. 1993) at tooth ablation wavelengths (9-11 µm). In contrast
to frequency-domain PTR, the pulsed mode has only a single channel available, formed from the
rapid temporal decay of the thermal pulse (Nicolaides et al. 2001). Earlier reports on frequency-
domain PTR measurements introduced a robust and complex fitting algorithm for the generation
of independent sets and simultaneous extraction of optical and thermal parameters and thickness
values for the each effective layer considered in the 3-layer tissue analysis (Matvienko et al.
2009a/b). The robustness of the algorithm i.e., its independence of the initial estimation of
parameters, is an extremely important parameter in defining a unique solution in the multi-
parameter fitting procedure (Matvienko et al. 2009b).
In summary, combining the depth profilometric nature of PTR and the extraction of optical and
thermophysical properties from PTR data, this technique has been proven advantageous to
resolve the internal structure as well as characterize tissues thermophysically, in a non-contact,
non-destructive fashion.
24
2 Rationale
Dental caries is an infectious bacterial disease affecting mineralized tooth tissues, enamel and
dentin, characterized by loss of inorganic structure in subsurface layers (demineralization). Early
detection of this disease prior to cavitation, allows preventive therapies to be instituted by
promoting inorganic ion re-uptake (remineralization). The most studied therapeutic agent is
fluoride, with voluminous literature on the effects of fluorides in enhancing the remineralization
process. The influx of light and laser based caries detection systems, all pride on the unique
optical properties of teeth. Furthermore, in order to augment the effectiveness of each laser based
system, optical and thermal interactions between the laser and tissue volume must be
meticulously explored as changes in these properties may be reflective of the overall tooth
health. As enamel is a structurally and chemically composite tissue, the process of optical and
thermal property extraction is both technically challenging and computationally extensive,
typically involving destructive analysis of thin tissue sections.
As an emerging non-destructive technique, frequency-domain photothermal radiometry (PTR) is
an established sensitive methodology to characterize pathological dental tissues. PTR is based on
the generation of diffuse-photon-density waves in turbid media by a harmonically modulated
laser beam to induce an oscillatory temperature thermal-wave field, which can be detected
remotely with mid-IR detectors. Modulated luminescence (LUM) monitors the optical-to-
radiative energy conversion, a complementary signal channel.
The purpose of the present study was to evaluate the ability of back-propagation and
transmission PTR-LUM to detect, longitudinally monitor and quantify simulated enamel
demineralized and remineralized lesions. The project is divided into 2 sections based on the
PTR-LUM detection mode. The first section is concerned with PTR-LUM in backscatter mode
where a combined theoretical formalism applied to the experimental PTR signals was used to
extract opto-thermophysical properties from the treated enamel. The second chapter investigates
PTR-LUM in transmission-mode where changes as a function of demineralization and
remineralization time were monitored in real-time without sample disruption.
25
Hypothesis:
The combined detection modes of PTR-LUM are efficacious in measuring and quantifying
mineralized layers generated through dissolution and mineral deposition reactions, characteristic
of demineralization and remineralization processes.
The objectives of the first study investigating PTR-LUM in backscatter mode are two-fold:
1. To identify a relationship between PTR-LUM amplitude and phase signals to histological
features of demineralized and remineralized lesions through microradiographic analysis
2. To develop a coupled diffuse-photon-density and thermal wave model for the extraction
of opto-thermophysical properties from PTR signals and relate these changes in
properties to morphological changes during de- and re-mineralization.
Overall, this study is important in establishing PTR-LUM as a novel combination analytical
technique for the non-destructive evaluation of mineralized multi-layered enamel and
quantitatively characterizing the fundamental processes governing enamel demineralization and
remineralization.
26
3 PTR-LUM Backscatter Mode: Materials and Methods
3.1 Sample Collection and Sterilization
Forty-two mature, permanent human molars extracted by dental professionals for orthodontic or
other surgical purposes were collected, debrided of all soft attached connective tissue and sealed
in plastic containers containing distilled water at 4oC until use. The study protocol was approved
by the University of Toronto Ethics Review Board (Protocol #25075). Samples were collected
from dental offices in the Greater Toronto Area. Samples were submitted to the University of
Toronto‘s Department of Environmental Nuclear Science Gamma Irradiation services for
sterilization prior to utilization. Irradiation took place in a gamma cell (type G.C.220) at a dose
of 4080 Gy and a gamma dose rate of 3.3 kGy/hr. Gamma radiation has been established as an
effective and acceptable sterilization method for the elimination of microbes from dental
specimens without significantly affecting demineralization and remineralization rates (Amaechi
et al. 1999).
3.2 Sample Preparation
All sterilized samples (n = 42) were mounted on LEGO® blocks in order to allow for precise
realignment of samples on the sample stage during subsequent measurements. A single region
per tooth (lingual/palatal surface) was selected and assessed visually to ensure no visible stains,
cracks or other surface imperfections were present. Lingual/palatal surfaces of maxillary and
mandibular molars were the selected sites for treatment and PTR-LUM measurements. These
surfaces were previously found to be more susceptible to acid dissolution with the least amount
of variability compared to labial/buccal surfaces (Tucker et al. 1998). All samples were sealed in
a chamber containing Petri dishes of distilled water to maintain ambient humidity conditions.
Samples were maintained in the humid chamber at all times, excluding the time when
measurements and treatments were being executed. Retaining the samples in the humid chamber
maintained ambient conditions in a thermodynamically stable state and preserved sample
hydration for the duration of the experiment. Prior to the first PTR-LUM scan, each sample was
covered in 2 coats of transparent, acid-resistant nail varnish on all surfaces excluding the enamel
surface delimiting a 6 mm X 6 mm treatment window.
27
3.3 Demineralization and Remineralization Treatments
Samples were randomly distributed into 4 treatment groups outlined in Table 3. The first
treatment group was only demineralized. Two samples, not included in the group 4 matrix, were
used strictly for theoretical analysis and were demineralized for 40 days. The demineralizing gel
was changed after 20 days. The remaining 3 treatment groups were subjected to a mineral
solution with variations in fluoride content; either no fluoride, low fluoride (1 ppm) or high
fluoride (1000 ppm), in the form of NaF. All demineralizing and remineralizing treatments were
conducted at room temperature and in sealed experimental tubes.
Table 3. Treatment groups for backscatter PTR-LUM (n = 40)
3.3.1 Demineralization
The demineralizing medium consisted of an acidified lactic gel containing 0.1M lactic acid
gelled to a thick consistency with 6% hydroxyethyl cellulose (HEC) and adjusted to pH 4.5 by
the addition of 0.1M NaOH (Amaechi et al. 1998). Samples incubated in demineralizing
solutions were left unagitated for the duration of the treatment period. Mounted samples were
inverted and immersed in individual Falcon tubes containing 30 mL of acidified gel. Samples
were demineralized for a period of up to 10 days, with sample interruption for PTR-LUM
measurements after 5 days and 10 days of acid exposure. Following demineralization, all
samples were rinsed under running distilled water for 2 minutes in order to remove any residual
adsorbed gel on the enamel surface. The teeth were dried in ambient air for 1 hour, followed by
incubation in the humid chamber until PTR-LUM scans were executed. The same procedure was
followed for PTR-LUM scans after 5 days of demineralization.
Treatment Group Demineralization treatment (days)
Remineralization treatment (days)
Sample Size
Group 1: No fluoride remineralization 10 28 10
Group 2: Low fluoride remineralization (1 ppm F)
10 28 10
Group 3: High fluoride remineralization (1000 ppm F)
10 28 10
Group 4: Demineralized only 10 ----- 10
28
3.3.2 Remineralization
The constituents of the remineralizing solution are outlined in Table 4 (Amaechi and Higham
2002). Individual experimental groups were exposed to a remineralizing solution for 4 weeks
formulated with different concentrations of fluoride, 0 ppm, 1 ppm and 1000 ppm F as NaF. The
pH of the remineralizing solution was adjusted to levels approximating natural saliva, pH 7.2.
Sodium carboxymethylcellulose (CMC) was added in order to increase the viscosity of the
remineralizing solution to a consistency comparable to natural saliva, while methyl-p-
hydroxybenzoate served as a preservative (Amaechi and Higham 2001). The remaining
compounds provided the inorganic components required for the remineralization process. The
main constituents of enamel mineral, calcium and phosphate, were added to the remineralizing
solution in the form of calcium lactate and phosphate complexes, respectively (Amaechi and
Higham 2001). Samples were inverted and immersed in 30 mL of solution, renewed every 5
days. Following individual treatments, samples were rinsed under running distilled water for 2
minutes and left to air dry for 1 hour. After drying, samples were placed in the humid chamber
until PTR-LUM scans were performed.
Table 4. Composition of the remineralizing solution.
3.4 PTR-LUM Experimental Setup
The experimental set-up is shown in Fig. 9. The PTR-LUM experimental setup is equipped with
2 semiconductor laser diodes mounted on a rotating stage, one emitting at 660-nm (Mitsubishi
ML101J27) and the other at 830-nm (Thorlabs, DL7032-001). A description of the laser
Remineralizing solution components Concentration (g/L)
MgCl 2 ·6H 2 O 0.03
K 2 HPO4 0.121
KH 2 PO4 0.049 KCl 0.625 Calcium lactate 3.85 Methyl - p - hydroxybenzoate 2.0 Sodium carboxymethylcellulose 0.4
Fluoride 0 or 1 or 1000 ppm NaF pH (adjusted with KOH) 7.2
29
parameters is given in Table 5. A diode laser driver (Thorlabs, LDC 210) triggered by the built-
in function generator of the lock-in amplifier (Stanford Research System, SR830) modulated the
laser current harmonically. The modulated infrared PTR signal from the tooth was collected and
focused by two off-axis paraboloidal mirrors (Melles Griot 02POA017, Rhodium coated) onto a
Mercury Cadmium Telluride (MCT) detector (Judson Technologies J15D12, spectral range: 2 to
12 μm, peak detectivity D* ≈ 5×1010
cm Hz1/2
W-1
at ca. 12 μm) operating at cryogenic
temperatures by means of a liquid-nitrogen cooling mechanism and with an active area of 1 mm2.
Before being sent to the lock-in amplifier, the PTR signal was amplified by a preamplifier
(Judson Technologies PA-101). For the simultaneous measurement of PTR and LUM signals,
under 660-nm irradiation, a lens (focal length: 100 mm) was placed above the two off-axis
paraboloidal mirrors such that there was no interference with infrared energy passage between
the off-axis mirrors. The collected modulated luminescence was focused onto a silicon
photodiode. A cut-on coloured glass filter (Oriel 51345, cut-on wavelength: 715 nm) was placed
in front of the luminescence photodetector to block laser light reflected or scattered by the tooth.
For monitoring the modulated luminescence, another lock-in amplifier (Stanford Research
System, SR850) was used. Both lock-in amplifiers were controlled by a computer via USB to
RS-232 port connections.
Table 5. Laser parameters for backscatter PTR-LUM measurements.
Laser wavelength
Optical output power (CW)
Operating current
Beam Size
659 nm 130 mW 140 mA 5.60 mm
830 nm 100 mW 200 mA 0.71 mm
670 nm* 500 mW 800 mA 0.59 mm
30
Figure 9. Experimental setup for backscatter-mode PTR-LUM study in experiment 1.
3.5 PTR-LUM frequency scans
Initial PTR-LUM scans were done before any treatment (baseline measurement) at the center of
each delineated window. Samples were removed from the humid chamber 20 minutes prior to
PTR-LUM frequency scans. A further 10 min elapsed with the sample placed under direct laser
incidence in order to achieve thermal stabilization. This standardized procedure was followed for
all PTR-LUM scans and based on the earlier observation that changes in optical properties,
shown as changes in fluorescence intensity, and the thermal properties, were negligible following
20-min of stabilization time for periods lasting less than 1 hour (Jeon et al. 2004; Al- Khatteb et
al. 2002; Gmur et al. 2006). The total drying time implemented in the present study was in-line
with previous in vitro reports employing a 30-min (Pretty et al. 2002a) to 45-min (Zhang et al.
2000). PTR-LUM frequency scans were completed for all samples at 2 different wavelengths,
660-nm and 830-nm. The de-focused 660-nm laser beam ensured one-dimensionality of the
induced photothermal field. A full frequency scan consisted of varying the laser modulation
frequency at a fixed sample position from 1 Hz to 1000 Hz. This frequency range was segmented
into 21 steps controlled by computer software (Labview, National Instruments, Austin, TX,
USA) to automatically increment frequencies sequentially. A total of 28 data points were
measured at each frequency for PTR. Only the latter 20 data points were averaged and recorded
by the computer program. The first 8 data points served as cut-off points allowing time for the
samples to stabilize following a change in modulation frequency. For LUM measurements, 40
data points were averaged and recorded with 15 cut-off points. A similar frequency scan
Pre-Amplifier
HgCdTe Detector
Lock-in Amplifier
Optical filter & Photodetector
Off-axis mirrors
Laser Driver Waveform
Sync. Signal
Laser Diodes (659nm, 830nm)
Slider
Rotational stage
Sample
3-axis translational stage
Computer
Internal
Generator Function
Amplifier Lock-in
31
procedure was performed for 830-nm laser irradiation; however, no LUM data were available.
Frequency scans were performed during the demineralization process after 5 and 10 days of
treatment, and after 2, 5, 10, 20 and 28 days from the start of remineralization.
To obtain meaningful information from PTR frequency scan data and remove any influence of
instrumental frequency response, the experimentally measured signals must be calibrated against
an opaque semi-infinite reference sample. The instrumental transfer function was calculated
using a thermally thick glassy carbon sample (diameter 40 mm, thickness 10 mm, Grade GC-
20SS Tokai Carbon Co., Ltd., Japan) with known thermal conductivity (κ) and diffusivity(α) (κ =
5.8 W/mK, α = 4.8 × 10-6
m2/s). The measured glassy carbon PTR frequency response,
Vcarbon(ω), was fitted to the theoretical signal calculated for the semi-infinite opaque solid
[Mandelis 2001]:
0carbon carbon
0 0 0 0
, exp
2 1
s
s s
s s
IV C T z dz C z dz
kk
k
where k is the thermal conductivity, σ the thermal-wave number and I0 is the incident laser
intensity. Subscripts 0 and S refer to air and carbon glass, respectively. Obtained from the fits
was the only unknown parameter in the equation above, the instrumental factor, C(ω), and
subsequently used to normalize PTR experimental data. Experimental PTR amplitude and phase
signals were subsequently divided and subtracted from the theoretically derived instrumental
normalization factor, respectively. LUM transfer function was determined by reflecting the
incident laser light directly onto the photodiode using a mirror. The reference was divided and
subtracted from experimental LUM amplitude and phase, respectively.
3.6 Theoretical Model
In the present study, experimental PTR amplitude and phase signals were fitted to the 3-layer
coupled diffuse-photon-density-wave and thermal-wave theoretical model using simplex
downhill algorithm developed for the investigation of multilayered sound and demineralized
enamel (Matvienko et al. 2009b, 2009c) (Fig. 10). The theoretical model consisted of 2
components, the optical field and thermal field. A list of parameters in the theoretical fitting
32
program is listed in Table 6. The following theoretical model was derived by Dr. A. Matvienko
in communication with Prof. A. Mandelis.
Table 6. The list of parameters fitted from the theoretical analysis.
3.6.1 Optical Field
The optical field is generated by incident laser radiation and induces both coherent and diffuse
photon density fields within enamel, which make up the total diffuse photon density field:
; ; ;i i it c dz z z (3)
where ic is the coherent photon density and
id is the diffuse photon density of the turbid
medium. The subscript i denotes the effective layers where layer 1 – intact surface layer, layer 2
– lesion body and layer 3 – sound enamel.
The one-dimensional coherent photon-density field takes into account the reduction of the
incident intensity due to scattering and absorption (Matvienko et al. 2009c):
Symbol Parameter Units
μa Absorption coefficient m-1
μs Scattering coefficient m-1
α Thermal diffusivity m2/s
κ Thermal conductivity W/mK
ηNR Non-radiative energy conversion efficiency
μIR Infrared absorption coefficient m-1
H Heat transfer coefficient W/m2K
g Cosine of the scattering angle
L Layer thickness μm
R2 Reflection coefficient (reflection at L1 - L2 interface)
R3 Reflection coefficient (reflection at L2 - L3 interface)
33
1 1
1
1
1 2 2
2
1 2
1
3
0 1 2 1
1 2 1
0 1 2 1 2 3 2 1
1 2 1 2 3 2
0 1 2 3 1
1 exp exp 2
1 exp 2
1 1 exp exp exp 2
1 exp 2 1 exp 2
1 1 1 exp exp
t t
c
t
t t t
c
t t
t
c
I R z R L z
R R L
I R R L z L R L z L
R R L R R L
I R R R L
2 3
1 2
2 1 2
1 2 1 2 3 2
exp
1 exp 2 1 exp
t t
t t
L z L L
R R L R R L
(4)
where I0 is the laser intensity, R1 , R2 and R3 are the reflection coefficients of the outermost
turbid medium, the second layer, and the third layer interface, respectively .
Furthermore,
i i it a s (5)
where t is the total attenuation coefficient of layer i, which includes the absorption and
scattering coefficients of the medium.
Figure 10. The 3-layer geometrical representation used for theoretical analysis and associated
optical and thermal parameters of each layer.
The dc form of the diffuse-photon-density field [Nicolaides et al. 2001]:
2
2
13 '
i i i id a t d i
i
dz z G z
dz D (6)
where the function Gi and the reduced attenuation coefficient (µt‘) are given by:
i i
i i
i i
t i a
i s c
t s
gG z
g
and
(7)
' 1t a sg
(8)
Laser beam
L1 L2
0 L1 z
L3
L1 + L2
μa1 μs1 α1 κ1
R2 R3 R1
μa2 μs2 α2 κ2
μa3 μs3 α3 κ3
L1 L2
34
The general solutions for the optical fields for each layer (i = 1,2,3), including coherent and
diffuse components, can be written as:
1
1 1
1 1 1 1
1 2 1
exp exp
1 exp exp 2
t
eff t t
z a Q z b Q z
I C z R L z
(9a)
2
1 2 2 2
2 2 1 2 2 1
2 1 3 2 1
exp exp
1 exp exp 2 (
t
eff eff t t
z a Q z L b Q z L
I I C z L R L z L
(9b)
3
1 2 3 3
3 3 1 2
3 1 2
exp
(1 )exp
t
eff eff eff t
z b Q z L L
I I I C z L L
(9c)
where the integration constants due to the coherent field solutions are given by:
1
1
1
2
2
3 2
2
0 1
1 2 1
2 1
2 3 2
3 2
3,
3 '
1
1 exp 2
1 exp
1 exp 2
1 exp
i i i
i
i i i
s t a
a t t
eff
t
t
eff
t
eff t
gC
I RI
R R L
R LI
R R L
I R L
(10)
In Eqs. (9) Qi are defined as 3 'i ii a tQ . The third-kind boundary conditions at the air-tooth
interface and the continuity of photon-density field and photon flux at the interfaces between
solid layers are applied:
1 1
1 2
1 2
1 1
2 3
2 3
1 2 1 2
0
1 1
1 2
1 2 1 2
2 3
) 0
)
)
)
)
d d
z
d d
d d
z L z L
d d
d d
z L L z L L
da A z
dz
b L L
d dc D z D z
dz dz
d L L L L
d de D z D z
dz dz
(11)
The constant A is defined as, 12
1
rA D
r
(12)
35
where r is the internal reflection of uniformly diffusing radiation, which depends on the index of
refraction of the sample.
Solving the system of the five equations of the boundary conditions using the photon diffusion
and coherent fields, one can obtain the coefficients a1, a2, b1, b2, b3:
1
1
2
1
1 1 1 12 12
1
12 12 1 1 12 12 1 1
1 1 1 1 1
2 2 22 2 2 2 12 1 1 1 12 1 1 1
12 1 1 1
2 12 1 1 1
2 ( exp 2 ) 1 2;
1 2 exp 1 2 exp
exp 2 ;
exp 2 exp exp
exp ;
exp
t
t
t
t
VF G f N L d P X VXa
X VX Q L M X VX Q L
b a M d P f N L
a b Y f L d X a Q L X b Q L
Y f d L
b VF VX a Q L V
2 2
12 1 1 1
3 2 23 2 2 2 23 2 2
23 2 2 23 2 2 33 3
exp ;
exp exp
exp exp ;t t
X b Q L
b a X Q L b X Q L
Y d L Y f L Y d
(13)
Here, the parameters M, N, P, X, Y, and d are defined as:
1 1
1 2 1
3 1 2
1
1 1 1
1 1 1 2 2 2 1
3 2 1 2
1 11, , ,
1 1 1
, ,
, , 1 exp ,
1 exp .
i
t t
i ti iij ij
j j j j
eff eff t
eff t t
A AQ AM N P
Q A Q A Q A
DD QX Y
D Q D Q
d C I f d R d C I R L
d C I R L L
(14)
The coefficients F, G and V are defined as:
2 2
2 1
1 1 2
2 23 2 23 3 33
2 2
2 2 23 2 2 23 2 2 23
22 2 2 2 12 1 1 1
1 12 1 1 12 1 2 22 2 22 2
23
23
exp 1 exp 1 1
exp 1 exp 1 exp 1
exp 2 exp exp ;
1 exp 1 exp 1 1 exp 2 ;
1
11 ex
1
t t
t t
t t t
L Y L Y d YF d f
Q L X Q L X Q L X
Y f L d Y f d L
G d Y L f Y L d Y f Y L
VX
X
2 2
;
p 2Q L
(15)
36
3.6.2 Thermal Wave Field
The total photon density field (ψt) is also the source for the thermal wave field which propagates
as thermal waves into the medium. The thermal wave field is given as:
2
2
2; ; ; 1,2,3i
i
a
i i i NR t
i
dT z T z z i
dz
(16)
where i
i
i
(17)
is the thermal wavenumber, [m-1
], which depends on the modulation frequency and on thermal
diffusivity of i-th layer.
The thermal-wave fields for each layer can be written in the form:
1 1
1 1 1 1 1 1 1 1 1
1 1 1
; exp exp exp exp
exp exp 2 ;t t
T z A z B z C Q z D Q z
E z F L z
(18a)
2 2
2 2 2 1 2 2 1 2 2 1
2 2 1 2 1 2 2 1
; exp exp exp
exp exp exp 2 ;t t
T z A z L B z L C Q z L
D Q z L E z L F L z L
(18b)
3
3 3 3 1 2 3 3 1 2
3 1 2
; exp exp
exp ;t
T z B z L L D Q z L L
E z L L
(18c)
The coefficients Ci, Di, Ei and Fi are defined as:
1 1
1
1
2 2
1 2
2
3 3
1 32
3
1 1
1
1
2 2
2 2
1 12 2
1 1
2 22 2
2 2
3 32 2
3 3
1 12 2
1 1
; 1, 2
; 1,2,3
1 ;
1 ;
1 ;
1
i i
i i
NR a
i i
i i i
NR a
i i
i i i
NR a
eff
t
NR a
eff eff
t
NR a
eff eff eff
t
NR a
eff
t
C a iQ
D b iQ
E I C
E I I C
E I I I C
F I C
2 2
1 2
2
2
2 2 32 2
2 2
;
1 .NR a
eff eff
t
R
F I I C R
(19)
37
To determine the coefficients Ai and Bi, the following boundary conditions are used:
1 1
1 2 1 2
1
1 1
0
1 1 2 1
1 2
1 2
2 1 2 3 1 2
2 3
2 3
,) 0; ;
) , , ;
, ,) ;
) , , ;
, ,) .
z
z L z L
z L L z L L
dT za HT
dz
b T L T L
dT z dT zc
dz dz
d T L L T L L
dT z dT ze
dz dz
(20)
The photothermal radiometric (PTR) signal represents the overall Plank radiation emission
integrated over the depth of the sample:
1 2
1 20
, , ,IR IR IR
L L
z z z
PTR IR
L L
V C T z e dz T z e dz T z e dz
(21)
Here, μIR is the spectrally averaged infrared absorption/emission of the medium. The IR
absorption spectrum for enamel is shown in Fig. 7B.
The measured PTR signal has an oscillating character and can be represented as:
exp PTRi
PTR PTRV V
, (22a)
where the amplitude and phase components are:
PTR PTR,PTR PTRAmp V Phase (22b)
3.7 Multiparameter Fitting of Experimental Curves
Experimental frequency scan data were fitted across the frequency range of 4 Hz – 354 Hz. At a
modulation frequency of 4 Hz the assumption of no dentinal involvement is based on the
determination of the thermal diffusion length of intact enamel. Given the lowest modulation
frequency of 4 Hz and a range of previously published values of the thermal diffusivity of sound
enamel (α = 4.0 - 4.69 x 10-7
m2/s) (Braden 1964), the thermal diffusion length (see equation 2)
is approximately μ (4 Hz) ≈184 µm, much smaller than the average thickness of lingual enamel
(≈0.84 – 2.04 mm) (Macho and Berner 1993). The assumption of one-dimensional heat diffusion,
38
i.e. heat loss due to lateral diffusion was negligible, was employed based on the size of the
illuminated area (Table 5) relative to the thermal diffusion length in the range of modulation
frequencies investigated. This was accomplished by defocusing and expanding the laser beam to
≈5.60 mm, a size much larger than the thermal diffusion length at the lowest modulation
frequency investigated (1 Hz).
For the theoretical representation of sound mature enamel, a 2-layer representation was assumed,
where layer 1 denoted a thin surface layer of finite thickness where mineral content and
optothermal properties vary from underlying sound enamel, henceforth referred to as
‗aprismatic‘ enamel, and layer 2 as semi-infinite enamel (Fig. 11). The properties of layer 2 and
layer 3 were equalized, as the underlying sound enamel substrate was considered semi-infinite.
By assuming a two-layer approximation of the three-layer model to fit the enamel data, the
complexity of the computational fits was significantly reduced in addition to the implementation
of the same mathematical description and software package for sound and demineralized enamel
(Matvienko et al. 2009b).
Figure 11. Schematic geometry of effective layers for multiparameter fittings of sound enamel.
All parameters were fitted between the limits defined in Table 7 for sound enamel or
de/remineralized enamel. The criterion for the best fits was defined by the residual, i.e. the
combined deviation between experimental and theoretical amplitude and phase curves. The
computational algorithm did not provide any restrictions to the fitting results and ‗selected‘ the
optimal values in the multi-parameter fitting procedure, such that values fell within the defined
limit ranges and did not converge to either the upper/lower limits in the range. The tolerance of
the fitting procedure represented the change in the residual corresponding to the change of a
single parameter. The fitting procedure continued until the change in any of the parameters
Aprismaticlayer
Sound enamel
0 L1 z
Laser beam
39
stopped decreasing the residual, defined by the tolerance, or until the maximum number of
iterations was reached, whereupon the algorithm stopped (Matvienko et al. 2009a).
The initial procedure involved fitting experimental frequency scan data from baseline
measurements, across the frequency range 4 Hz to 354 Hz, to the upper and lower limits of the
parameter ranges for sound enamel, as derived from literature (Table 7). A broader range of
upper and lower limits was fixed for layer 1 (aprismatic layer) as the physical characterization of
this layer has been omnipresent, however, in terms of optical and thermal parameters, properties
are undefined in the literature (Kodaka 2003; Ripa et al. 1966; Xue et al. 2009; Gwinnett 1967;
Whitaker 1982).
The initial fit on untreated enamel was performed by segmenting the range of limits into 20 - 30
equal steps and best fits were performed for all combinations and all parameters. The derived set
parameters from the initial fitting across the 20 – 30 division range were averaged and used as
input values (± S.D.) to perform a second fit across the same number of divisions (20 – 30)
between the limits. This averaging process was performed 3 times in order to derive a set of
optical and thermal parameters, independent of the number of divisions between the limits. It
was previously shown (Matvienko et al. 2009a) that the initial range of the parameters can be
significantly reduced (up to 100 times) through the averaging process to generate a narrow range
of unique values, from the best fits of the experimental data, which accurately describes the
properties of the sound enamel sample.
40
Table 7. Fixed upper and lower limits of the fundamental parameters defined for the
multiparameter fitting of untreated, sound enamel and de- and remineralized enamel.
Physical parameters Layer
Sound enamel De- and remineralized
enamel
Lower
limit
Upper
limit
Lower
limit Upper limit
Absorption coefficient
[µa, m-1
] L1 + L2 1 100
a 1 150
Scattering coefficient
[µs, m-1
]
L1 110b 6 000
a 110
b 157 000
d
L2 1 000c
6 000a 1 000
c 157 000
d
Thermal conductivity
[κ, W/mK]
L1 0.10 0.93e 0.10 0.93
e
L2 0.77f 0.93
e 0.10 0.93
e
Thermal Diffusivity
[α, m2/s]
L1 2.0 x 10-7 f
7.7 x 10-7
2.0 x 10-7 f
7.7 x 10-7
L2 4.2 x 10-7 g
7.7 x 10-7
2.0 x 10-7 f
7.7 x 10-7
Aprismatic layer
thickness [L1, µm] L1 5 60
* -------
a Fried et al.,[1995];
b Zijp, [2001] and references therein;
c Spitzer and ten Bosch, [1975];
d
Spitzer and ten Bosch, [1977]; e Craig and Peyton, [1961];
f Minesaki, [1990];
g Braden,
[1964]; * Kakaboura et al., [2005]. Sound enamel: L1 = aprismatic layer and L2= semi-
infinite sound enamel; Demineralized enamel: L1 = intact surface layer and L2= lesion body
The final derived set of sound enamel optical and thermal parameters was then fixed as
representing the properties for Layer 3 in subsequent fittings of demineralized and remineralized
treatment curves. Thus, the sound enamel curve was always the first curve fitted. From the first
exposure of the sample to the acid demineralizing gel, the 2-layer configuration of sound enamel
was no longer valid. As a result, the 3-layer profile was considered, where Layer 1 denoted the
intact surface layer overlying the demineralized lesion body (Layer 2), followed by semi-infinite
sound enamel (Layer 3) (Fig. 12).
Figure 12. Schematic structure of effective layers used for fits of demineralized and
remineralized enamel. L1 = surface layer; L2 = lesion body; L3 = sound enamel.
Surface layer
Lesion body
Sound enamel
0 L1 L1 + L2 z
Laser beam
41
The final demineralized PTR curve was fitted with thicknesses derived from TMR mineral
content profiles (Fig. 13). The lower limit for thickness of layer 1 was defined as the depth at
maximum content of the surface layer (MSL) and upper limit defined as the median between the
maximum content of the surface layer and minimum content of the lesion body (SLMAX). The
thickness limits of layer 2 were defined as the difference between LM (the median between the
lesion depth, LD, and the depth at the minimum content of the lesion body, MLB) and SLMAX, as
the lower limit; and difference between LD and the depth at MSL as the upper limit.
Figure 13. Schematic mineral content profile for the theoretical determination of layer
thicknesses. MSL denotes the maximum mineral volume of the surface layer. MLB refers to the
minimum mineral content in the subsurface lesion body. SLMAX refers to the upper limit of the
surface layer thickness, defined as the median between MSL and MLB. LM refers to one boundary
for the lower limit determination of lesion body thickness and defined as the median between
MLB and LD. LD refers to the TMR defined lesion depth at 95% of the sound enamel calibration
level (SE) at 87 vol%. L1 = intact surface layer and L2 = subsurface lesion.
After deriving layer thicknesses for layers 1 and 2, they were fixed as the upper limits for fitting
intermediate treatment curves. Therefore, all parameters, excluding thicknesses, were allowed to
vary between the limits defined in Table 7. The lower limit for thermal diffusivity was fixed at
the value determined for dentin (2.0 x 10-7
m2/s) (Minesaki 1990), whereas the upper limit was
fixed at the product of an 85% increase in the average thermal diffusivity from Table 2 (≈ 4.2 x
10-7
m2/s). The latter value was determined from preliminary investigations of parameter limits,
where fitted parameters varied freely between the defined limits without converging to the upper
limit. For the thicknesses the major assumption was that intermediate thicknesses were no larger
than the thicknesses derived at the treatment end point.
SE
87
vol.
%
LD SLMAX LM
Sample depth (μm)
MSL
MLB
L2 L1
42
For remineralized samples, final PTR curves following remineralization were fitted first in the
same manner as PTR curves following the last demineralization. All parameters were allowed to
vary between the limits defined in Table 7, except thicknesses, which were determined by TMR
mineral content depth profiles as described above and shown schematically in Fig 10. From
fitting the final remineralized PTR curve, the TMR-measured thicknesses of layer 1 and 2 were
fixed as the lower limit and upper limit for fitting the final demineralized PTR curve,
respectively. In this way, two treatment end points can be defined in fitting the intermediate
remineralized curves. The lower limit for layer 1 was set at 5 μm, which was the lower limit of
the aprismatic layer for fitting sound enamel. This was done in order to account for large
aprismatic layers that may decrease in thickness during the demineralization period. The upper
limit for layer 2, the lesion body, was set as the difference between the sound enamel level (SE)
and the depth at the surface layer maximum (Fig. 13). Following the derivation of parameters
corresponding to the 2 treatment end-points, with the final thicknesses based on TMR values,
thicknesses of both layers were fixed for fitting intermediate remineralization PTR curves.
The uniqueness of the fitted parameters is essential in verifying the reliability and robustness of
the theoretical algorithm. The uniqueness-of-fit to the multi-parameter computational algorithm
following de- and remineralization is demonstrated in Appendix 4.
3.8 Transverse microradiography (TMR) and image analysis
Following completion of all PTR-LUM measurements all samples were subjected to transverse
microradiography (TMR) analysis to determine the mineral loss and depth of the artificially
demineralized and remineralized lesions. Sample preparation and image analysis was completed
at the University of Texas Health Science Center at San Antonio, under the supervision of Prof.
B.T. Amaechi. The samples were sectioned using a water-cooled diamond-coated wire saw
model 3242 (Well, Le Locle, Switzerland), to produce a thin enamel slice approximately 100-µm
from the lesion area. A thin section was taken from the treated enamel region across the center of
the laser beam irradiation spot (≈5 mm). The slice, together with an aluminum step wedge (10
steps of 24.5 µm thickness), was microradiographed on type lA high resolution glass X-ray
plates (IMTECH CA, USA) with a Phillips x-ray generator system equipped with a nickel
filtered Cu-Kα target, producing monochromatic radiation of wavelength appropriate for
43
hydroxyapatite (l84 Ǻ). The plates were exposed for 10 minutes at 20kV/10 mA, and processed.
Processing consists of a 5-minute development in a developer (Kodak HR) and 15 min fixation
in a Rapid-fixer (Kodak) before a final 30-minute wash period. After drying, the
microradiographs were visualized using a DMR optical microscope (Leica) linked via a CCTV
camera (Sony, XC-75CE) to a personal computer (90 MHz Dell™ Pentium). The enhanced
image of the microradiograph was analyzed under standard conditions of light intensity and
magnification and processed, along with data from the image of the step wedge, using the TMR
software (TMRW version 2.0.27.2, Inspektor Research Inc., Amsterdam, Netherlands) (de
Josselin de Jong et al. 1987) to quantify the lesion parameters of integrated mineral loss (∆z,
vol%.µm) and lesion depth (LD, µm). The implementation of the latest dedicated TMR software
package utilized a new algorithm developed to mathematically flatten curved tooth surfaces, by
completing several scans for each microradiographed section. The mineral loss was computed as
the difference in volume percent of mineral between sound and demineralized tissue integrated
over the lesion depth. The mineral content plateau in deeper regions of the enamel section,
representative of sound tissue, was preset at the 87 vol% level (de Josselin de Jong et al. 1987).
The lesion depth was determined as the distance from the measured sound enamel surface to the
location in the lesion where mineral content was 95% of the sound enamel mineral volume.
Lesion parameters were determined by averaging several scans over the distance of the thin
section taken from the center of the treated area and corresponding to the irradiated beam size in
PTR-LUM experimental measurements.
3.9 Statistical analysis
Data obtained from TMR image analysis were analyzed statistically. Significant differences
between demineralized and remineralized lesions in terms of lesion depth and mineral loss in the
presence of variable fluoride concentrations were examined using ANOVA and post hoc Tukey‘s
test (p < 0.05). All statistical analysis was done using statistical analysis software (SPSS v. 14.0
for Windows, SPSS Inc., Chicago, IL).
44
4 Results
4.1 Sound enamel
4.1.1 Microradiographic Analysis and PTR-LUM signals
Intact, sound and untreated enamel surfaces exhibited a shiny, lustrous and semi-transparent
appearance, characteristic of healthy enamel (Fig. 14a). From the sound enamel samples (n= 4)
sectioned for TMR analysis average mineral loss and lesion depth parameters (± s.d.) were 322.5
± 162.8 vol%.µm and 19.8 ± 18.5 µm, respectively. Microradiographic images and associated
densitometric tracings for a representative sound enamel sample are presented in Fig. 15.
Microradiographs revealed uniform high radiodense enamel in all sound enamel samples. The
mineral volume profiles showed a thin surface layer of lower mineral volume, distinctly
identifiable from underlying bulk enamel. A gradual increase in mineral volume from the
anatomical enamel surface to bulk enamel occurred at a depth below the enamel surface, ranging
from approximately 4.4 µm to 44.0 µm.
Figure 14. Visual appearance of (a) sound enamel and (b) white-spot appearance after 10-days
of acid treatment.
PTR amplitude decreased across the entire frequency range from 1 Hz to 1 kHz with a notable
decrease in slope at frequencies above ≈44 Hz (Fig. 15). PTR phase exhibited near-linear
frequency dependence at modulation frequencies in the low-intermediate range (≈1 – 100 Hz).
The presence of phase maxima became apparent at modulation frequencies above 100 Hz. LUM
amplitude exhibited characteristic curvature and large slope changes in the mid-frequency range
(≈ 63 – 100 Hz) which were associated with the corresponding phase minimum.
(a) (b)
45
Figure 15. PTR-LUM amplitudes and phase curves for a representative sound enamel sample
under 660-nm laser excitation. The densitometric tracing (top right) and microradiographic
image (bottom right) are presented in the adjacent figures. Error bars, when not visible, are of
the size of the symbols.
4.1.2 Theoretical analysis of untreated enamel samples
Multiparameter fits of amplitude and phase signals generated from a representative sound enamel
sample showed very good agreement between experimental and theoretical curves (Appendix 2;
Fig. A.2.1). Theoretical fitting of sound enamel curves of multiple samples (n = 10) revealed a
narrow range of optical and thermal parameters of each layer as well as thicknesses of the
aprismatic enamel layer. A list of the central parameters derived from multi-layered sound
enamel is presented in Table 8. The mean derived thickness of layer 1 (aprismatic layer) was ≈13
µm. A derived set of parameters for layer 2 showed that they were distinct from layer 1 and
within the range of values derived from literature. While the range and standard deviations for
the aprismatic layer were rather high, there was a high degree of inter-sample reproducibility in
the derivation of sound enamel parameters (Table 8).
100µm
1 10 100 1000
1E-4
1E-3
PTR Amplitude
Am
plit
ude (
a.u
.)
Frequency (Hz)1 10 100 1000
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
LUM Phase
LUM Amplitude
Am
plit
ude (
a.u
.)
Frequency (Hz)
1 10 100 1000
-95
-90
-85
-80
-75
-70
-65
-60
-55 PTR Phase
Phase (
Deg)
Frequency (Hz)1 10 100 1000
-18
-16
-14
-12
-10
-8
-6
-4
-2
Phase (
Deg)
Frequency (Hz)
46
Table 8. Mean (± s.d.) derived optical and thermal parameters of intact enamel layers (n = 10)
4.2 Demineralization Group
4.2.1 Microradiographic Analysis and PTR-LUM Signals
Acid gel demineralization of intact samples produced visible white spot enamel lesions (Fig.
14b). Lesions were created with an average mineral loss and lesion depth of 1247 ± 502
vol%.µm and 90 ± 12 µm, respectively, determined from TMR measurements. A representative
demineralized lesion treated for 10 days is shown in Fig. 16. The lesion characteristics were
those of a classical subsurface lesion in which microradiographs defined a uniform, clear and
distinct lesion body underlying a superficial intact enamel surface layer with mineral volume
near, but lower than, the sound enamel level. Layer thickness determined by TMR analysis,
averaged at several points across the microradiographed lesion, were 3.6 µm for the surface
layer, and 85 µm for the lesion width, defined as the distance between the peak mineral of the
intact surface layer to the point where mineral volume is 95% of sound enamel level (Fig. 16).
PTR-LUM frequency scans are also shown in Fig. 16. PTR behaviour to subsurface lesion
formation exhibited increases in amplitude across the entire modulation frequency range. A large
PTR amplitude increase after 5 days resulted in slope changes in the frequency response and the
onset of curvature predominately at high modulation frequencies, as expected from the depth-
profilometric character of this technique (high modulation frequencies correspond to short
thermal diffusion lengths). After the 10-day treatment period, amplitude curvature shifted to
lower modulation frequencies. Inflection of the PTR phase of the demineralized curves with the
healthy curve occurred initially at higher modulation frequencies and shifted to lower
frequencies as treatment progressed. This resulted in a larger phase lag at high modulation
frequencies. As demineralization progressed, PTR amplitude increased in a monotonic fashion
while PTR phase curves exhibited a shift in maxima to lower modulation frequencies. This
resulted in an increase in phase lag at high frequencies and decrease in phase lag at lower
Sound enamel
Parameters Layer 1:
Aprismatic enamel Layer 2:
Sound enamel
Absorption coefficient (µa ; m-1) 65 ± 15 44 ± 23
Scattering coefficient (µs ; m-1) 763 ± 1113 5399 ± 847
Thermal conductivity (κ ; W/mK) 0.48 ± 0.23 0.87 ± 0.05
Thermal diffusivity (α; m2/s) 5.29 x 10-7 ± 1.45 x 10-7 4.41 x 10-7 ± 8.94 x 10-9
Layer thickness (µm) 13.41 ± 7.79 —
47
modulation frequencies. Trends similar to those detailed above were also observed in the
frequency response under the 830-nm laser (Appendix 1; Fig. A.1.1). The LUM signal channel
also exhibited consistent trends with treatment time for demineralized enamel. A monotonic
depression in both amplitude and a decrease in phase minima were evident. It is important to
note the opposite trends in the PTR and LUM signals during demineralization, where PTR signal
amplitudes increase with demineralization and LUM signals decrease.
An additional sample, not included within the demineralized control sample matrix, was treated
for an extended period of time (40 days) for theoretical analysis and is displayed in Fig. 17. The
40-day lesion exhibited similar histology to the 10-day lesion, although presented a deeper lesion
body and thicker intact surface layer (lesion depth = 114.8 µm). A distinct radiodense surface
layer is visible above a radiolucent body of the lesion. The same trends in PTR and LUM
identified above for the lesion displaying classical subsurface behaviour are also evident in the
current sample. Amplitude monotonically increased and phase lag monotonically decreased at
low modulation frequencies. After ≈10 days of treatment, PTR amplitude at high modulation
frequencies did not change much until the end of treatment, whereas an increase and pronounced
curvature were marked at lower modulation frequencies. A phase maximum shift to lower
modulation frequencies was also clearly evident over the duration of treatment. Interestingly,
after about 20 days of acid exposure, a secondary phase peak at ≈200 Hz became apparent.
Similar PTR amplitude and phase behaviour was noted in the frequency response under the 830-
nm laser (Appendix 1; Fig. A.1.1).
48
Figure 16. PTR-LUM amplitudes and phase curves for a 10 day demineralized sample under
660nm laser excitation. Error bars, when not visible, are of the size of the symbols. The
densitometric tracing (top right) and microradiographic image (bottom right) of the lesion are
presented in the adjacent figures. Mineral loss = 1310 vol%.μm; Surface layer thickness = 3.6
μm; Lesion width= 85.0 µm. Error bars, when not visible, are of the size of the symbols.
Enamel
100µm
1 10 100 1000
1E-5
1E-4
1E-3
0.01
Before Treatment
Demin - 5 days
Demin - 10 days
Am
plit
ude
(a.u
.)
Frequency (Hz)1 10 100 1000
0.1
0.15
0.2
0.25PTR Amplitude LUM Amplitude
Am
plit
ude (
a.u
.)
Frequency (Hz)
1 10 100 1000
-100
-95
-90
-85
-80
-75
-70
-65
-60
PTR Phase
Phase (
Deg)
Frequency (Hz)1 10 100 1000
-18
-16
-14
-12
-10
-8
-6
-4
-2
LUM phase
Phase (
Deg)
Frequency (Hz)
49
Figure 17. PTR-LUM signals for the 40 day demineralized lesion under 660-nm. Error bars,
when not visible, are of the size of the symbols. The densitometric tracing (top right) and
microradiographic image (bottom right) of the lesion are presented in the adjacent figures.
Mineral loss =1420 vol%.μm; Surface layer thickness = 2.6 μm; Lesion width= 112.2 µm. Error
bars, when not visible, are of the size of the symbols.
4.2.2 Theoretical analysis of demineralized enamel samples
Of the 17 parameters extracted from the theoretical fitting of demineralized samples, a select
group of parameters were found to change with treatment time, while no specific trends were
identified in the remaining parameters (See Appendix 3). The central parameters which changed
as a function of treatment time were the main optical and thermal transport properties, as
expected, including optical scattering and absorption coefficients, thermal conductivity and
diffusivity and layer thicknesses and are presented in Figs. 18 – 20. A sample exposed to the
demineralizing solution for 10 days, similar to substrates used for remineralization studies is
shown in Figs. 18 and 19a. A second sample was incubated for an extended period (40 days) in
the demineralizing agent, with PTR-LUM measurements at 0, 5, 10, 15, 20, 30 and 40 days
(Figs. 19b and 20). Both samples showed a good fit of experimental data to 3-layer theory at all
treatment times (Appendix 2; Fig. A.2.2).
1 10 100 1000
1E-4
1E-3
0.01
Am
plit
ude (
a.u
.)
Frequency (Hz)1 10 100 1000
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
Before Treatment
Demin- 5 Days
Demin- 10 Days
Demin- 15 Days
Demin- 20 Days
Demin- 30 Days
Demin- 40 Days
PTR Amplitude LUM Amplitude
Am
plit
ude (
a.u
.)
Frequency (Hz)
1 10 100 1000
-78
-75
-72
-69
-66
-63
-60
-57 PTR Phase
Phase (
De
g)
Frequency (Hz)100
-19.5
-19.0
-18.5
-18.0
-17.5
-17.0
LUM phase
Phase (
De
g)
Frequency (Hz)
1 10 100 1000
1E-4
1E-3
0.01
Am
plit
ude (
a.u
.)
Frequency (Hz)1 10 100 1000
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
Before Treatment
Demin- 5 Days
Demin- 10 Days
Demin- 15 Days
Demin- 20 Days
Demin- 30 Days
Demin- 40 Days
PTR Amplitude LUM Amplitude
Am
plit
ude (
a.u
.)
Frequency (Hz)
1 10 100 1000
-78
-75
-72
-69
-66
-63
-60
-57 PTR Phase
Phase (
De
g)
Frequency (Hz)100
-19.5
-19.0
-18.5
-18.0
-17.5
-17.0
LUM phase
Phase (
De
g)
Frequency (Hz)
100µm
50
The change in optical parameters (absorption and scattering coefficients) and thermal parameters
(diffusivity and conductivity) as a function of demineralization time for the 10 day treated
sample is shown in Fig. 18a-d. Over the 10 day period, absorption (Fig. 18a) and scattering (Fig.
18b) increased in the surface layer (layer 1). Within the lesion body (layer 2), little change in
absorption was found, whereas scattering increased linearly from the onset until the end of the 10
day period. Thermophysical properties of both layers, conductivity (Fig. 18c) and diffusivity
(Fig. 18d), became poorer with demineralization, excluding the thermal conductivity of the
surface layer which increased at day 10.
Figure 18. The change in optical absorption (a) and scattering (b) coefficients and thermal
conductivity (c) and diffusivity (d) parameters as a function of time, over the 10 day
demineralization period. Layer 1 = surface layer; Layer 2 = lesion body.
0 5 10
0
20000
40000
60000
80000
100000
120000
140000
160000
Sca
tte
rin
g C
oe
ffic
ien
t (m
-1)
Treatment Time (days)
Layer 1
Layer 2
0 5 10
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
The
rmal
Co
nd
uct
ivit
y (W
/mK
)
Treatment Time (days)
Layer 1
Layer 2
0 5 10
3.5x10-7
4.0x10-7
4.5x10-7
5.0x10-7
5.5x10-7
6.0x10-7
6.5x10-7
Th
erm
al D
iffu
sivi
ty (
m2/s
)
Treatment Time (days)
Layer 1
Layer 2
(c) (d)
(a) (b)0 5 10
70
80
90
100
110
120
130
Ab
sorp
tio
n C
oef
fici
ent
(m-1
)
Treatment Time (days)
Layer 1
Layer 2
51
Changes in layer thicknesses of the 10-day and 40-day demineralized samples are shown in Fig. 19. Over
the 10-day period, the thickness, or depth, of the growing subsurface lesion increased nearly linearly. At
later treatment times (Fig. 19b), the thickness of layer 2 continued to increase until the end of the 40-day
treatment period. In terms of layer 1, opposite behaviours were observed in the 2 presented samples. The
thickness of the surface layer decreased from 0 days over the 10 day period in Fig. 19a. In the 40 day
demineralized lesion (Fig. 19b), the thickness was stable for the first 15 days followed by an increase
until the end of the treatment period. The final thickness of layer 1 and layer 2 determined theoretically
for the 10 day sample were 6.6 µm and 81.2 μm, respectively. Final thicknesses determined theoretically
for the 40 day sample were 18.7 µm and 92.1 µm, respectively.
Figure 19. Changes in the thickness of layer 1 and layer 2 as a function of time for the 10 day (a)
and 40 day (b) demineralized samples. The inset in (b) shows the details of layer 1 thickness over
time on an expanded scale. Layer 1 = surface layer; Layer 2 = lesion body.
Optical and thermophysical properties plotted as a function of time for the 40-day demineralized
sample are shown in Fig. 20a-d. As in the 10-day demineralized sample, absorption and
scattering coefficients of layer 1 had an increasing trend over the demineralization period (Fig.
20a). After the 30-day period the scattering properties of the demineralized lesion body were
dominant and superseded the scattering properties of the surface layer. Thermal conductivity
(Fig. 20c) and diffusivity (Fig. 20d) of layer 2 decreased rapidly for the first 10 days of
demineralization reaching a minimum at 15 days after which properties slightly increased and
0 5 10
0
10
20
30
40
50
60
70
80
Layer 1
Layer 2
Laye
r Th
ickn
ess
(m
)
Treatment Time (days)0 10 20 30 40
0
10
20
30
40
50
60
70
80
90
0 10 20 30 40
10
12
14
16
18
20
Laye
r Th
ickn
ess
(m
)
Treatment Time (days)
Layer 1
Layer 2
(b)(a)
52
stabilized for the remainder of the treatment period. Thermal conductivity and diffusivity of layer
1 behaved similarly; a decrease over the first 15 days was followed by an increase above the
baseline level at day 0. Thermal diffusivity after 20 days increased nearly linearly until the end
of treatment.
A summary of the typical trends in the core physical parameters following demineralization are
presented in Table 9. The trends were evaluated from the 10-day (Figs. 18, 19a) and 40-day
(Figs. 20 and 19b) demineralized samples, discussed above, as well as the demineralization
phase of the remineralized samples (Figs. 24-26, 29-31 and 34-36).
Figure 20. The change in optical absorption (a) and scattering (b) coefficients and thermal
conductivity (c) and diffusivity (d) parameters as a function of time over the 40 day
demineralization period. Layer 1 = surface layer; Layer 2 = lesion body.
0 10 20 30 400
20
40
60
80
100
120
140
Ab
sorp
tio
n C
oe
ffic
ien
t (m
-1)
Treatment Time (days)
Layer 1
Layer 2
0 10 20 30 40
0
25000
50000
75000
100000
125000
150000
Scat
teri
ng
Co
effi
cien
t (m
-1)
Treatment Time (days)
Layer 1
Layer 2
0 10 20 30 40
0.4
0.5
0.6
0.7
0.8
The
rmal
Co
nd
uct
ivit
y (W
/mK
)
Treatment Time (days)
Layer 1
Layer 2
0 10 20 30 40
2.0x10-7
3.0x10-7
4.0x10-7
5.0x10-7
6.0x10-7
7.0x10-7
The
rmal
Dif
fusi
vity
(m
2 /s)
Treatment Time (days)
Layer 1
Layer 2
(a) (b)
(d)(c)
53
Table 9. General trends in the main physical parameters following short and long term
demineralization. The ↑, ↓ and ↔ arrows refer to an increase, decrease or no change,
respectively, in parameters over time.
† A decrease was noted in the 40-day demineralized and high fluoride samples
ffi An increase was observed in the fluoride-free sample
* An increase was observed in the high fluoride sample
¥ Differed depending on initial thickness of the aprismatic layer
4.3 Remineralization Treatment Groups
4.3.1 Microradiographic analysis and visual appearance
Compared to sound enamel, remineralized enamel maintained white-spot enamel appearance
(Fig. 21) induced following demineralization (Fig. 14b). After the 4-week immersion in the low
fluoride and fluoride-free remineralizing solution, there was no difference in the macroscopic
appearance of enamel from after demineralization. A marked change in macroscopic enamel
appearance was evident in samples of the high fluoride group. These changes included enhanced
enamel opacity and chalky-white appearance affecting the entire exposed treatment window (Fig.
21c).
Figure 21. Visual appearance of representative samples from each remineralization treatment
group. (a) Remineralized in the absence of fluoride; (b) remineralized in the presence of low
fluoride; (c) remineralized in the presence of high fluoride levels.
Physical Parameters
Demineralization
0 – 10 days 15 – 40 days
µa1 ↑ ↑
µa2 ↓ ↑
µs1 ↑ ↑
µs2 ↑ ↑
κ1 ↑† ↑
κ2 ↓ ↑
α1 ↓‡ ↑
α2 ↓* ↔
L1 ↑/↓¥ ↑
L2 ↑ ↑
54
Average mineral loss and lesion depths of remineralization and demineralization treatment
groups are presented in Table 10. Analysis of variance of the treatment groups with respect to
mineral loss and lesion depth revealed no significant differences in mean mineral loss (p > 0.05),
and a significant difference in mean lesion depth (p < 0.05). Post hoc statistical analysis (Tukey
test) revealed a significant difference in mean lesion depth between all 3 remineralization groups
relative to the demineralized control (p < 0.05). No significant differences were found between
the 3 remineralization treatment groups with respect to mineral loss or lesion depth.
Table 10. Average mineral loss and lesion depth of remineralization and demineralized
treatment groups. Means with different letters are significantly different at p < 0.05.
4.3.2 Fluoride-free remineralization group
4.3.2.1 Microradiographs and mineral content depth profiles
An exemplary sample from the fluoride-free group revealed a heavily mineralized superficial
enamel layer, overlying the intact surface layer and body of the lesion (Fig. 22). This typical
mineral volume distribution was the most frequently observed occurring in 80% of remineralized
samples. Hypermineralized surface layers, with respect to the intact surface layer and lesion
body, were present at mineral volume approaching sound enamel and higher than the mineral
volume of the subadjacent intact surface layer. In these samples, mineral volume peaks occurred
directly at the enamel surface and in microradiographic images displayed as thin radiopaque
surface layers discernible from underlying mineral layers.
Treatment Group Mean mineral loss (vol%.µm) Mean lesion depth (µm)
No fluoride 1055 ± 257 65 ± 7a
Low fluoride (1 ppm) 1087 ± 452 64 ± 11a
High fluoride (1000 ppm) 932 ± 373 60 ± 15a
Demineralized only 1247 ± 502 90 ± 12b
55
Figure 22. Microradiographic image and mineral volume profile for an exemplary sample from
the fluoride-free treatment group.
4.3.2.2 PTR-LUM frequency response
PTR and LUM amplitude and phase signals for the sample microradiograph shown in Fig. 22 are
presented in Fig. 23. In order to enhance the trends of signal behaviour following
remineralization, amplitude ratios and phase differences with respect to the final
demineralization amplitude and phase signals are presented. Amplitude ratios greater or less than
unity indicate an increase or decrease in amplitude above or below the final demineralization
curve, respectively, across the probed modulation frequency range. Positive and negative phase
differences refer to smaller and larger phase lags, respectively. Following demineralization until
the final remineralization treatment point there was a trend of increasing PTR amplitude across
the entire frequency range and decrease in PTR phase lag, particularly at low modulation
frequencies. The PTR amplitude shifted very slowly during the first 10 days of exposure to the
mineralizing solution, however, following 10 days the curves rapidly and consistently increased
in amplitude until the end of the exposure period. An increase in phase lag occurred at all
treatment points except day 10, where a drastic and transient decrease in phase lag was observed
at high modulation frequencies. Amplitude ratios and phase differences under 830-nm radiation
revealed similar PTR features as noted under 660-nm (Appendix 1; Fig. A.1.2).
LUM amplitude ratios, normalized with respect to the final demineralized curve, revealed
relatively flat frequency dependence. Over the course of both de- and remineralization
treatments, the flat appearance was maintained, whereas vertical shifts in amplitude dominated.
LUM phases exhibited minima in the modulation frequency range 63 - 100 Hz. Large decreases
100 µm
56
in LUM amplitude and phase minima occurred over the first 10 days. A slight increase in
amplitude and phase after 20 days was followed by a reversal and decrease at 28 days.
Figure 23. PTR-LUM amplitude ratios and phases differences with respect to the final
demineralization state for a sample in the fluoride-free treatment group, under 660nm laser
excitation. The corresponding microradiograph and mineral volume profile is shown in Fig. 22. Error bars, when not visible, are of the size of the symbols.
4.3.2.3 Theoretical analysis of fluoride-free remineralized samples
An excellent fit between PTR experimental data and 3-layer theory was found for the fluoride-
free sample (Appendix 2; Fig. A.2.3). The change in optical parameters (absorption and
scattering coefficients), thermophysical parameters (diffusivity and conductivity) and layer
thicknesses as a function of demineralization time (10-day) and subsequent remineralization (4-
week) in the fluoride-free solution is shown in Figs. 24-26. Plots of the additional parameters
over treatment time are given in Appendix 3. Trends in optical and thermal properties over the
1 10 100 10001.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
Am
plit
ude R
atio (
V/V
0)
Frequency (Hz)1 10 100 1000
0.80
0.84
0.88
0.92
0.96
Remin- 2 Days
Remin- 5 Days
Remin- 10 Days
Remin- 20 Days
Remin- 28 Days
PTR Amplitude LUM Amplitude
Am
plit
ude R
atio (
V/V
0)
Frequency (Hz)
1 10 100 1000
-4
-2
0
2
4
6
8 PTR Phase
Phase D
iffe
rence (-
0)
Frequency (Hz)10 100
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0.0
0.1LUM phase
Phase D
iffe
rence (-
0)
Frequency (Hz)
57
10 day demineralization period are consistent with those described in the previous section for
demineralized samples. At the onset of remineralization, a decrease in optical absorption
coefficient of both layers for 10 days was followed by a linear increase in the properties of layer
1 until the end of the 4-week period (Fig. 24A). Optical scattering coefficients continued to rise
within the lesion body and within the surface layer a marked increase in surface scattering was
noted at the 10 day remineralization period (Fig. 24B). This is consistent with the smaller phase
lag at high modulation frequencies and increase in slope of the amplitude ratio of the 10-day
remineralized curve (Fig. 23).
Figure 24. Change in optical absorption (A) and scattering coefficients (B) over treatment time
for a sample in the fluoride-free treatment group. Vertical dashed lines separate demineralization
and remineralization treatments. Layer 1 = surface layer; Layer 2 = lesion body.
At the onset of remineralization, a large decrease in thermal diffusivity (Fig. 25B) of the surface
layer occurred with a small increase in the subsurface layer. Surface layer diffusivity increased
over the remineralization period and saturated after 10-day remineralization. The diffusivity of
the lesion body changed in a similar fashion, decreasing monotonically after the first day of the
10-day remineralization. Thermal conductivities (Fig. 25A) exhibited similar trends to
diffusivities: that of the surface layer also increased with increasing remineralization time,
whereas small increases were only found after 5 days of remineralization.
0 10 20 30 40
0
25000
50000
75000
100000
125000 Demin Remin
Scat
teri
ng
Co
eff
icie
nt
(m-1
)
Treatment Time (days)
Layer 1
Layer 2
(A) (B)
0 10 20 30 40
20
40
60
80
100
120
Demin Remin
Ab
sorp
tio
n C
oef
fici
ent
(m-1
)
Treatment Time (days)
Layer 1
Layer 2
58
Figure 25. Change in thermal conductivity (A) and diffusivity (B) over treatment time for a
sample in the fluoride-free treatment group. Vertical dashed lines separate demineralization and
remineralization treatments. Layer 1 = surface layer; Layer 2 = lesion body.
Changes in layer thicknesses (Fig. 26) were dominant within the first 10 days of
remineralization, where a significant decrease in thickness of the lesion body occurred with a
concomitant increase in thickness of the surface layer. Minimal changes in thickness occurred
after the 10 day remineralization period.
Figure 26. Change in layer thicknesses over treatment time for a sample in the fluoride-free
treatment group. The vertical dashed line separates de- and remineralization treatments. Layer 1
= surface layer; Layer 2 = lesion body.
0 10 20 30 40
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
ReminDemin
Ther
mal
Co
nd
uct
ivit
y (W
/mK
)
Treatment Time (days)
Layer 1
Layer 2
0 10 20 30 40
2.0x10-7
3.0x10-7
4.0x10-7
5.0x10-7
6.0x10-7
7.0x10-7
ReminDemin
Ther
mal
Dif
fusi
vity
(m
2 /s)
Treatment Time (days)
Layer 1
Layer 2
(A) (B)
0 10 20 30 40
0
10
20
30
40
50
60
70
80
90
ReminDemin
Laye
r Th
ickn
ess
(m
)
Treatment Time (days)
Layer 1
Layer 2
59
4.3.3 Low fluoride (1 ppm) remineralization group
4.3.3.1 Microradiographs and mineral content depth profiles
An exemplary sample from the low fluoride group revealed a more discernible approximate 3-
layer geometrical lesion structure, outlining a radiodense intact surface layer overlying the lesion
body and followed by bulk sound enamel (Fig. 27).
Figure 27. Microradiographic image and mineral volume profile for an exemplary sample from
the low fluoride treatment group.
4.3.3.2 PTR-LUM frequency response
PTR-LUM frequency response is shown in Fig. 28. A consistent trend of increasing PTR
amplitude across the entire modulation frequency range and decrease in phase lag at low
frequencies from the final demineralization curve to the final remineralization curve was evident.
However, marked differences were evident at earlier and later mineral solution exposure times.
Large separation appeared between measurements in the first 10 days and subsequent
measurements at later periods. Furthermore, trends of increased slope in PTR amplitude and
phase with a marked approximately linear increase in amplitude from low to high modulation
frequencies were evident at later remineralization times. Significant amplitude depression at the
inception of the exposure period was noted and continued for 10 days. Amplitude ratios and
phase differences under 830-nm radiation revealed a near-monotonic increase in amplitude with
a notable change in slope increasing toward the high modulation frequencies (Appendix 1; Fig.
A.1.3).
LUM amplitude and phase behaviour mirrored changes in PTR where a reversal in direction in
both amplitude and phase occurred for the first 10 days of remineralization. It is also important
100 µm
60
to note that LUM trends are opposite those of PTR, such that higher PTR signal amplitude
correlated with lower LUM signal amplitude and vice versa.
Figure 28. PTR-LUM amplitude ratios and phases differences with respect to the final
demineralization state for a sample in the low fluoride treatment group, under 660nm laser
excitation. The corresponding microradiograph and mineral volume profile is shown in Fig. 27. Error bars, when not visible, are of the size of the symbols.
4.3.3.3 Theoretical analysis of low fluoride remineralized samples
An excellent fit was found between experimental and theoretical data for the final demineralized
PTR curve and all remineralized curves (Appendix 2; Fig. A.2.4). Changes in optothermal
properties during the initial demineralized phase are consistent with the previous description of
demineralized control samples. An increase in absorption coefficient of the lesion body occurred
over the 10-day remineralization period (Fig. 29A) with an increase in the surface layer from day
20 until day 38. A marked decrease in the scattering properties of the lesion body occurred over
1 10 100 1000
0.85
0.90
0.95
1.00
1.05
1.10
1.15
1.20
1.25
Am
plit
ude R
atio (
V/V
0)
Frequency (Hz)1 10 100 1000
0.80
0.84
0.88
0.92
0.96
1.00
1.04
1.08
Remin- 2 Days
Remin- 5 Days
Remin- 10 Days
Remin- 20 Days
Remin- 28 Days
PTR Amplitude LUM Amplitude
Am
plit
ude R
atio (
V/V
0)
Frequency (Hz)
1 10 100 1000
-3
-2
-1
0
1
2
3
4
5 PTR Phase
Phase D
iffe
rence (-
0)
Frequency (Hz)10 100 1000
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0.0
0.1
0.2 LUM phaseP
hase D
iffe
rence (-
0)
Frequency (Hz)
61
the first 5 days of remineralization (Fig. 29B). From the 5-day remineralization period onward
scattering properties began to rise and increased above the final demineralized state at the 30-40-
day overall treatment period. Scattering properties of the surface layer were insignificant
compared to the dominant changes which occurred within layer 2.
Figure 29. Change in optical absorption (A) and scattering coefficients (B) over treatment time
for a sample in the low fluoride treatment group. Vertical dashed lines separate demineralization
and remineralization treatments. Layer 1 = surface layer; Layer 2 = lesion body.
At the onset of remineralization, an increase in the thermal properties of the lesion body was
noted over the first 10 days (Fig. 30A, B), which then began to decrease at prolonged treatment
times consistent with the PTR amplitude and phase frequency response (Fig. 28). The
improvement in the thermal conductivity of the lesion body occurred with the generation of a
transient poorer conductivity of the surface layer (Fig. 30A). After 20 and 28 days of
remineralization an increase in the thermal conductivity of the surface layer was evident.
Diffusivities followed a similar pattern; however, properties of the surface layer remained higher
than the subsurface layer and increased from day 5 - 28 (Fig. 30B).
0 10 20 30 40
0
25000
50000
75000
100000
125000
150000
Demin Remin
Scat
teri
ng
Co
effi
cien
t (m
-1)
Treatment Time (days)
Layer 1
Layer 2
(A) (B)
0 10 20 30 400
20
40
60
80
100
120
140
Demin Remin
Ab
sorp
tio
n C
oef
fici
ent
(m-1
)
Treatment Time (days)
Layer 1
Layer 2
62
Figure 30. Change in thermal conductivity (A) and diffusivity (B) over treatment time for the
low fluoride sample. Vertical dashed lines separate de- and remineralization treatments. Layer 1
= surface layer; Layer 2 = lesion body.
A large reduction in the thickness of the lesion body occurred over the first 10 days of
remineralization with almost no change in thickness thereafter (Fig. 31). The large decrease in
thickness of the lesion body occurred with a simultaneous increase in thickness of the surface
layer from day 0 to day 10 of remineralization.
Figure 31. Change in layer thicknesses over treatment time for a sample in the low fluoride
treatment group. The vertical dashed line separates de- and remineralization treatments. Layer 1
= surface layer; Layer 2 = lesion body.
0 10 20 30 40
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
ReminDemin
The
rmal
Co
nd
uct
ivit
y (W
/mK
)
Treatment Time (days)
Layer 1
Layer 2
0 10 20 30 40
2.0x10-7
3.0x10-7
4.0x10-7
5.0x10-7
6.0x10-7
7.0x10-7
ReminDemin
Th
erm
al D
iffu
sivi
ty (
m2 /s
)
Treatment Time (days)
Layer 1
Layer 2
(A) (B)
0 10 20 30 40
0
10
20
30
40
50
60ReminDemin
Laye
r T
hic
kne
ss (
m)
Treatment Time (days)
Layer 1
Layer 2
63
4.3.4 High fluoride (1000 ppm) remineralization group
4.3.4.1 Microradiographs and mineral content depth profiles
An exemplary sample from the high fluoride group showed a hypomineralized layer above the
intact surface layer which reached a maximum mineral volume at a depth below the enamel
surface (≈14 µm) (Fig. 32). Surface hypomineralization was evident in 70% of the high fluoride
group. Poorer distinction between the intact surface layer and the subsurface lesion was due to
reduced subsurface radiolucency.
Figure 32. Microradiographic image and mineral volume profile for an exemplary sample from
the high fluoride treatment group.
4.3.4.2 PTR-LUM frequency response
PTR-LUM frequency responses for a sample from the high fluoride group are presented in Fig.
33. A decrease in low-frequency amplitude up to the 10-day exposure period was evident.
Onward from the 10-day exposure period a substantial increase in PTR amplitude across all
modulation frequencies and decrease in PTR phase lag at low modulation frequencies occurred.
The low frequency PTR phase pattern was accentuated with increasing exposure time: smaller
phase lag and more pronounced curvature of phase maxima. A trend consistent among all
samples remineralized in the presence of high fluoride concentrations was the decrease in PTR
amplitude across the entire modulation frequency range from the 20 day to the 28 day exposure
period. This occurred with a concomitant increase in phase lag at low modulation frequencies.
Nearly identical trends were evident for frequency scans with the 830-nm laser (Appendix 1; Fig.
A.1.4). As noted for the other 2 treatment groups, poor SNR of the 830-nm laser made small
phase changes due to remineralization difficult to discern. LUM amplitude and phase signals did
not exhibit trends consistent with treatment time; however, they reflected changes in PTR
100 µm
64
amplitude at high frequency. Larger PTR amplitude at high frequencies correlated with lower
LUM amplitude and vice versa. Little change in amplitude accompanied a decrease in phase at 2
days of remineralization. Marked reduction in LUM amplitude occurred after 5 and 10 days of
remineralization in both samples. This was followed by a large increase in amplitude and phase
at 20 days of remineralization, which continued to increase at day 28.
Figure 33. PTR-LUM amplitude ratios and phase differences with respect to the final
demineralization state for a sample in the high fluoride treatment group, under 660nm laser
excitation. The corresponding microradiograph and mineral volume profile is shown in Fig. 32.
Error bars, when not visible, are of the size of the symbols.
4.3.4.3 Theoretical analysis of high-fluoride remineralized samples
Optical and thermal properties and thicknesses of the high fluoride sample are shown in Figs. 34-
37. The depth profiles were constructed from the fitting of 3-layer theory to experimental PTR
1 10 100 1000
1.0
1.2
1.4
1.6
Am
plit
ude R
atio (
V/V
o)
Frequency (Hz)1 10 100 1000
0.84
0.88
0.92
0.96
1.00PTR Amplitude LUM Amplitude
Am
plit
ude R
atio (
V/V
o)
Frequency (Hz)
1 10 100 1000
-4
-2
0
2
4
6
8
Remin- 2 Days
Remin- 5 Days
Remin- 10 Days
Remin- 20 Days
Remin- 28 Days
PTR Phase
Phase D
iffe
rence ( -
0)
Frequency (Hz)10 100 1000
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0.0
LUM phase
Phase D
iffe
rence ( -
0)
Frequency (Hz)
65
curves, which showed an excellent fit with a small residual (Appendix 2; Fig. A.2.5). Optical
properties (Fig. 34) during the demineralized phase followed trends identified for the 10-day
treated samples of the demineralized control group; however a different thermophysical profile
was evident (Fig. 35). It is noteworthy to point out a marked increase in the scattering coefficient
of layer 1 after 5 days of demineralization (Fig. 34B). A large decrease in conductivity (Fig.
35A) of both layers occurred at 5 days of demineralization while smaller changes in diffusivity
occurred (Fig. 35B).
An increase in the scattering coefficient of the lesion body after 2 days of remineralization
rapidly declined at prolonged exposure times. A slight reversal in the scattering properties led to
an increase at the 28-day remineralization period. Changes in the scattering properties of the
surface layer were small compared to the dominant changes in the lesion body.
Figure 34. Change in optical absorption (A) and scattering coefficients (B) over treatment time
for a sample in the high fluoride treatment group. Vertical dashed lines separate demineralization
and remineralization treatments. Layer 1 = surface layer; Layer 2 = lesion body.
Remineralization enhanced the thermophysical properties of both layers over the 10-day
remineralization period. At longer layer exposure periods to the fluoride solution, a marked
decrease in both thermal conductivity (Fig. 35A) and thermal diffusivity (Fig. 35B) was
observed.
0 10 20 30 40
20
30
40
50
60
70
80
90
100
110
120
Demin Remin
Ab
sorp
tio
n C
oe
ffic
ien
t (m
-1)
Treatment Time (days)
Layer 1
Layer 2
0 10 20 30 40
0
20000
40000
60000
80000
100000
120000
140000
160000
Demin Remin
Scat
teri
ng
Co
eff
icie
nt
(m-1
)
Treatment Time (days)
Layer 1
Layer 2
(B)(A)
66
Figure 35. Change in thermal conductivity (A) and diffusivity (B) over treatment time for a
sample in the high fluoride treatment group. Vertical dashed lines separate de- and
remineralization treatments. Layer 1 = surface layer; Layer 2 = lesion body.
A significant decrease in thickness occurred within the lesion body over the 10-day
remineralization period, following a steep increase during the early demineralization period (Fig.
36). Little-to-no change was observed in the thickness of the surface layer over the 4 week
remineralization period.
Figure 36. Change in layer thicknesses over treatment time for a sample in the high fluoride
treatment group. The vertical dashed line separates de- and remineralization treatments. Layer 1
= surface layer; Layer 2 = lesion body.
A summary of the typical trends in the core physical parameters following demineralization and
remineralization are presented in Table 11. The trends during demineralization are the same as
those outlined in Table 9. The trends evaluated in Table 11 are compiled from Figs. 24-26, 29-31
0 10 20 30 40
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
ReminDeminTh
erm
al C
on
du
ctiv
ity
(W/m
K)
Treatment Time (days)
Layer 1
Layer 2
0 10 20 30 40
2.0x10-7
3.0x10-7
4.0x10-7
5.0x10-7
6.0x10-7
7.0x10-7
ReminDemin
The
rmal
Dif
fusi
vity
(m
2 /s)
Treatment Time (days)
Layer 1
Layer 2
(B)(A)
0 10 20 30 40
0
10
20
30
40
50
60
70
ReminDemin
Laye
r Th
ickn
ess
(
m)
Treatment Time (days)
Layer 1
Layer 2
67
and 34-36 and separate short-term trends (0 – 10 days) from long-term trends (20 – 28 days)
during remineralization.
Table 11. General trends in the main physical parameters following demineralization and short
vs. long term remineralization. The arrows indicate trends where ↑, ↓ and ↔, refer to an increase,
decrease or no change, respectively, in parameters over time.
† A decrease was noted in the 40-day demineralized and high fluoride samples
ffi An increase was observed in the fluoride-free sample
* An increase was observed in the high fluoride sample
¥ Differed depending on initial thickness of the aprismatic layer
5 Discussion
A common limitation in in vitro cariology research is that a direct measure of mineral content
during de- and remineralization evaluation is destructive in nature. Furthermore, frequently used
substrates for de- and remineralization experiments include thin, polished enamel sections rather
than whole teeth. It has been repeatedly demonstrated that abraded enamel samples demineralize
and remineralize at different rates and extent than enamel that has its natural surface intact (Xue
et al. 2009). As a result, studies implementing polished, ground or abraded enamel sections cause
a significant removal of surface structural features (i.e. aprismatic enamel), precluding the
identification of surface enamel influences and diverging farther from in vivo relevancy.
5.1 PTR-LUM signals and multiparameter fits of sound enamel
Since the natural enamel surface modulates the chemical reactions between the internal sound
enamel structure and external oral environment, proper characterization of its properties are
Physical Parameters
Demineralized Remineralized Remineralized Remineralized
No fluoride Low fluoride High fluoride
0 – 10 days 0 – 10 days
20 – 28 days
0 – 10 days
20 – 28 days
0 – 10 days
20 – 28 days
µa1 ↑ ↓ ↑ ↑ ↑ ↔ ↑
µa2 ↓ ↓ ↑ ↑ ↓ ↑ ↑
µs1 ↑ ↑ ↓ ↓ ↑ ↑ ↓
µs2 ↑ ↑ ↑ ↓ ↑ ↓ ↑
κ1 ↑† ↑ ↑ ↓ ↑ ↑ ↓
κ2 ↓ ↑ ↔ ↑ ↓ ↑ ↓
α1 ↓‡ ↓ ↔ ↓ ↑ ↑ ↓
α2 ↓* ↓ ↔ ↑ ↓ ↑ ↓
L1 ↓¥ ↑ ↔ ↑ ↔ ↔ ↔
L2 ↑ ↓ ↔ ↓ ↔ ↓ ↔
68
essential. To date, there have been few reports on the surface characterization of aprismatic
enamel, mainly a result of its removal in in vitro studies coupled with the fact that its
infinitesimal thickness lies at the lower detection limit of current evaluation techniques and is
often presented as continuous and indistinct from the underlying bulk enamel. Initial attempts to
fit experimental PTR amplitude and phase to diffuse photon density and thermal wave theory
based on 2 effective layers where L1 and L2 were enamel and dentin, respectively, yielded poor
quality fits. However, recently, the introduction of a layer at the enamel surface with optothermal
properties different from bulk enamel, termed the aprismatic layer, resulted in a good fit of
experimental data to theory (Matvienko et al. 2009b). In the present study, the 2-layer
approximation of sound enamel, where layer 1 is the aprismatic layer and layer 2 is semi-infinite
sound enamel was implemented (Fig. 11). Although aprismatic enamel is more prevalent in
unerupted and deciduous dentition (primarily molars), it has also been observed in about 70% of
permanent human molars at varying thickness depending on the tooth surface (Gwinnett 1967),
and therefore its presence was considered to play an integral role at the superficial aspect of
sound enamel. Furthermore, evidence that the surface layer indeed represented a layer of
aprismatic enamel could not be discerned from microradiographic analysis (Fig. 15). Earlier
studies documented that the outermost layers of enamel were occasionally hypermineralized and
appeared as radiodense zones on microradiographs (Gwinnett 1967; Darling and Crabb 1956).
The described structurally dense crystallite packing and reduced pore volume of aprismatic
layers (Gwinnett 1967) are in contrast to the surface hypomineralization observed in the
microradiographs of sound teeth in the present study (Fig. 15). However, it has also been stated
that to date there is no evidence to suggest that the aprismatic layer is hypo- or hyper-mineralized
compared to underlying enamel (Ripa et al. 1966; Gwinnett 1967). The characterization of a thin
surface layer in the theoretical representation of sound enamel may likely be influenced by a
combination of factors, including: the presence of a residual aprismatic layer, surface mineral
loss due to the storage conditions of the extracted samples prior to experimentation (distilled
water at 40C), abrasion of the enamel surface during the tooth‘s lifetime in the oral cavity and
lastly the higher fluoride concentrations that may form a distinct mineral phase (F-OHAp) at the
outermost enamel (Weatherell et al. 1974). All of the aforementioned sources may contribute to
the generation of a surface layer with variable optothermal properties. For the purposes of the
69
present investigation, all of these sources were classified under the general term ―aprismatic‖,
even though the surface layer is not likely to be completely devoid of prisms.
Analysis of the optothermal parameters derived from the theoretical fittings of PTR amplitude
and phase signals of untreated teeth revealed highly variable opto-thermophysical properties of
layer 1 (aprismatic enamel) whereas the properties of layer 2 (sound prismatic enamel) fell
within a narrow range (Table 8). As expected, the values derived for sound enamel were
consistent with the values found in earlier literature (Tables 1 and 2), since the literature values
were used as guidelines to define the initial limits of the fitting program. The computational
program optimally selected parameter values between the literature-defined limits without
converging to the upper/lower parameter ranges in order to yield the smallest residual between
experimental data and theory. In terms of the optical properties, the diffuse prism-packing and
the presence of amorphous mineral deposits within the aprismatic surface layer has been
suggested to act as optical gatherers (Gwinnett 1967; Odor et al 1996). A higher PTR-derived
mean optical absorption coefficient in the aprismatic layer (65 m-1
) compared to sound enamel
(44 m-1
) supports this earlier observation (Table 8). The largest deviation in optothermal
properties in sound enamel was observed in the thermal properties of the aprismatic vs. prismatic
enamel (Table 8). A slightly higher thermal diffusivity and poorer thermal conductivity of the
aprismatic layer compared to the underlying enamel, may suggest a much lower density of this
layer as diffusivity due to the relationship between density and diffusivity (See equation 1). This
is further supported by the evidence of surface hypomineralization in mineral content depth
profiles (Fig. 15). The presence of surface discontinuities, such as the aprismatic layer, resulted
in poorer thermal properties, thereby generating a thermally impeding layer at the most
superficial aspect of sound enamel. This would effectively reduce thermal diffusion lengths as
near-surface thermal-wave confinement dominates. From PTR scans of untreated teeth, phase
frequency responses were near-linear at lower modulation frequencies, however exhibited phase
peaks in the high frequency range. The observation of phase maxima at higher modulation
frequencies is consistent with the generation of a thermal-wave interference pattern occurring
closer to the anatomical surface. Given the frequency at which phase maxima appear, above 100
Hz, and the mean thermal diffusivity values of sound enamel from the fitted data (4.41 x 10-7
m2/s) (Table 8), the corresponding mean thermal diffusion length is approximately µ (100 Hz) ≈
70
37.5 µm. This thermal diffusion length corresponding to the phase maxima at 100 Hz, the lowest
frequency where maxima appeared in sound enamel was similar to the largest thickness of the
aprismatic layer determined from TMR analysis (44 µm). The theoretically derived thickness of
the aprismatic layer was on average ≈13 µm, with a maximum thickness of ≈22 µm. These
thickness values are in line with previous investigations by Ripa (1966) and Gwinnett (1967)
who observed average surface layer zones of 30 µm and 20 µm, respectively. Whittaker (1982)
found that the most frequently observed (42 - 53%) width of the aprismatic layer in lingual
enamel of permanent molars and 3rd
molars was between 16 – 45 μm.
The overall characterization of the outermost enamel surface layer is vital as all chemical attacks
on enamel, be it caries or erosion, are modulated by surface enamel properties. This layer may be
an important determinant in the overall caries susceptibility of enamel; its incidence has been
previously related to lower caries rates (Jackson 1971). As the occurrence of thick aprismatic
surface layers was low, a causal relationship between aprismatic layer thickness and the depth of
the subsequently demineralized lesion could not be established. Analysis of sound enamel in this
study illustrated the capability of PTR-LUM to non-destructively evaluate and quantify the
properties of untreated intact enamel surfaces. This would be invaluable in dental research in
terms of selecting more uniform enamel substrates for in vitro or in vivo demineralization and
remineralization studies. This is supported by the fact that the properties of the untreated sample
can have a marked response on subsequent de- and remineralization (Groeneveld et al. 1975;
Brudevold et al. 1968; Xue et al. 2009). Extrapolation to clinical environments may allow for the
identification of caries susceptible sites on enamel and further, may be implicated in time-
dependent enamel etching and bonding techniques.
5.2 PTR-LUM signals during short and long-term demineralization
At the first exposure of the sound enamel to the demineralizing solution, the 2-layer theoretical
approximation was no longer valid, and instead a 3-layer theoretical approximation was
considered, where layer 1, 2 and 3 were the intact surface layer, lesion body and sound enamel,
respectively (Fig. 12). The intact surface layer may be a combination of aprismatic and
remineralized enamel; however, since the first PTR measurement occurred after 5 days of
demineralization it is likely that the latter dominated. During demineralization a consistent trend
71
of increasing PTR amplitude with a concomitant decrease in PTR phase lag and a shift of the
phase peaks to lower frequencies were observed (Figs. 16 and 17). These trends suggest the
formation of a depth-wise growing subsurface lesion as documented elsewhere (Jeon et al. 2008;
Jeon et al. 2004a; Nicolaides et al. 2002). Changes in PTR phase with demineralization are
attributed to thermal-wave interference patterns within the demineralized lesion, which increases
in thickness during the demineralization period.
5.2.1 Multiparameter fits of PTR signals during demineralization
PTR amplitude and phase trends can be explained based on the generated set of optical and
thermal properties of each effective layer (Figs. 18 - 20). Prior to the discussion of the trends in
opto-thermophysical properties during demineralization it is important to highlight the
uniqueness of the fitted parameters, which is examined in detail in Appendix 4. The convergence
of the fitted parameters, in particular thicknesses, to similar values under different parameter
ranges (Appendix 4; Table A.4.2) illustrates the robustness of the computational algorithm and
the reliability of the derived values from the best fits of experimental data. Opto-thermophysical
properties of demineralized samples were generated from the theoretical fitting program by first
fitting the final demineralized PTR amplitude and phase curve, using thicknesses from the final
TMR as guidelines (Fig. 13). Given the PTR amplitude and phase curve fittings and parameters
extracted from the untreated state and from the final demineralized state, the thicknesses from
the 2 treatment end points were used to fit frequency scan data from intermediate treatment
points. Optical absorption coefficients for layer 1 increased with demineralization time, while no
trend was evident in the absorption properties of the lesion body (Figs. 18a and 20a). Larger
absorption coefficients result in a stronger thermal wave field and consequently larger amplitude.
The higher spatial rate of photon absorption within layer 1 results in the confinement of the
subsurface extent of the thermal wave to a narrower region causing a higher amplitude and
smaller phase lag (Matvienko et al. 2009b). An earlier study that determined optical properties
from enamel slabs revealed that there was no evidence of an increase or decrease in optical
absorption coefficients between sound and carious enamel, however large errors (>40%) in the
calculated absorption coefficients were observed (Spitzer and ten Bosch 1977). Higher scattering
coefficients of the developing lesion (layer 1 and 2) are also consistent with the aforementioned
PTR trends (Figs. 18b and 20b). Upon demineralization, changes in the scattering coefficient
72
were more dominant than changes in the absorption coefficient, as has been documented
previously (Spitzer and ten Bosch 1977). This was caused by crystalline disintegration of the
enamel structure and the generation of small pores which can act as scattering centers (Darling et
al. 2006; Angmar-Månsson and ten Bosch 1987; Zijp 2001). Higher scatter of the diffuse-photon
density field results in shorter optical path lengths within enamel (Angmar-Månsson and ten
Bosch 1987) which photothermally is tantamount to a higher absorption coefficient and increases
the generated PTR signal. The previously documented higher reflectance and poorer
transmission properties of carious lesions would likely contribute to the localization of the
optical field to a narrow region, lessening the influence of the underlying layers (Ko et al. 2000;
Spitzer and ten Bosch 1977). As photons are localized to a narrower surface region, a higher
probability for absorption and non-radiative conversion processes would confine the thermal
wave centroid closer to the enamel surface (Matvienko et al. 2009a; Mandelis and Feng 2002).
The scattering properties of both layer 1 (surface layer) and layer 2 (lesion body) increased
substantially from the start of demineralization, with greater scattering contribution from the
demineralized lesion body near the end of the treatment period (Figs. 18b and 20b). Considering
only the first 20 days, a rapid increase in the scattering coefficient of the lesion body from day 0
– 10 followed a more gradual change from 10-20 days (Fig. 20b). Replacement of the
demineralizing medium with a fresh aliquot after 20 days most likely dissipated the chemical
gradients in the gel surrounding the enamel accelerating the demineralization rate until near-
saturation conditions were restored. This may explain the linear increase in scattering coefficient
of the lesion body from the 20 – 40-day period, as well as the concomitant increase in lesion
depth over the same period (Fig. 19b). As incipient lesions typically generate an increasingly
inhomogeneous medium, a higher scattering coefficient was expected relative to sound enamel.
Substantial increases in the scattering coefficient of demineralized enamel relative to sound
enamel have been documented. Darling et al. (2006) found an increase in the scattering
coefficient by 1 or 2 orders of magnitude over the scattering coefficient of sound enamel. Spitzer
and ten Bosch (1977) found a scattering coefficient of white spot lesions to be 5 -10 times higher
than sound enamel. Higher scattering within the lesion body may occur since this zone occupies
the largest volume within a carious lesion, decreasing in mineral volume from 87 vol% at the
sound enamel level to ≈60 vol% in the subsurface minimum. Subsurface demineralization is
73
characterized by the enlargement of the micropores through central core dissolution of crystals
(Palamara et al. 1986). The enlargement of the micropores and the spaces at the prism
boundaries make the tissue more penetrable to the influx of acids from the demineralizing
medium and the out-diffusion of dissolved minerals from the dissolution reaction. The enhanced
scattering properties of the intact surface layer may be due to the composite nature of this layer
during demineralization. The outer surface layer maintains a level of porosity greater than sound
enamel and less than the lesion body (Figs. 16 and 17). Furthermore, the intact surface layer
most likely acquired minerals along its inner surface, deposited from the out-diffused flux at the
advancing lesion front, as well as outer surface, from mineral deposition from the demineralizing
solution at later treatment times as the buildup of mineral ions in solution increased and the
driving force (degree of saturation) for demineralization was reduced. Miake et al. (2003) and
Tohda et al. (1990) showed that demineralized surface layers contained a large number of
irregularly shaped crystals of various sizes. The presence of unstructured crystals in the surface
layer would augment the scattering properties of layer 1 by creating additional crystal grain
boundaries, as seen in Fig. 20B.
The generation of layers with poorer thermal properties was supported by theoretical analysis
which consistently showed a decrease in thermal conductivity and diffusivity of the lesion body
(layer 2) (Figs. 18c, d and 20c, d). The creation of small micro-channels within the
demineralized lesion may generate an impedance to heat propagation resulting in a poorer
thermal conductivity. A reduction in the thermal conductivity would result in a corresponding
reduction in thermal diffusivity based on equation 1. The poorer thermal diffusivity of the lesion
body may impede back-propagation of thermal wave contributions from much deeper regions
from reaching the enamel surface and as a result confine the region of thermal wave field within
the growing demineralized layer. Investigations on the extraction of thermal properties, namely
conductivity and diffusivity, from carious and sound enamel are scarce. A reduction in the
thermal properties in artificially demineralized lesions relative to sound enamel was observed by
El-Brolossy et al. (2008) using photoacoustic spectroscopy; however, the extent of reduction in
conductivity (0.72 W/mK) and diffusivity (3.81x10-7
m2/s) values for demineralized enamel were
far less than those observed in the present study (Figs. 18c, d and 20c, d). Intuitively, it might be
expected that thermal diffusivity would increase with demineralization due to its inverse relation
74
to density (equation 1), which decreases within a lesion. However, the increase in lesion porosity
results in filling of the enamel pores with a combination of air and water, both of which have a
poorer thermal conductivity than sound enamel (≈0.87 Wm-1
K-1
), 0.026 and 0.598 Wm-1
K-1
,
respectively, as well as a higher specific heat capacity (Almond and Patel 1996; Vargaftik et al.
1994). For comparative purposes, dentin, which has intrinsically higher porosity and mineral
volume occupied by water, conductivity (0.577 - 0.623 Wm-1
K-1
) and diffusivity (1.87 - 2.6 x10-7
m2s
-1) values are much lower than enamel (Brown et al. 1970; Braden 1964). This indicates that
the balance between the ratio of thermal conductivity to the product of the density and heat
capacity will ultimately determine the outcome of the thermal diffusivity, as well as the fact that
residual water within the enamel pores may have a significant influence on thermal diffusion
properties of enamel.
Contrasting processes were resolved in the thermal profiles for the surface layer which may be
related to the dynamic dissolution – reprecipitation processes during lesion formation. A rapid
decrease in thermal diffusivity and conductivity of layer 1 for the first 15 days of
demineralization was followed by an increase and stabilization of values (Fig. 20c, d). Initial
exposure of enamel to the demineralizing medium likely results in superficial mineral loss of the
most acid soluble mineral phases. As a result, changes in surface enamel structure would have a
significant impact on the optical and thermal energy generation and propagation. The decreasing
thermal properties of the surface layer may be related to the superficial removal of mineral
phases which may include the ―priming‖ of the enamel surface (Moreno and Zahradnik 1974)
and/or etching of the aprismatic layer. A decrease in thickness of layer 1 in the 10-day
demineralized sample may support the breakdown of the aprismatic layer toward the formation
of the intact surface layer with the concomitant progression of the lesion body inwards (Figs.
19a, 26 and 36). The initial dissolution of enamel has been shown elsewhere (Groenhuis et al.
1980), where a slightly larger surface roughness was induced over a 7 days acid exposure period.
Other studies have found surface erosion within the first 120 – 245 hrs of demineralization
followed by a period of substantial subsurface demineralization (Anderson and Elliott 1992;
Anderson et al. 1998; Zhang et al. 2000a). An increase in roughness would increase the
photothermal signal due to the larger surface area to volume ratio yielding a higher probability
for absorption events and higher thermal wave fields due to confinement at the rough spots. As
75
the thermal properties of the surface layer became poorer for the first 15 days, there would be
effective confinement of optical – thermal energy conversion reactions within this thermally
insulating surface layer, preventing contributions from deeper thermal wave sources (Fig. 20c,
d). After 15 days, the properties increased which may be the result of the well-defined, built up,
intact surface layer overlying the lesion body. The improved thermal properties of layer 1 may
facilitate thermal energy propagation and enhance the interference between forward and L2
interface-interacted (back-propagating) thermal waves. A similar trend was observed in the
optical reflection coefficient between the first 15 days and latter 25 days, where the increase in
the latter 25 days may be the result of optical reflection within the well-defined layers of the
built-up surface layer and lesion body (Appendix 3; Fig. A.3.2c). An increase in optical
reflectivity during demineralization was observed by Baumgartner et al. (2000) using the PS-
OCT system and Ko et al. (2000) with a CCD camera and image-processing software. Surface
dissolution may dominate earlier demineralization times, generating poorer thermal properties in
the surface layer; whereas at later demineralization times the enhancement of the thermal
properties may indicate mineral reprecipitation to restore surface enamel crystallinity. This
reaction mechanism is further supported by the changes in thickness of the intact surface layer
(layer 1) (Fig. 19b-inset), which paralleled the change in thermal properties. Little change in the
thickness of layer 1 was evident for the first 15 days, whereas substantial mineral gains were
found thereafter. Growth of the intact surface layer over time was also observed by Gray and
Francis (1963), Groeneveld et al. (1975) and Gao et al. (1993). This is in contrast to Featherstone
et al. (1978) who observed that after 5-days of demineralization the surface layer formed and its
thickness remained relatively constant thereafter. The stabilized parameters of layer 2 after a
period of time may be explained based on the fact that once the lesion front has passed a certain
depth only minor changes in the mineral volume of the lesion body follow. Thus, the minimum
mineral volume of the lesion body will remain about constant and rather the lesion body acts as a
transport medium for the diffusion of ions to and from the advancing from of the lesion (Arends
et al. 1997). Slight improvements in the thermal properties from the 15-day period onward (Figs.
20c, d) may be attributed to mineral deposition throughout the depth of the lesion as dissolved
mineral ions are transported from the advancing front toward the enamel surface and/or a result
of errors in the theoretical extraction of thermal properties (See Appendix 4).
76
The overall trends in the core physical parameters following short and long-term
demineralization are displayed in Table 9. Of particular note from Table 9 is the fact that the
thickness of the surface layer did not consistently change over the demineralization period.
However, when large aprismatic layers were calculated in the untreated state, the thickness of the
surface layer decreased over time (Fig. 19a). The opposite was found in the case of smaller
aprismatic layers (Fig. 31). In the case of intermediate aprismatic layers, there appeared to be an
initial dissolution, evident in the decrease in surface layer thickness after 5 days, followed by
reprecipitation, evident in the increase in surface layer thickness after 10 days (Fig. 36). Small
deviations from the typical trends, indicated by the symbols in Table 9, may be attributed to
inter-sample biological variability reflected in the different rates and extent of lesion
development. For example, as noted above for the 40-day demineralized sample, the thermal
diffusivity of the surface layer (layer 1) exhibited a two- stage behaviour, where a decrease was
observed following the 10-day demineralization and an increase occurred at later periods, from
day 20 – 40 (Fig. 20d, Table 9). In the fluoride-free sample (Fig. 25B) an increase in the thermal
diffusivity of the surface layer was observed at day 10 of the demineralization phase and may
suggest a more rapid formation and enhanced crystallinity of the surface layer compared to the
40-day demineralized sample. Furthermore, in the high fluoride sample, non-monotonic changes
in thermal properties were observed during demineralization (Fig. 35). The non-monotonicity of
the trends in the physical properties of the high fluoride sample, that cannot be determined from
Table 9, may be related to the deviation of the lesion (Fig. 32) from the assumed 3-layer
geometrical structure due to poorer differentiation between surface and subsurface layers. This
may be explained by the fact that the present study employs a relatively crude 3-layer physical
model representing reality when in fact the lesion is often more complex. Both of the above-
mentioned facts point toward the power of the algorithm used, where the derived opto-
thermophysical parameters change as a function of the lesion structure and not the computational
algorithm.
The change in thermal properties (Fig. 20c, d) and layer thickness (Fig. 19b) paralleled trends in
the PTR amplitude and phase frequency response (Fig. 17). Phase maxima for the first 10 days of
demineralization exhibited a downward shift toward the mid-frequency range with small changes
at higher modulation frequencies. The shifting phase peak toward lower modulation frequencies
77
is indicative of a thermal wave interference pattern forming as a result of the growing subsurface
lesion. As the subsurface lesion increased in thickness, the distance over which the interference
pattern occurred broadened. In the theoretical analysis of the 40-day demineralized sample, no
changes were observed in the thickness of layer 1 for the first 15 days, whereas the lesion body
continued to grow deeper (Fig. 19b). From the 15-day period onward, the emergence of a second
phase peak at higher modulation frequencies (> 100 Hz) (Fig. 17) was concomitant with a large
increase in the derived thickness for layer 1, which reached a maximum thickness of ≈19 µm at
the 40-day demineralization period (Fig 16b - inset). The phase peak at higher modulation
frequencies may indicate a second interference pattern generated by the thickening intact surface
layer. The absence of the aforementioned phase peaks in the 10-day demineralized samples was
most likely due to the smaller surface layer thicknesses (Fig. 16).
Theoretical analysis of PTR amplitude and phase curves allows for the non-destructive
evaluation of mineralization kinetics. Changes in lesion thickness over time for 2 demineralized
samples present different trends as a function of treatment time. From the 10-day demineralized
sample (Fig. 19a) a near-linear relationship between lesion width (thickness of layer 2) and the
demineralization period was evident. A linear relationship between lesion depth and
demineralization time would infer that the rate-controlling process during demineralization is the
reaction at the crystallite level. This means that the dissolution process at the advancing front of
the lesion is slow relative to the diffusion of acid species and mineral ions into and out of the
lesion. Several studies have reported a linear relationship between lesion depth and time
(Anderson et al. 2004; Gao et al. 1993; Arends et al. 1997). When a demineralization gel was
used, a linear relationship between lesion depth and time was observed and attributed to an
inhibitor - controlled dissolution process at the crystallite level (Arends et al. 1997). In the
present acidified gel system, the inhibitor controlled dissolution would be ascribed to the
crystallite adsorption of HEC macromolecules. In the 40-day demineralized sample, a non-linear
rate of lesion growth with time was observed (Fig. 19B). This relationship suggests diffusion-
limited rate of enamel demineralization. Enamel demineralization as a diffusion-controlled
process has been proposed by several earlier studies (Gray 1962; Featherstone 1983; Higuchi et
al. 1965; Wu et al. 1976; Poole et al. 1981; Wong et al. 1987; ten Cate and Arends 1980;
Featherstone et al. 1979; Featherstone and Mellberg 1981). Furthermore, lesion depth varying
78
with the cube root of demineralization time has also been proposed (Groeneveld and Arends
1975; Featherstone et al. 1979), and indicated that diffusive transport of acids and/or dissolved
mineral ions throughout the depth of the lesion to and from the advancing lesion front is the rate
controlling processes. The variance in lesion progression between the 2 samples further
illustrates that the demineralization process is not merely one-dimensional, as changes in
structural gradients, chemical driving forces and porosity, among other factors, complicate the
process and significantly affect the rate of lesion development. A non-linear rate of lesion
progression with time may have been expected due to the mechanism of demineralization in the
present acidified gel system. Gellation of the demineralizing medium generated stagnation
conditions which crudely mimics in vivo dental plaque conditions, in places such as the bottom
of occlusal fissures, gingival margins or approximal sites. The maintenance of ‗sink‘ conditions
within the demineralizing medium means that rapid initial enamel dissolution to remove soluble
mineral phases results in a decrease in the solubility rate with time due to the accumulation of
reaction products and the lower driving force for demineralization (Gray 1962). In the present
study, the entire enamel surface measured was left exposed while other tooth surfaces were
coated in an acid-resistant varnish. As relatively large windows were used in the present study,
mineral concentration at the enamel – gel interface will be much larger than the corresponding
concentrations expected for smaller treatment windows and as a result the rate of lesion
progression will decrease over time (Ruben et al. 1999). Given the above-mentioned mechanism
of demineralization in the acidified gel, the linear relationship in the 10-day demineralized
samples may be influenced by inter- and intra-sample variability due to structural and chemical
gradients, particularly with respect to the utilization of natural enamel surfaces in addition to
error in the derivation of layer thickness for the intermediate demineralization periods.
Determining the lesion depth of the intermediate demineralization times was important for the
longitudinal monitoring of lesion progression and identifying layer thicknesses as destructive
TMR data was unavailable. Calculated mean lesion depth for intermediate demineralized curves
(5 days) was 56.7 ± 18.8 µm, which was within the range of lesion depths from other studies
implementing the same or similar demineralizing system. Boyle et al. (1998) found a mean
lesion depth after 7 days of ≈48 µm, whereas a 7-day immersion in a 0.05 mM lactic acid gel
produced a 38 µm lesion in a study by Issa et al. (2003). ten Cate and Arends (1980) using the
79
same demineralizing solution as the present study found lesion bodies ranging from 15 – 60 µm
with surface layers approximately 15 µm thick after a 4-day treatment period.
5.2.2 LUM signal generation during demineralization
As a complementary signal channel, modulated LUM, which monitors optical radiative
processes, produced consistent trends with treatment time. LUM amplitude decreased
monotonically and phase decreased at the frequency of phase minima (≈89 Hz) with
demineralization time (Figs. 16 and 17). These trends and the poorer contrast between sound and
demineralized enamel are consistent with earlier reports of modulated LUM behaviour to
artificial demineralized lesions (Jeon et al. 2008; Jeon et al. 2004a). The reduction in LUM with
demineralization can fundamentally be explained based on light scattering and absorption
properties of sound relative to demineralized enamel. Lower scattering properties of sound
enamel result in longer photon path lengths and a higher probability of photon absorption and
fluorescence emission from the entire enamel volume, DEJ and/or dentin (Angmar-Månsson and
ten Bosch 1987; Mujat et al. 2003). Alteration of the scattering coefficient in demineralized
enamel may have 2 effects on the level of fluorescence detected. Both scattering of the excitation
light before reaching a fluorophore and/or multiple scattering of the converted fluorescent light
within the tooth before exiting and being collected by the detector, will result in a lower overall
fluorescence collection from demineralized lesions (Girkin et al. 2000). From the onset of
demineralization, internal reflection sites are created as the enamel crystalline structure is broken
down which culminates in the higher optical scattering coefficients relative to sound enamel, as
demonstrated earlier. The higher scattering properties reduce photon path lengths causing a
proportional reduction in the total light path before it emerges at the enamel surface (Angmar-
Månsson and ten Bosch 1987). Borsboom and ten Bosch (1983) found the reduction in the mean
photon path length was about 5 times smaller in carious enamel. Assuming that enamel is not the
only source of fluorescence, the intense scattering properties of demineralized enamel can also
act as a barrier preventing incident photons from interacting with chromophores that lie deeper
toward the DEJ and in dentin (Mujat et al. 2003). These aforementioned mechanisms have been
proposed as explanations for the observed fluorescence loss using the QLF device. Additional
explanations for the loss of autofluorescent properties have been attributed to the loss of
chromophores during the demineralization process and the quenching of fluorescence by a
80
change in the molecular environment of the chromophores (Angmar-Månsson and ten Bosch
2001; Hafstrom-Bjorkman et al. 1992). Stable and high SNR normalized LUM phase curves
exhibited minima in the mid-frequency range characteristic of optical relaxation times
determined for enamel (Nicolaides et al. 2000). In a fluorophore LUM emission rate dynamic
model developed by Nicolaides et al. (2000), LUM emission was assumed to occur through
molecular or electronic radiative processes. From the model, optical relaxation lifetimes were
extracted, the longer of which (on the ms scale) was associated with the phase minimum at ≈30 –
200 Hz, and insensitive to the overall defect state of the tooth. Further insensitivity to the
treatment process was observed in the present study outside the frequency range of phase
minimum, i.e. <30 Hz and >200 Hz. If chromophore lifetimes were altered with demineralization
a shift in the LUM phase extrema to different frequencies would most likely be observed. This
was not the case in the present study, but rather phase minima decreased monotonically. The
decrease at full-width-at-half-maximum may be indicative of the smaller number of excited
chromophores, a result of the intense scattering properties and acid-induced apatite crystal
collapse yielding a reduced radiative conversion processes and lower signal magnitude. This is
evident in the monotonic decreases in LUM amplitude as a function of demineralization time.
Although the exact source of fluorescence has not been identified, it is most probable the
fluorescent properties are derived from both the organic components, such as protein
chromophores, as well as the inorganic mineral components of apatite (Spitzer and ten Bosch
1976). The intense fluorescent properties of dentin have also been proposed to be the result of
multi-source organic and inorganic constituents (Armstrong 1963). This may factor into the
fluorescent properties of enamel as described above, since optical path lengths are much larger in
sound teeth. In contrast to the lower LUM amplitude and phase minima in demineralized vs.
sound enamel and the similar fluorescence intensity reduction described by QLF analyses, the
DIAGNOdentTM
device has demonstrated that carious tissue emitted much stronger fluorescence
intensity relative to sound tissue, at a similar excitation wavelength (λ = 655 nm) as the present
study, increasing by more than one order of magnitude (Hibst and Gall 1998). The source of this
fluorescence has been related to porphyrins derived from bacterial metabolism, which occur at
later stages in caries development where bacterial infiltration and accumulation occur in the
dentin. Moreover, the poor sensitivity for lesions confined to enamel (Shi et al. 2000) coupled
81
with the insensitivity to in vitro created lesions (Pretty 2006) indicates the unlikely influence of
the described fluorescence mechanism to explain the LUM behaviour in the present study.
Modulated LUM has previously shown a strong sensitivity to the degree of (de)-hydration of the
teeth (Jeon et al. 2008) which may be imbedded within the monotonicity of the LUM trends with
demineralization and remineralization and difficult to uncouple from actual changes in
mineralization. Baseline shifts in LUM amplitude were observed merely as a result of extended
storage conditions in the humid chamber in between treatments as shown in Appendix 5 (Fig.
A.5.1) and has been shown previously in laser fluorescence signal generation in different storage
media over time (Francescut et al. 2006). Similarly, QLF fluorescence intensity varied depending
on the degree of hydration, where light scattering was strongly influenced by the presence of
water or air within the pores. Since scattering is dictated by the enamel crystallites in relation to
their immediate environment, i.e. air or water, the refractive index contrast increases, and hence
scattering increases, as a function of drying time since a greater contrast exists between enamel
and air vs. enamel and water (Amaechi and Higham 2002; Al-Khateeb et al. 2002). It is also
important to point out the trends in LUM amplitude and phase that paralleled those of PTR,
where the largest increase in PTR amplitude yielded the largest decrease in LUM amplitude.
This effect can be explained based on the abovementioned light scattering properties and also on
the photophysical vs. photothermal processes in the trade-off between radiative and non-radiative
energy conversion efficiency, respectively. In terms of the thermal signal, enhanced light
scattering coefficients would confine the thermal centroid closer to the enamel surface yielding a
higher amplitude and smaller phase lag. In contrast, in terms of LUM, the higher optical
scattering coefficients will result in less fluorescence generation. Crystalline destruction during
demineralization will enhance the non-radiative component as excited chromophores lose their
radiative energy conversion pathways and channel the absorbed energy into non-radiative
thermal emissions. Thus, the outcome of a reduced radiative component and increased non-
radiative is a smaller LUM signal and larger PTR signal, respectively. This illustrates the
complementary nature of the generated PTR and LUM signals.
82
5.3 PTR-LUM signals during short and long-term remineralization
All three remineralization treatment groups exhibited reductions in lesion depth compared to the
demineralization control group (ANOVA, p < 0.05), illustrating significant remineralization
induced by all 3 mineral solutions (Table 10). Surprisingly, no significant differences in mineral
loss were observed between the 4 treatment groups (p > 0.05). The large variance in mineral loss
of the demineralized control group may have precluded the identification of reduction in mineral
loss following remineralization. Large variation in the extent of demineralization and
remineralization was noted elsewhere (Gao et al. 1993) and attributed to small differences in
structure and mineral content between and within sections. With respect to remineralization, the
possible influence of inhibitors of crystal growth was proposed which would effectively reduce
and/or limit crystal growth on pre-existing demineralized crystallites. In the present study, the
latter effect may be enhanced by the presence of carboxymethylcellulose (CMC) in the
remineralizing solution, a common artificial saliva substitute to increase viscosity and moisten
the oral mucosa (Vissink et al. 1985). The effects of CMC on remineralization have been
associated with its ability to adsorb onto OHAp (Arèas and Galembeck 1991) and form
complexes with calcium and/or phosphate ions, the latter of which reduces the available free
mineral ions and remineralization capacity (Vissink et al. 1985). Across all 3 treatment groups
differences in PTR amplitude and phase were noted between earlier and later treatment times.
Specifically, after the 10 day exposure period in all treatment groups, amplitudes significantly
increased and phase lag decreased monotonically until the end of the treatment period. This
behaviour may be related to the known process of remineralization, the rate of which depends on
the availability of growth sites within a lesion and porosity of the intact surface layer (ten Cate
1990; Larsen and Fejerskov 1989). Furthermore, remineralization has been known to proceed via
3 main routes: the restoration of partially demineralized crystallites, growth of residual crystals
and de novo crystal formation (Yanagisawa and Miake 2003). Early on following exposure of the
demineralized lesion to supersaturated mineralizing solutions enamel porosity is high and the
surface area available for crystal growth is also large resulting in precipitation on residual enamel
crystallites (Koulourides et al. 1974; Ingram and Edgar 1994). However, once the pores in the
surface layer become occluded with precipitated mineral, diffusion fluxes across the surface
layer are retarded and remineralization of the inner lesion body is inhibited or reduced
83
(Silverstone et al. 1981). Rapid initial remineralization followed by slower rates at prolonged
treatment times has been documented in the literature for decades. Johanssen (1965) found rapid
remineralization over the first 24 hours, a reduced rate over 48 hours and no further change
thereafter for 3 weeks. Gao et al. (1993) found mineral gains occurred over an 8 week period and
after 14 weeks remineralization essentially stopped. When fluoride was added to an artificial
remineralizing solution at levels between 1 – 10 ppm, maximum changes were found to occur
over a 10 day period (Silverstone 1972; Silverstone 1977). Lastly, Al-Khateeb et al. (2000)
found that remineralization varied within the first week between the different treatment groups,
with fluoride enhancing the process; however a plateau was reached thereafter. In a recent study
implementing a similar artificial remineralizing solution, preferential initial mineralization
occurred in deeper layers after 1-day of exposure, whereas after 1 week, enhanced mineralization
was noted in the outermost mineralized layers (Tanaka et al. 2009). The different rates of
remineralization in the aforementioned studies are most likely related to the use of different
remineralizing agents, enamel substrates, biological variability, and the variance in artificial
lesion production.
Although there were no significant differences between the 3 remineralization treatment groups
in terms of mineral loss and lesion depth, thicknesses derived from the theoretical fittings
pointed toward preferential remineralization of subsurface layers in the presence of fluoride. It
follows that in the absence of fluoride, surface mineral deposition was dominant, which can be
seen in the increase in surface layer thickness in layer 1 relative to layer 2 in the fluoride-free
sample (Fig. 26). The presence of a surface mineralized layer in the microdensitometric tracing
may act as a diffusion barrier preventing mineral deposition in the lesion body (Fig. 22). In the
presence of both low and high fluoride, the theory-derived thicknesses indicated that subsurface
remineralization was the dominant process and may confirm the fluoride-enhancing effect on the
remineralization process described in earlier literature (ten Cate and Arends 1977; Silverstone et
al. 1981) (compare Fig. 26 to Figs. 31 and 36). An interesting trend in remineralized samples was
the presence of phase peaks at high modulation frequencies which was manifest predominately
in the fluoride-free treatment group (Fig. 23). The large decrease in phase lag at high frequencies
occurred with a concomitant increase in amplitude across the same frequency range. In all
samples exhibiting the aforementioned peaks, mineral content depth profiles showed a high
84
mineral volume in the most superficial part of the lesion (Fig. 22). Not only did the presence of
the surface mineralized layer correlate with the appearance of a smaller phase lag at high
frequencies but also correlated with the theoretical calculation of a higher scattering coefficient
in the surface layer (See Figs. 22, 23 and 24B). This is consistent with a recent PS-OCT study on
the remineralization of enamel where a highly scattering apatitic layer above the existing lesion
surface was observed after 20 days of remineralization (Can et al. 2008). Therefore, changes in
the PTR frequency response could be related to changes in the histological appearance of the
lesion and further may be useful in determining lesion activity. The LUM channel did not exhibit
a similar sensitivity at the 10 day remineralization period (Fig. 23). The high frequency of
occurrence of the surface mineralized layer in the fluoride-free group (70%) compared to the low
fluoride (40%) and high fluoride (20%) group supports the fluoride-enhanced deposition of
mineral initially in deeper layers without preferential mineralization of the outer surface,
however, further theoretical analysis of additional remineralized samples is required to validate
this conclusion.
In the present study, theoretical fitting of the final remineralized PTR amplitude and phase
curves based on thicknesses determined from densitometric tracings (Fig. 13) was used to
extrapolate and predict the demineralized lesion characteristics prior to remineralization as well
as follow the changes in opto-thermophysical properties as a function of time of exposure to the
mineralizing solutions. A summary of the main trends in opto-thermophysical parameters during
remineralization in all 3 treatment solutions is outlined in Table 11. In the presence of low-
fluoride levels (Fig. 28), a rapid decrease in amplitude across the entire modulation frequency
range and increase in phase lag at low frequency was observed for the first 10 days. A set of
derived optical and thermal parameters for the surface layer and lesion body presented different
trends as a function of treatment time (Figs. 29 – 31). The initial rapid decrease in amplitude and
increase in phase lag (Fig. 28) may be attributed to the large decrease in scattering coefficient of
the lesion body (Fig. 29B). The decrease in optical scattering coefficient may be related to the
restoration of enamel crystallinity within the lesion body, thereby reducing the acid-induced
porosity. At the crystalline level, this is consistent with the previously described mechanisms of
enamel remineralization, where the restoration of pre-existing, residual enamel crystals partially
dissolved during the demineralization process and the growth of surviving crystals are the
85
favoured processes (Tohda et al. 1990; Yanagisawa and Miake 2003; Silverstone and Wefel
1981). Mineral deposition was found to account for changes in the optical properties using
polarized light microscopy by the deposition of suitably oriented OHAp crystallites (Silverstone
and Poole 1969). Crystal growth is further enhanced by the presence of low levels of fluoride in
solution (1 ppm) due to the elevated driving force for mineral deposition, in the form of FAP or
F-OHAp, in the surface and subsurface regions (Silverstone et al. 1981; ten Cate et al. 1981).
Furthermore, fluoride has been shown to have a strong affinity for apatite crystals and is
incorporated in much greater amounts in porous demineralized layers compared to sound enamel
(Koulourides et al. 1974). Low incident-photon scattering (Fig. 29B) combined with the
improvement of the thermal properties of the lesion body (Fig. 30) up to 10 days following
remineralization treatment, may support the enhanced crystallinity of the lesion body, driving the
thermal centroid deeper into the enamel. This trend is supported by the abovementioned trends in
PTR frequency response as a decrease in PTR amplitude across the entire modulation frequency
range and increase in phase lag at lower modulation frequencies (Fig. 28). The validity of the
proposed mechanism may be enhanced by the observation that LUM amplitude and phase for the
sample exposed to the low fluoride solution were strongly correlated with the derived scattering
coefficient. The rapid decrease in scattering coefficients of the lesion (Fig. 29B), a result of
enhanced crystallinity would induce longer optical path lengths and greater fluorescence
generation as is seen in the larger LUM amplitude (Fig. 28). The observed increase in the
absorption coefficient would tend to have the opposite effect, decreasing optical path lengths,
however, given the magnitude of change in the scattering coefficient compared to absorption
coefficient it is clear that the scattering depression is dominant. The larger fluorescence signal
from remineralized enamel, however, was found to be rather complex and not proportional to
total mineral uptake, a result likened to the fact that remineralization is never fully complete (Al-
Khateeb et al. 2000). LUM signals from the fluoride-free sample also exhibited trends that
correlated with the scattering coefficients. However, this was not the case in the high fluoride
group. It is important to note that remineralization of demineralized lesions may not result in
fluorescence gain equal to that lost from the enamel during lesion formation (de Josselin de Jong
et al. 2009). This is most likely attributed to the heterogeneity in the demineralized enamel which
will induce scattering properties unique to individual samples. Furthermore, this is supported by
86
the fact that in some cases mineral is not deposited in a crystalline prismatic form as in sound
enamel, but rather in amorphous deposits which can influence scattering properties. An
additional influence to overall fluorescence generation is the hydration level of the sample as
described earlier for demineralized lesions. The dehydration rate of highly remineralized enamel
was found to occur at a much lower rate than sound and demineralized enamel (van der Veen
and de Josselin de Jong 2000). As drying times for all samples remained constant irrespective of
treatment, it may be possible that residual water left within the enamel pores influence the
overall fluorescence generation. Replacing water-laden interprismatic pores with air increases
the refractive index difference between the enamel prisms and the surrounding environment
which in turn increases the scattering coefficient. Therefore, the hydration state of enamel is an
important confounding factor that significantly affects LUM signal generation, manifested as
baseline shifts. The hydration state may have a similar effect on PTR signal generation. The high
infrared absorption coefficient of water (Fig. 7) compared to air would significantly attenuate the
emitted photothermal signal.
During prolonged treatment times in all 3 mineralizing solutions (i.e. after 10 days), PTR
amplitude and phase trends reversed direction resulting in a monotonic increase in amplitude
with a smaller phase lag (Figs. 23, 28 and 33). This may be attributed to the random orientation
of deposited crystals as well as de novo mineral precipitation within the surface layer and lesion
body, particularly within the interprismatic regions, a function of the lower specific surface area
of the crystalline mineral phase (Yonese et al. 1981; Amjad et al. 1981; Palamara et al. 1986).
An amorphous mineral phase would generate additional scattering centers through the larger
number of crystal grain boundaries effectively increasing the scattering coefficient and creating
poorer thermal properties as the density increases, both trends which were observed in the
theoretical fittings (Figs. 24b, 25, 29b and 30). Furthermore, in Figs. 25, 30 and 35, a decrease in
thermal properties at the final demineralization point or during the first 2 days of
remineralization, was followed by a transient increase, and a further decrease at prolonged
treatment times, an effect likened to competing thermal processes during the remineralization
period. Higher absorption coefficients calculated after the 10 day remineralization period in the
fluoride-free (Fig. 24A) and low fluoride group (Fig. 29A) may substantiate the proposed
87
amorphous mineral precipitation, and has been documented in an earlier study (Krutchkoff and
Rowe 1971). Longer exposure periods in a CMC-containing remineralizing solution were found
to significantly impede remineralization compared to non-CMC control solutions, an effect
related to the complexation of solution calcium and phosphate ions (Tschoppe et al. 2008). A
similar finding was observed by Amaechi and Higham (2001) where a remineralizing solution
without CMC, and identical in all other components, showed a greater remineralizing capacity.
Therefore, the documented effects of CMC, as described earlier, may roughly mimic intraoral
remineralization processes, as many salivary proteins have a high affinity for OHAp and
significantly regulate the available calcium and phosphate levels (Hicks et al. 2003). At later
remineralization times in the fluoride-free and low fluoride group, a greater thermal mismatch
was evident between the surface layer and subsurface layer (Figs. 25 and 30). The same trend
was also evident in the 40-day demineralized sample (Fig. 20d). As the surface layer becomes a
better conductor and diffuser, thermal energy will propagate rapidly toward the L1 - L2 interface.
However, since the properties of the subsurface layer are so poor, thermal fluxes are significantly
impeded and as a result, thermal energy will accumulate at the boundary. Effective confinement
of the thermal centroid will then occur within the surface layer thereby resulting in the observed
increase in amplitude and smaller phase lag as the reaction moves closer to the enamel surface.
This may suggest an amplification of the dominant surface reaction whereby the effects of the
underlying enamel at later remineralization times are concealed. Furthermore, this may imply
that once the surface layer reaches a critical thickness its properties dominate the thermal
response at the expense of the underlying enamel, which may require lower frequencies (longer
thermal wavelengths) to probe deeper regions. The observed surface deposited mineral which
was most prevalent in the fluoride-free group (Fig. 22) may chemically amount to a diffusion-
barrier generating an impedance of inorganic mineral ion diffusion into deeper layers, while at
the same time driving the thermal-wave centroid closer to the enamel surface, apparent in the
increasing amplitude and smaller phase lag. After the first 10 days of remineralization, changes
in thickness were minimal (Figs. 26, 31 and 36) but rather significant changes in the optothermal
transport properties were dominant. These changes may indicate the shifting thermal centroid
from deeper within the enamel during earlier exposure periods, when ingress and precipitation of
mineral ions to restore crystallinity and reduce thicknesses of deeper layers is enhanced, toward
88
the enamel surface at prolonged treatment time. Larger absorption coefficients at later exposure
periods in the surface layer (layer 1) may further support surface dominated reactions as the
major contributor to the PTR signal (Figs. 24A and 29A).
PTR-LUM frequency responses and theoretical fittings of the high fluoride sample displayed
trends that deviated from the low and fluoride-free samples. In the latter samples, rapid changes
in thermal properties occurred over the first 10 days which was followed by an inversion and a
large thermal mismatch at later remineralization times (>20 days) (Table 11). A decrease in
amplitude following the first 10 days of remineralization, predominately at low modulation
frequencies, was accompanied by a near-monotonic decrease in phase lag across the same
frequency range (Fig. 33). This may suggest significant mineral deposition within the lesion
body such that the regression in the thickness of the subsurface layer progresses from the
advancing front of the lesion toward the surface layer. If the lesion following demineralization
had a certain thermal centroid at a given frequency and during subsequent remineralization the
thickness of the subsurface layer decreased with minimal changes in the thickness of the surface
layer, as was shown in the thicknesses derived from the theoretical model (Fig. 36), then as the
subsurface layer becomes thinner the thermal centroid will be maintained within the layer as it
shifts closer to the surface. Klinger and Wiedemann (1985) found that after a 93 hour immersion
of demineralized lesions in a calcium phosphate solution, the bottom of the lesion moved toward
the enamel surface. In contrast, the low fluoride sample showed mineral gains in both surface
and subsurface layers over the first 10 days (Fig. 32). The higher fluoride concentrations would
be expected to diffuse rapidly into deeper areas within the porous demineralized enamel thereby
increasing total fluoride within the lesion to enhance mineral accumulation. After 10 days of
exposure to the high fluoride solution a marked reduction in the thermal properties of both layers
was evident (Fig. 35). This was accompanied by an increase in the absorption coefficient (Fig.
34A) and an increasing scattering coefficient (Fig. 34B) which was expected since an opaque
and chalky macroscopic appearance of enamel was observed after the 4-week immersion in the
high fluoride solution (Fig. 21c). The surface hypomineralization observed in the mineral content
depth profile of the high fluoride group may be a result of surface mineral loss during the
demineralization process, the fluoride-enhanced driving force for subsurface remineralization
and/or the deposition of a surface material of a different phase. The latter 2 processes may be
89
likely due to the high fluoride concentrations implemented (1000 ppm), where a calcium
fluoride-like phase may precipitate (Larsen and Fejerskov 1978). The formation of calcium
fluoride-like material on enamel surfaces, a reaction mechanism between fluoride and enamel
known to break up the crystal lattice, may occur; however this process is enhanced under acidic
pH conditions (Kidd and Joyston-Bechal 1980). CaF2 is a relatively less stable phase compared
to FAP and F-OHAP under normal oral conditions (Mellberg and Mallon 1984). In the presence
of phosphate ions the CaF2-like deposits may be hydrolyzed to F-OHAp (Larsen and Richards
2001; Ogaard 2001). Additionally, a HF-induced etch of the enamel surfaces may occur,
however, given the neutral pH of the remineralizing solution, the concentration of HF would be
rather small. The chemical behaviour in the presence of high fluoride, manifested as a surface
hypomineralization, is supported by the minimal change in PTR-derived thickness of the surface
layer thickness over the remineralization period. Furthermore, the poorer thermal properties
generated at prolonged remineralization times may reflect a surface precipitate. If a precipitate
was formed at the enamel surface following fluoride exposure, the level of integration of this
layer with the underlying enamel could not be determined from microradiographic analysis.
5.4 Errors and Limitations in the extraction of opto-
thermophysical properties
Historical methods for extracting optical and thermal properties from enamel require the use of
thin sections of known thickness. In terms of thermal diffusivity measurements, it has been
shown that the different preparation methods of samples resulted in significantly different results
(Panas et al. 2003). Furthermore, sectioning enamel can disrupt its crystalline ultra-structure and
induce micro-cracks which can significantly affect the optical properties determined from
transmission and reflection measurements. Thus, sample manipulation on its own can induce
significant changes in optical and thermal properties of the tissue, adding a source of variability
on top of the inherent intra and inter-sample local differences in structure (Panas et al. 2007).
Spitzer and ten Bosch (1977) determined the absorption and scattering coefficients of enamel
slabs as a function of demineralization time, however, large errors in the absorption coefficient
(> 40%) were observed. Furthermore, they estimated that error from the determination of the
optical properties from one sample is about 60%. Scattering and absorption coefficients
90
determined by comparing the scattering data with Monte Carlo light scattering simulations of
enamel sections in a study by Fried et al. (1995) found an error of 30% for all scattering
coefficients. Ko et al. (2000) used reflection and transmission measurements from enamel
sections calculated according to Kubelka-Munk equations to derive optical scattering
coefficients. The authors found a 28% variation in the scattering coefficients. In terms of the
latter 2 studies, variations in the optical coefficients were attributed to lesion heterogeneity,
including the influence and composite nature of the intact surface layer, and intra- and inter-
sample variability. An estimation of the error in the theoretical program due to experimental
error of PTR signal measurement is presented in Appendix 4. The percentage difference in the
scattering coefficient is on the order of those described in the aforementioned literature (Table
A.4.1). Additional sources of error in the theoretical extraction of opto-thermophysical
parameters may be influenced by the variable rates of sample de/hydration, small microstructural
differences within and between samples and also by the crude 3-layer representation of the
complex caries lesion. In an earlier study implementing standardized drying times, lesions
varying in severity also exhibited different de-hydration rates (Lagerweij et al. 1999). Thus,
larger lesions and/or lesions with remineralized surface layers, which may dry at slower rates
compared to smaller lesions, may result in more residual water occupying enamel porosities
thereby influencing PTR frequency response and optical and thermal property extraction (van der
Veen and de Josselin de Jong 2000). The delineated layers of the caries lesion are not perfectly
reflecting and transmitting interfaces, but rather based on the accumulation or depletion of
thermal energy. In reality, 3 effective layers may not approximate the multi-layered caries lesion,
however, the introduction of additional layers is computationally intensive and the lengthy
calculation time currently imposed for the 3-layer system would be further increased. Earlier
studies attempting to indirectly model physical phenomena during demineralization processes are
generally restricted to a 2-layer approximation (Mujat et al. 2003; Zijp 2001). The surface layer
was neglected as its thickness was typically small compared to the thickness of the demineralized
lesion. As discussed early on, attempts to fit the present demineralized and remineralized PTR
data to a 2-layer approximation yielded a very poor fit of the experimental and theoretical curves.
Thus, the contribution from the intact surface layer was significant enough so that optical and
thermal properties of the layer were considerably different from the lesion body and contributed
91
to the overall PTR amplitude and phase frequency response. The present study illustrates the first
account where demineralized and remineralized enamel could be resolved into 3 effective layers,
where the non-destructive extraction of opto-thermophysical parameters of each layer allowed
for depth-profile reconstruction as a function of treatment time.
5.5 Comparison of irradiation wavelengths and future directions
A comparison of the PTR frequency response under both irradiation wavelengths (660-nm and
830-nm) revealed similar trends across the entire modulation frequency range. The fact that
similar trends were evident under 830-nm laser radiation (Appendix 1) may implicate the longer
wavelength laser light as an effective light source for evaluating sound, de- and remineralized
enamel. Thus, the 830-nm laser may be used as a viable alternative to the 660-nm laser and/or
used as a dual wavelength PTR probe, which would further enhance the depth profilometric
character of PTR by yielding additional amplitude and phase signal channels. The advantage of
the longer wavelength laser light is the deeper penetration of the optical field as absorption and
scattering coefficients are lower than smaller wavelengths (see Table 1). As a result, deeper
subsurface features may be resolved with the longer wavelength light. However, a disadvantage
of the longer wavelength light is the lower SNR, as can be seen in the PTR frequency responses
(Appendix 1).
Qualitative evaluation of PTR frequency response may provide a rough estimate of lesion size.
The PTR phase peak at low modulation frequencies, of the 10-day (Fig. 16) and 40-day
demineralized samples (Fig. 17), indicate thermal wave interference patterns occurring within
the demineralized lesion. Calculating the thermal diffusion length at the frequency of the phase
maxima may therefore be a rough estimate of lesion depth. For example, in the 10-day
demineralized sample phase maxima appear at ≈8 Hz, and in the 40-day demineralized sample at
≈5 Hz. Given the thermal diffusivity of the lesion body for the 10-day and 40-day samples, 3.3 x
10-7
and 2.3 x 10-7
m2/s, respectively, thermal diffusion lengths are ≈114 µm for the 10-day
sample and 121 µm for the 40 day sample. These estimates are close to the values of the 10-day
lesion (88.6 μm) and 40-day lesion (114.8 μm) determined by TMR. However, following a
single frequency scan, the thermal diffusivity is an unknown parameter and as a result thermal
diffusion length cannot be predicted as easily. In the present study, layer thicknesses were
92
validated using TMR where microdensitometric tracings were used as guidelines to define the
thicknesses limits in the theoretical program. Clinically, final thicknesses are clearly unavailable
and therefore the robustness of our theoretical/computational program would beneficially yield
good estimates of lesion parameters, irrespective of the initial set of parameter value ranges.
Given the reliability of the calculated thicknesses outlined in the previous paragraph, in the
future refinement of the theoretical program and fitting procedure, different programs may be
designed based on the overall size of the lesion. Thus, the PTR phase, which has emerged as the
most sensitive channel to the depth-dependent changes, may be used in vitro or in vivo as an
initial screening channel in order to roughly estimate lesion depth, after which the specific
theoretical program can be implemented based on the aforementioned phase behaviour to extract
the relevant opto-thermophysical parameters.
5.6 Summary
In summary of the trends described above, PTR and LUM in backscatter mode were sensitive to
the formation of mineralized layers during de- and remineralization. At the initial state, PTR
signals and the combined theoretical model indicated that sound enamel is a complex multi-
layered substrate where an aprismatic layer at the surface of the enamel with different optical and
thermal properties must be considered. At the onset of demineralization, an increase in PTR
amplitude and decrease in phase lag in was observed with a monotonic depression in LUM
signals. Multi-parameter fits of PTR experimental data revealed a marked increase in optical
scattering coefficients and the generation of poorer thermophysical properties during
demineralization consistent with crystalline disintegration and formation of subsurface
microporosities. Changes in LUM signals could be explained based on the enhanced light
scattering properties of demineralized enamel and the channelling of radiative to non-radiative
energy sources caused by the loss of inorganic structure. Trends in opto-thermophysical
parameters during demineralization indicated that artificial caries lesions involve a dynamic
surface dissolution– reprecipitation mechanism during subsurface lesion formation. Trends in
PTR signals and opto-thermophysical parameters during the remineralization phase indicated a
multi-factorial and complex repair process. This was considered as interplay between shifting
thermal centroids as mineral gains in surface and subsurface regions alter the opto-
thermophysical properties of the effective layers as a function of remineralization time. The
93
theoretical model pointed to a fluoride-enhanced remineralization of the lesion body, however,
no statistically significant differences in TMR defined mineral loss and lesion depth were noted
between the remineralization treatment groups. Lastly, the high fidelity and uniqueness of the
fitted parameters illustrates the effectiveness of the computational algorithm and its potential
applicability toward the non-destructive quantification of lesion thicknesses and the
reconstruction of opto-thermophysical depth profiles.
94
CHAPTER 2: Transmission mode PTR – LUM
6 Rationale
In the first chapter of this project PTR and LUM were investigated in back-propagation mode
where sample treatment was interrupted periodically during the test period in order to be
analyzed. Sample interruption, coupled with the washing and drying periods and the storage in
the humid box may add additional sources of variability to the experimental protocol,
predominately as changes in water content can significantly influence PTR and LUM signal
generation. The influence of external water-based solutions on infrared emissions was initially
observed during preliminary measurements where the ability of backscatter-mode PTR was
assessed to monitor changes in enamel in real-time during continuous exposure to the treatment
solutions. The results of this satellite experiment revealed the inability of mid-IR photons to
penetrate the water-based demineralizing and remineralizing solutions; a result attributed to
strong water absorption bands in the mid-IR spectral range (Fig. 7). This prompted realignment
of the experimental setup to consider PTR-LUM transmission experiments. The advantage of the
transmission PTR-LUM system is that indirect measurements of mineral loss can be monitored
during the treatment process without sample interruption.
An extensive review of in vitro de- and remineralization literature revealed only a limited
number of reports, by a single research group, concerned with directly monitoring enamel and/or
dentin demineralization and/or remineralization continuously in real-time (Gao et al. 1991; 1993;
1993a; Anderson and Elliott 1992; Anderson et al. 1998, 2004). An ingenious set of experiments
conducted by the aforementioned research group, modified the conventional microradiographic
system to include a photon-counting system, which allowed for the increased frequency of
mineral-content measurements on relatively thick (≈350 µm) sections for direct real-time mineral
quantification during demineralization and remineralization treatments (Anderson et al. 1998).
Typically, the protocol employed in vitro to study demineralization-remineralization phenomena
often involves the use of multiple thin enamel sections each delineated with nail varnish to
maintain a region of sound tissue, and interrupted repeatedly for analysis. Thus, direct or indirect
measures of mineralization are evaluated when the sections are in a static state. As both
demineralization and remineralization are kinetic, time-dependent processes, significant sample
95
interruption can affect both the rate and extent of lesion formation. Furthermore, the use of
multiple sections introduces additional biological variability where each sample may exhibit
independent rates; a function of the composite nature of enamel and the diverse history in the
complex oral environment. Consequently, the ability to monitor changes in a tooth as a function
of time with minimal disturbance would be ideal, as both reliability and controllability of the in
vitro treatment method would be improved. However, due to technical difficulties in the
experimental setup and validation techniques, real-time studies have been challenging and not
often explored.
The combined PTR - LUM system has demonstrated sensitivity to characterize the disease state
of dental tissue in the backscatter-mode. The objective of the present study is to assess the ability
of the PTR-LUM system to monitor changes in real-time during de- and re-mineralization
processes.
7 PTR-LUM Transmission-Mode: Materials and Methods
7.1 Sample Preparation
Five intact and sterilized samples (gamma irradiated, 4 kGy) that were visually identified as
sound without the presence of defects, caries lesions or stains were sectioned in half
mesiodistally. The experimental protocol was approved by the University of Toronto Ethics
Review Board (Protocol #25075). The anatomical surface of the sectioned enamel was left intact
while the cut side of the section was ground sequentially using successively finer grits of
waterproof SiC paper under constant water exposure (320-b to 1200-b waterproof SiC paper).
Sections were ground to an average thickness of about 1.25 mm.
An opening was created on one wall of a container (25 cm3) matching both the size and shape of
the cut section. The enamel section was inserted into the cut wall and fixed in position with
utility wax, such that the enamel surface is in constant contact with de- and re-mineralizing
solutions. The intact enamel surface was positioned facing laser irradiation and the polished
surface oriented facing the off-axis mirrors (Fig. 2.1). Containers were fixed on LEGO bricks
and placed on the micrometer stage such that the intact enamel surface was at the focal plane of
96
the incident laser light. The size of the delimited window exposed to the treatment solutions was
approximately 2 mm x 2 mm.
Figure 2.1. Experimental apparatus for transmission experiments. Laser beam is incident on the
enamel surface.
7.2 PTR-LUM frequency scans
Frequency scans similar to those in experiment 1, described in detail above, were run initially on
intact enamel samples under 670-nm laser radiation. The modulation frequency range was from 1
Hz to 1000 Hz. The number of data and cut off points for PTR were increased to 25 and 12,
respectively. Corresponding data and cut-off points for LUM measurements were 40 and 15,
respectively. LUM data acquisition was alternated between the transmission and backscatter
mode. PTR Measurements commenced at the first exposure to the demineralizing gel. Two
samples were treated simultaneously; alternating frequency scans every 3 hrs, allowing 20 min of
thermal stabilization time prior to scanning. Immediately following the initial immersion of
demineralizing gel and remineralizing solution, measurements were performed every 30 min for
7 hours. As the MCT detector sensing element requires liquid nitrogen cooling, samples were
neither scanned overnight, nor over weekends. Sections remained under constant solution
exposure during these periods and the total time was factored into overall treatment time.
Individual samples were left undisturbed and containers were sealed during the treatment and
measurement processes.
97
7.3 Demineralization and Remineralization Treatments
Demineralization and remineralization treatment groups are shown in Table 2.1. Enamel sections
were demineralized in 25 mL of the same acidified gel solution as described in Experiment 1
(Section 1.4.1). Total demineralization time was 15 days. After the final PTR-LUM
measurement during demineralization, the gel was carefully suctioned out of the container using
a syringe, while the sample container remained on the sample stage. Containers were washed
through with 20 rinses of distilled water to remove gel particulates adsorbed on the enamel
surface and container walls. After a drying period of 1 hour, 25 mL of a remineralizing solution,
containing 1 ppm fluoride, was decanted and measurements resumed for an additional 20 days.
The remineralizing solution constituents are the same as presented in Table 4.
Table 2.1. Treatment groups for transmission-mode PTR-LUM study (n = 10).
7.4 PTR-LUM Experimental Setup
A diagrammatic representation of the experimental setup for transmission measurements of
enamel sections is illustrated in Fig. 2.2. This setup is modified from backscatter PTR-LUM
setup by the addition of a higher power laser diode (500 mW; 800 mA; Sony SLD 1332V) fixed
in a laser diode mount (Thorlabs TCLDM9) and positioned behind the 3-axis translational
sample stage. The laser beam was focused to a spot size of 590 μm. An opening created where
the polished surface contacts the container walls allowed transmitted infrared emissions to be
collected by the off-axis paraboloidal mirrors and focused on a MCT detector. Transmitted
modulated luminescence signals were collected using the same photodetector setup as the
backscatter PTR-LUM experimental mode. An additional photodetector was positioned on the
same side of laser irradiation in order to collect back-scattered LUM emissions through the
treatment solution.
Treatment Group Demineralization treatment (Days)
Remineralization treatment (Days)
Sample size
Demineralization 15 ----- 5
Mineral solution (1 ppm fluoride)
15 25 5
98
Figure 2.2. Experimental setup for transmission-mode PTR-LUM.
7.5 Transverse microradiography (TMR) and image analysis
At the completion of all transmission PTR-LUM measurements all samples were
microradiographed, in the same manner as detailed earlier, to determine the mineral loss and
depth of the artificially demineralized and remineralized lesions.
8 Results
8.1 Time-series demineralization experiments
An exemplary artificial caries lesion, characterized by a thin, intact surface layer, superficial to a
deep subsurface lesion body was created following demineralization of enamel sections and is
shown in Fig. 2.3. Mineral loss and lesion depth of the presented lesion was 3750 vol%.μm and
98.5 µm, respectively. PTR and LUM amplitude and phase signals at 1 Hz, plotted as a function
of time, for the demineralized lesion in Fig. 2.3 revealed monotonicity with treatment time (Fig.
2.4). A decrease in PTR amplitude with a concomitant increase in PTR phase lag occurred from
the initial acid gel exposure period over the course of 15 days. Following an initial transient time
lag lasting approximately 15 - 20 hrs PTR amplitude decreased and phase lag increased linearly
up to about 75 hrs. A reduced rate and a slope change followed 75 hrs of demineralization until
the treatment conclusion. Time-series changes in transmission-mode LUM amplitude and phase
signals at 89 Hz, the frequency of LUM phase minima, are displayed in Fig. 2.4. LUM signals
mirrored those of PTR throughout the duration of the treatment period.
Photodiodes and optical filters
SampleLaser Diode Module
Off- axis Mirrors
IR Detector
Off-axis mirrors
Pre-Amplifier
HgCdTe Detector
Lock-in Amplifier
Optical filter & Photodetector
Laser Driver Waveform
Sync. Signal
Sample Apparatus
Computer
Internal
Generator Function
Amplifier Lock-in
Optical filter & Photodetector
Laser Diode and Module (670 nm)
99
Figure 2.3. Exemplary microradiograph (a), densitometric tracing (b), and visible light
transmission image (c) of a demineralized enamel section. Mineral loss and lesion depth were
determined as 3750 vol%.μm and 98.5 µm, respectively.
Figure 2.4. Time-series transmission-mode PTR-LUM amplitude and phase signals at 1Hz
(PTR) and 89Hz (LUM) for a sample demineralized for 15 days.
100µm
(A) (B) (C)
0 50 100 150 200 250 300 3500.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
0.022 PTR Amplitude
Am
plit
ud
e (
a.u
.)
Time (hrs)
0 50 100 150 200 250 300 350
-140
-135
-130
-125
-120
-115
-110
-105
-100
-95 PTR Phase
Ph
ase
(D
eg
)
Time (hrs)
0 50 100 150 200 250 300 350
0.20
0.22
0.24
0.26
0.28
0.30LUM Amplitude
Am
plit
ud
e (
a.u
.)
Time (hrs)
0 50 100 150 200 250 300 350
-16.4
-16.0
-15.6
-15.2
-14.8
-14.4 LUM Phase
Ph
ase
(D
eg
)
Time (hrs)
100
8.2 Time-series remineralization experiments
At the onset of the remineralization treatment period, a step-decrease in phase lag at 1 Hz was
accompanied by a concomitant increase in amplitude (Fig. 2.5). This behaviour occurred for a
period of ≈200 hrs since the start of the mineral solution exposure, after which amplitude and
phase signals reversed direction and reached stable values after ≈650 hrs until the end of the
exposure period. LUM amplitude and phase at 89 Hz, the frequency where LUM phase minima
appear, reveal similar trends as PTR however, exhibited less marked changes at the onset of
mineral solution exposure. Visible light transmission images reveal the non-uniform appearance
of remineralized enamel, with regions of mineral restoration intertwined with more opaque
regions of demineralization. Microradiographs and mineral content depth profiles of an
additional demineralized and remineralized sample is presented in Fig. 2.6; corresponding PTR-
LUM signals are shown in Fig. 2.7. The lesion displayed a relatively thick remineralized intact
surface layer of ≈15 µm superficial to a deep subsurface lesion ≈124 µm with an exceedingly
large amount of mineral loss (5370 vol%μm) (Fig. 2.6). Following the exposure to the
mineralizing solution, PTR amplitude at 1 Hz (Fig. 2.7A) exhibited a step-increase for ≈300 hrs
which occurred with a concomitant decrease in phase lag. After ≈300 hrs of remineralization
amplitude signals decreased below those of the final demineralization and remained near-
constant for the rest of the treatment period excluding a slight increase in the final ≈100 hrs.
Transmission LUM signals at 89 Hz revealed similar trends as the PTR at 1Hz during the
demineralization phase (Fig. 2.7B). At the onset of the remineralization phase, a slight increase
in amplitude occurred for the first ≈100 hrs with no discernible changes in phase. At later
remineralization times a gradual decrease in amplitude and phase were noted until the end of the
treatment period. Time-series LUM in backscatter mode at 89Hz monitored the effects at the
enamel surface through the demineralizing and remineralizing solutions. During the
demineralization phase, LUM amplitude and phase decreased for the first ≈175 hrs and increased
thereafter (Fig. 2.7C) consistent with the slope changes in both transmission PTR and LUM
signals (Fig. 2.7A, B). At the onset of remineralization, an increase in amplitude and phase
paralleled the increase in PTR amplitude and decrease in phase lag (Fig. 2.7C). This was
followed by a marked decrease in both LUM amplitude and phase after ≈600 hrs. A slight
101
increase was observed again for the last ≈100 hrs as noted for PTR amplitude and phase and
opposite the trends in transmission LUM.
Figure 2.5. Time-series transmission PTR-LUM amplitude and phase signals at 1Hz (PTR) and
89Hz (LUM). Vertical dashed lines divide de-and remineralization treatments. The visible light
transmission image (top right) and microradiographic image (bottom right) are presented in the
adjacent figures. Mineral loss = 1010 vol%.μm; Lesion depth = 61 μm.
Figure 2.6. Microradiograph and mineral content profile of a de- and remineralized sample. The
corresponding PTR-LUM signals are presented in Fig. 2.7. Mineral loss = 5730 vol%.μm;
Lesion depth = 139.4 μm.
100 µm
102
Figure 2.7. Time-series transmission PTR signals at 1 Hz (A) and transmission LUM at 89Hz
(B). (C) Time-series LUM signals at 89 Hz viewed in backscatter mode. Vertical dashed lines
divide de-and remineralization treatments. The corresponding microradiograph and mineral
content depth profile is presented in Fig. 2.6.
9 Discussion
9.1 PTR-LUM signals and time-series demineralization
Exposure to the demineralizing solution generated a mineral distribution with a large mineral
loss to lesion depth ratio, as depicted in the microradiograph (Fig. 2.3). Both PTR and LUM
signal channels exhibited monotonic decreases over time (Fig. 2.4). The decrease in PTR
amplitude over time can be explained based on the generation of a near surface layer, most likely
the demineralized lesion, with poorer thermal properties and higher scattering coefficients, as
was shown in Chapter 1. In transmission-mode, thermal confinement closer to the enamel
surface means non-radiative conversion reactions are occurring farther away from the IR detector
and the result is a smaller amplitude and larger phase lag of the generated photothermal response.
Furthermore, higher scatter of the photon density field from the onset of demineralization
effectively impedes photon path lengths, confining the thermal-wave centroid to a narrower
surface region. This will have a marked effect on both PTR and LUM signal generation, as less
thermal and optical energy is available for transmission through the thin enamel section. As
0 200 400 600 800 1000
0.23
0.24
0.25
0.26
0.27
0.28
0.29
0.30
0.31
Demin Remin
LUM Amplitude
Am
plitu
de
(a
.u.)
Time (hrs)
0 200 400 600 800 1000
-16.2
-16.0
-15.8
-15.6
-15.4
-15.2
-15.0
-14.8
-14.6
-14.4
Demin Remin
LUM Phase
Ph
ase
(D
eg
)
Time (hrs)
0 200 400 600 800 1000
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
Demin Remin
LUM Amplitude
Am
plit
ud
e (
a.u
.)
Time (hrs)
0 200 400 600 800 1000
-6
-5
-4
-3
-2
-1
0
1
2
3
4
Demin Remin
LUM Phase
Ph
ase
(D
eg
)
Time (hrs)
0 200 400 600 800 1000
0.010
0.011
0.012
0.013
0.014
0.015
0.016
PTR Phase
PTR Amplitude
Remin
ReminDemin
Demin
Am
plit
ud
e (
a.u
.)
Time (hrs)
0 200 400 600 800 1000
-122
-120
-118
-116
-114
-112
-110
-108
Ph
ase
(D
eg
)
Time (hrs)
(A) (B) (C)
103
described earlier, sound enamel contains a unique structural alignment of inorganic prisms
running almost perpendicular to the enamel surface. Where the prisms run perpendicular to the
enamel surface, incident light shines directly on enamel crystallites and is transmitted deeper into
the tooth in a manner analogous to optical fibres (Odor et al. 1996). From the onset of
demineralization, ultrastructural changes in enamel, such as the widening of interprismatic
spaces, result in larger surface and subsurface layer porosity which act as scattering centers,
while at the same time the small microchannels are significant impedances to heat flow. A
combination of both effects, which manifest themselves as a marked increase in the scattering
coefficient in the lesion and poorer thermal properties, result in a shift of the thermal centroid
closer to the enamel front surface. At early demineralization times the optical and thermal
contrast between the surface layer, lesion body and sound underlying enamel would be minimal.
As a result, surface mineral loss combined with subsurface mineral loss would confine the
thermal centroid closer to the near surface, while allowing significant thermal-wave propagation
beyond the lesion body - sound enamel interface. However, once the lesion grows to the size
evident in Fig. 2.3, a marked thermal discontinuity would be expected between the lesion zones
and as a result inter-reflections within the surface and lesion body would increase the density of
photons within the lesion body, confining both optical diffuse photon field and the thermal-wave
centroid to this region, and reducing the amount of thermal-wave transmission beyond the lesion
body – sound enamel interface.
A rapid decrease in amplitude and phase of both PTR and LUM occurred for about the first ≈75
hrs following exposure to the demineralizing solution. The initial rapid rate of decline in both
signals may be attributed to surface and subsurface mineral loss under the acid challenge. The
initial dissolution may occur directly at the enamel surface causing an increase in surface
roughness, which would effectively confine photons and thermal waves to a narrower surface
region reducing the efficiency of the transmitted signals. Following the first rapid decline in
PTR-LUM amplitude and phase signals, a level of saturation appeared; however, following ≈125
hrs, a second rapid decrease in PTR and LUM signals was evident (Fig. 2.4). This trend may be
analogous to that described in earlier studies where an initial sigmoidal-shaped distribution of
mineral loss over time was evident over the first 45 - 78 hrs (Anderson et al. 1998). Furthermore,
Gao et al. (1993a) also found similar trends, however, those authors found that the initial
104
demineralization rate was much higher, which is more consistent with the initial rapid rate of
PTR-LUM signal decline over the first ≈50 - 75 hrs (Fig. 2.4). Initial rapid demineralization was
followed by a slope decrease after ≈44 hrs, which they interpreted as the initial surface layer
formation, and further followed by a lower rate of demineralization related to the progression of
the subsurface lesion without any further change in the thickness of the surface layer. As a result,
the initial mineral loss distribution over time was sigmoidal in shape. It may be likely that the
initial rapid decrease in PTR-LUM signals followed by a transient decrease in the rate of signal
progression until about ≈125 hrs represents the sigmoidal-shaped distribution of mineral loss
described above. The initial demineralization period was observed in several studies and
explained based on a combination of factors, including natural enamel surface properties, which
maintain higher fluoride levels than underlying enamel, the formation of the subsurface lesion
and/or the dynamic process of intact surface layer formation (Anderson et al. 1998; Gao et al.
1993a; Anderson et al. 2004; Yamazaki et al. 2007). Therefore, it is likely that the same initial
period, where PTR and LUM signals rapidly decreased is characterized by a combination of
processes including the dynamic mineral loss-mineral gain of the intact surface layer occurring
with concomitant mineral loss from the subsurface lesion, as was evident in the results of
Chapter 1. Interestingly, the duration of the initial demineralization period was very similar in all
demineralized sections, particularly with respect to the PTR phase signal channel. Comparing the
3 demineralized sections in Figs. 2.4, 2.5 and 2.7A, the duration of the initial demineralization
period ranged from ≈75 - 125 hrs. This range was typical among all other demineralized enamel
sections not displayed. The reproducibility of the initial demineralization period was also
observed by Anderson et al. (1998), where a similar duration of the sigmoidal-shaped period of
mineral loss was found for all samples. A discrepancy in the duration of this initial period in the
present study compared to the earlier studies is presumably due to the stronger acid challenge
and constant-composition conditions in the earlier study. This would explain why the duration of
the initial period in the present study appeared much longer. The fact that this initial
demineralizing period has only been observed in few studies may be due to the nature of the
investigative technique in the present study, where real-time indirect measures of mineral content
were employed over short (≈20-min) intervals during the demineralization process in a non-
invasive manner. Following the initial demineralization period PTR and LUM signals decreased
105
in a near-linear fashion. This is similar to the observations in the earlier studies, where following
the initial sigmoidal-shaped mineral loss over time a linear distribution of mineral loss over time
was observed (Anderson et al. 1998; Gao et al. 1993a; Anderson et al. 2004). The authors
purported the linear period to subsurface mineral loss, which continued until the end of the
treatment period. Furthermore, the observation of a linear period indicated that reactions at the
dissolving crystal surfaces of the advancing lesion front were the rate-limiting processes in lesion
formation (Anderson et al. 1998). The rapid decrease in PTR signals following the initial
demineralization period may also suggest that the dominant mechanism is the mineral loss
occurring primarily from the subsurface regions. This is supported by the fact that a marked
decrease in mineral volume can be seen in the subsurface layer of demineralized enamel
sections. However, significant deviations from linearity were observed in the PTR signal
decrease over time. These deviations may suggest that lesion formation in enamel sections
cannot strictly be attributed to a single controlling mechanism, i.e. surface or diffusion –
controlled, but rather a combination, or continuum, of rate controlling processes (Elliott et al.
2008). Furthermore, in the present study, a reduction in the rate of PTR and LUM signal
decreases occurred near the end of the demineralization period, which is in contrast to the earlier
studies where the linear period extended until the end of the acid challenge (Anderson et al.
1998; Gao et al. 1993a; Anderson et al. 2004). This is most likely a function of the increased
mineral accumulation within the demineralizing gel over time which may cause an increase in
the degree of saturation of the demineralizing gel with respect to enamel mineral and lower
driving force for subsurface mineral loss. Furthermore, PTR signal deviation from linearity at
later demineralization times may be related to the local structural differences in enamel within
one section and between sections (Gao et al. 1993a; Anderson et al. 1998). This is particularly
evident in the present study where natural enamel surfaces remained intact and significant
variation in the structural and chemical properties of the surface enamel layer compared to the
underlying enamel are expected. The overall transition from the ‗initial‘ demineralizing period to
the ‗later‘ demineralization period may be related to shifting thermal centroids during the process
of lesion formation, where initial rapid mineral loss may shift the thermal centroid closer to the
surface while at later times the centroid is maintained within the growing demineralized lesion.
106
9.2 PTR-LUM signals and time-series remineralization
At the onset of remineralization, a transient step-decrease in PTR phase lag was accompanied by
an increase in amplitude (Figs. 2.5 and 2.7A). After ≈300 hrs amplitude and phase signals
reversed direction and reached stable values after ≈650hrs until the end of the treatment period
(Fig. 2.5). The second remineralized sample shown in Fig. 2.6, showed a less marked decrease in
phase lag and increase in amplitude over a similar period (Fig. 2.7A). This behaviour may reflect
the mechanism of fluoride enhanced remineralization, promoting rapid uptake and precipitation
of mineral ions from solution within the entire lesion depth. Subsurface remineralization
processes may only occur for a transient period, i.e. the first 10 days, whereas surface mineral
deposition may be dominant at later times. This also indicates a shifting thermal centroid
between the lesion body at earlier times due to enhanced restored crystallinity and at later
periods when the surface layer build up shifts the thermal centroid toward the surface.
Restoration of subsurface lesion crystallinity during earlier remineralization times would
improve the thermal properties of the lesion body, providing a better conduction medium for
thermal propagation and as a result a greater amount of thermal energy will be transmitted
through the enamel section. As sound enamel prisms may act as optical waveguides of the
incident laser light (Odor et al. 1996), enhanced subsurface crystallinity would restore optical
paths and improve light propagation and transmission. However, enhanced remineralization
within the porous demineralized lesion body is likely to occur with a concomitant deposition at
the enamel surface. According to the abovementioned interplay mechanisms, when subsurface
remineralization is dominant over surface mineral deposition, an amplitude increase occurs with
a phase lag decrease, as the quantity of transmitted energy increases. However, when mineral
deposition becomes dominant within the intact surface layer such that the increase in mineral
volume creates a diffusion barrier for mineral ion flux deeper into the lesion and/or the thermal
mismatch between the surface layer and deeper layers becomes large, the thermal centroid shifts
in the opposite direction and the transmitted signal intensity decreases. It may also be possible
that once the surface layer reaches a critical thickness, its properties become dominant at the
expense of the deeper layers. Transmission-LUM signals did not exhibit the same sensitivity
upon remineralization as did PTR. This is likely related to the fact that the LUM signal channel
is not depth-selective. Insensitivity of the LUM signal channel to depth-dependent processes
107
occurs as higher lesion scattering coefficients will scatter the initial laser radiation as well as
scatter the LUM photons, preventing the photons from transmitting through the enamel section.
However, better contrast was observed in backscatter LUM signals, as processes were monitored
from the sample surface through the remineralizing solution. A large increase in backscatter
LUM following remineralization may be due to restoration of surface crystallinity. This increase
was transient, and after ≈200 hrs began to decrease over time. The decrease in the LUM at later
treatment times is consistent with the increase in scattering coefficient noted in chapter 1, at
prolonged remineralization times, and is also consistent with the observed transient PTR signal
changes as scattering coefficients have a significant influence on thermal centroid localization
within enamel. A less marked decrease in phase lag and increase in amplitude may be a result of
the large demineralized lesion body that persisted following remineralization. The lack of
remineralization within the lesion body of the sample presented in Fig. 2.6 may be explained by
the extremely low mineral volume of the lesion body. With a mineral volume level around 20 -
30 vol%, complete dissolution of subsurface enamel crystallites may occur. As a result, upon
remineralization, fewer partially demineralized crystallites are available as scaffolds for mineral
reprecipitation and growth. In this case, de novo mineral precipitation is the mechanism required
for restoring the demineralized lesion, which requires larger driving forces for remineralization
within the lesion body. As the hypomineralized surface layer would maintain partially
demineralized crystallites, it would therefore be expected that remineralization of the surface
layer be the dominant mechanism, which was demonstrated in the increase in back-scatter LUM
signal at earlier remineralization times.
9.3 Errors and limitations of transmission PTR-LUM measurements
A possible source of error in the transmission measurements may be due to the presence of the
treatment solutions. The influence of the demineralizing gel and remineralizing solution on PTR
and LUM signal generation are shown in Appendix 5 (Figs. A.5.2 - A.5.4). Since the
demineralizing gel is slightly yellow in colour, its mere presence will induce a slight increase in
absorption and as a result, reduce the laser intensity delivered to the enamel surface (Fig. A.5.3).
However, regardless of the initial effects of the treatment solutions, the relative signal channels,
which were monitored in the present study, would not be affected. Furthermore, during
demineralization when the concentration of out-diffused mineral ions accumulates in the gel
108
layer surrounding enamel, a cloudy and diffuse accretion of particles is evident and showed a
slight influence on PTR (1 Hz) and LUM (89 Hz) at the probed frequencies (Fig. A.5.4). As
slight absorption was noted in the presence of the demineralizing gel (Fig. A.5.3), the switch
from the yellowish demineralizing medium to the transparent remineralizing medium induced
additional minor changes at 1 Hz (Fig. A.5.2) that may alter the laser energy distributed at the
surface of the enamel section.
9.4 Future directions
Future directions in the analysis of transmission measurements will include the application of a
modified 2-layer theoretical model to the PTR data. This will allow for the extraction of opto-
thermophysical parameters and the change in these parameters can be evaluated over time.
Quantifying the formation and regression of layer thicknesses in thin enamel sections may be
essential in determining the effectiveness of various demineralizing agents in producing
reproducible substrates for remineralization studies, as well as efficacy of remineralizing agents,
topical solutions and/or artificial saliva analogues. This study has shown that LUM can be used
for back-propagation interface studies and this channel is more sensitive than transmission LUM.
Furthermore, PTR, even in transmission mode, is very sensitive to front surface demineralization
and remineralization processes.
109
10 Significance
The results of the present study were generated in a highly-controlled environment which is
clearly not the situation in vivo. A likely factor expected to limit the potential application of the
photothermal radiometric technique to clinical settings is the presence of saliva. Saliva is 99.9%
by composition water and as depicted in Fig. 7 it has a strong absorption band in the mid IR.
Therefore, the collection of IR emissions through a water based solution would be significantly
attenuated, as was observed in the real-time PTR-LUM experiment. PTR was unable to monitor
changes in real-time as the demineralizing and remineralizing solutions were predominately
water-based. However, modulated LUM would not be plagued by strong water absorption bands
as the spectral range of the emitted luminescence (≈750 - 830-nm) peaks at a smaller wavelength
where water absorption is minimal. Nevertheless, modulated LUM, as an optical phenomenon is
highly sensitive to light scattering processes which change based on the hydration level of the
tooth, as seen earlier, and as all other optical techniques, such as QLF and DIAGNOdent would
be affected by the presence of stains and plaque (Angmar-Månsson et al. 2000). The presence of
plaque can be easily overcome by debriding the tooth surfaces prior to measurements. While the
overall magnitude of the PTR amplitude may be altered by the presence of stain and plaque, PTR
phase has established itself as the more sensitive channel due to its relative insensitivity to
surface features, which highlights the significant advantage of the multi-channel PTR technique.
A laser fluorescence (DIAGNOdent) study found a significant influence of in vitro storage
conditions and tooth hydration level on the fluorescence response (Francescut et al. 2006). These
same challenges of hydration have been confronted by other caries technologies such as QLF
(Pretty et al. 2004) and electrical conductance measurements (Ricketts et al. 1997). In order to
overcome the influence of hydration on the generated PTR-LUM signals, a reliable and clinically
reproducible drying technique is required for maintaining stable conditions for longitudinal
monitoring. This may include cotton roll isolation and/or chair-side compressed air for quick
drying of the samples, the rate of which is affected by room temperature and individual humidity
levels. In terms of QLF measurements, compressed air was found to be the most effective and
expeditious dehydration method for reproducible measurements over time. A drying time of 15
seconds was found to be sufficient for the abovementioned QLF measurements, which is an
110
established and clinically reasonable drying period (Pretty et al. 2006). A critical future test of
the PTR-LUM system must identify and evaluate the efficacy of a standardized drying technique
in vivo, which is vital in order to minimize errors and prove the device reliability toward the
future clinical application of the device. An additional limitation in the present study was that
caries was simulated without bacterial involvement and at room temperature. The collected
thermal signal should not be affected by physiological temperatures within the oral cavity as the
use of lock-in detection operates by locking onto the phase of the source frequency and therefore
removing all other component frequencies. As noted above, the phase signal channel is relatively
insensitive to such variations, while the overall PTR amplitude signal may change under
different temperature and hydration conditions.
The lesions created in the present study, using the acidified gel system, were small in size (mean
depth = 90 μm) (Table 10). A clinical diagnosis based on the detection of such small lesions less
than 100 µm would most likely involve patient education in optimizing plaque control and
salivary flow as well as decreasing the intake frequency of fermentable carbohydrates in order to
favour lesion repair without clinician intervention (Murdoch-Kinch and McLean 2003).
Monitoring these incipient lesions over time will ultimately determine whether the lesion
progresses to the point where therapeutic agents such as topical fluorides might be encouraged to
reverse the non-cavitated lesion or to the point of cavitation where restorative intervention is
necessary. Furthermore, this emphasizes the distinction between detection and diagnosis where
the former is only one component of a clinician‘s arsenal in deciding the overall diagnosis and
treatment option.
A substantial limitation in the application of the PTR-LUM system in the present study is the
restriction of the quantification of simulated caries to smooth surfaces, given that the majority of
caries lesions are presently found on occlusal and approximal tooth surfaces. Nevertheless, this
study is vital in the advancement of quantitative, non-destructive evaluation of the caries process.
As enamel and dentin are turbid media, with complicated structural geometries varying as a
function of depth it is important to start out with the simplest approximation of reality, in this
case, smooth surfaces, which maintain a relatively flat geometry with near-one-dimensional
diffusion processes during lesion formation. Only after gaining a complete understanding of the
111
PTR-LUM signals and combined theoretical formalism to the general cases, can one progress to
more complicated geometries and clinically relevant case studies such as intricate occlusal
fissures or approximal surfaces. The transmission-mode PTR-LUM sensitivity to simulated
caries in thin enamel sections, however, may prove efficacious in detecting and monitoring the
caries process at approximal contact points where the thinnest enamel layer can be found with no
dentinal involvement. The merits of a non-destructive early detection system relate to the
avoidance of high levels of damaging ionizing radiation as well as the detection of caries in tooth
surfaces not readily visible, such as hidden caries in the occlusal fissures and approximal
surfaces, where the caries incidence is high and current radiographic detection methods suffer.
The most feasible methods of clinically assessing caries progression, at present, are bitewing
radiographs combined with visual and/or tactile sensation (Benn 1994; Angmar-Månsson and ten
Bosch 1993). However, considering both the sensitivity and specificity of bitewing radiographs
in the detection of approximal caries lesions, 0.66 and 0.95, respectively, and the sensitivity and
specificity of visual-tactile examination for the same lesions, 0.52 and 0.98, respectively, poor
sensitivity for such lesions is clearly evident (Bader et al. 2001; Baelum 2010). In terms of
occlusal caries, the sensitivities of radiographic and visual-tactile examination are also poor at
≈30% and ≈18%, respectively (Bader et al. 2001). Thus, there is a strong driving force toward
the development of a sensitive caries detection technique and instrumentation applicable to
challenging assessment regions and provide additional quantitative information in the
augmentation of the disease characterization process. Although there have been numerous caries
detection systems investigated in vitro and evaluated clinically, at present, there is no single
detection system capable of reliably detecting caries on all tooth surfaces (Ferriera Zandona and
Zero 2006).
Several advantages and applications from the present study and in the development of a non-
invasive quantitative caries detection system which includes patient education in allowing the
longitudinal monitoring of caries activity without exposure to ionizing radiation, the specific
tailoring of remineralization therapies to the individual level, assessment of lesion activity in
order to distinguish active vs. arrested lesions, detailed in vitro, in situ and/or in vivo
investigations of de- and remineralization mechanisms without sample interruption and
destruction, and lastly the assessment of the efficacy of various remineralization agents for
112
clinical trials. In terms of the latter, continuous monitoring of enamel sections in transmission-
mode PTR-LUM without sample disruption would be ideal to establish effectiveness of topical
solutions, pastes, varnishes or rinses. In addition, future studies may consider modifying the
transmission-mode PTR-LUM experimental setup in order to include continuous flow chambers
analogous to in vitro artificial mouth setups in order to more closely simulate demineralization
and remineralization process occurring in vivo. It may be likely, sometime in the near future, that
direct quantitative information will be obtained through simple chair-side measurements in such
a manner that sound enamel and enamel caries structural geometry as a function of depth can be
reconstructed. Furthermore, this may provide insight into lesion activity, i.e. discerning between
active and arrested lesions, which may assist in tailoring remineralization therapies toward active
lesions and preventing unnecessary overtreatment of the arrested-type.
113
11 Summary
As a non-destructive technique, the combination of PTR and LUM along with the theoretical
model provides 4 distinct signal channels along with a comprehensive theoretical formalism to
yield quantitative information regarding lesion severity and the change in severity over time.
Although the results of the present study cannot be directly extrapolated to clinical environments,
it undoubtedly advances the discipline of quantitative dental diagnostics and forms the widest
parameter basis for future research in the field. Furthermore, quantitative extraction of optical
and thermal properties from intact, whole teeth, rather than prepared thin sections, allows for the
investigation of opto-thermophysical parameters under conditions more reflective of the natural
oral environment. The promising results from the present investigation places the quantitative
PTR and LUM technique at the forefront of non-destructive caries evaluation in vitro, above
existing purely optical systems, in terms of the total information extracted from the generated
signals. In light of the results of the present study the hypothesis is accepted in that the combined
detection modes of PTR-LUM proved to be efficacious in measuring and quantifying
mineralized layers generated during de- and remineralization processes.
12 Conclusions
From the combined backscatter and transmission-mode PTR-LUM experiments the following
conclusions could be drawn:
1. PTR and LUM signals in backscatter-mode were effective in detecting and monitoring
the formation, progression and regression of simulated enamel demineralized and
remineralized lesions. The complementary nature of the PTR trends under 830-nm as
observed under 660-nm laser radiation, illustrates the dual-effectiveness of either laser
wavelength in detecting incipient demineralized and remineralized enamel lesions.
2. The theoretical formalism developed to explain PTR signal trends proved to be effective,
reliable and reproducible in characterizing opto-thermophysical parameters of sound
enamel.
114
3. The high fidelity of the developed theoretical/computational model illustrates its
effectiveness and applicability to non-destructively quantify lesion thicknesses and
reconstruct opto-thermophysical parameters as a function of depth. Furthermore, the
fitting procedure implemented in this work, increased the robustness of the computational
algorithm, providing a unique solution for the multiparameter fits of multi-layered sound
enamel and enamel caries lesions.
4. Real-time acquisition of PTR and LUM signals in transmission-mode proved effective in
detecting and monitoring simulated demineralized and remineralized lesions in thin
enamel sections. Modulated LUM in backscatter was also sensitive in monitoring lesion
progression without sample disruption in the treatment solutions.
5. From both experiments it is clear that remineralization entails a multi-factorial and
complex process involving the interplay between shifting thermal centroids as mineral
gains in surface and subsurface regions alter the opto-thermophysical properties of the
effective layers. The theoretical model pointed to enhanced effectiveness of subsurface
lesion remineralization in the presence of fluoride, however, no statistically significant
differences in TMR defined mineral loss and lesion depth were noted between the
remineralization treatment groups.
115
13 Appendices
13.1 Appendix 1
13.1.1 PTR frequency response (830-nm laser)
Trends in the amplitude and phase frequency response under the 830-nm laser were similar to
those detailed for the smaller wavelength light (660-nm). Amplitude and phase for the 10-day
and 40-day demineralized samples are shown in Fig. A.1.1. Amplitude ratios and phase
differences, normalized with respect to the final demineralization curve, of the exemplary
samples from the fluoride-free, low fluoride and high fluoride group are presented in Figs. A.1.2
- A.1.4. Overall, similar trends were evident between the 2 wavelengths investigated. However,
trends in the phase behaviour under the 830-nm laser were difficult to discern, across the entire
modulation frequency range, due to the poorer SNR of the longer wavelength light.
Figure A.1.1. PTR amplitude and phase curves under the 830-nm laser for (A) the 10-day
demineralized sample and (B) the 40-day demineralized sample. The corresponding
116
microradiographs and mineral content depth profiles for (A) and (B) are presented in Fig. 16 and
Fig. 17, respectively. Error bars, when not visible, are of the size of the symbols.
Figure A.1.2.PTR amplitude ratio and phase difference with respect to the final demineralization
state for a sample in the fluoride-free treatment group under 830-nm laser excitation.
Corresponding microradiograph and mineral volume profile are shown in the adjacent figures.
Error bars, when not visible, are of the size of the symbols.
1 10 100 1000
1.2
1.4
1.6
1.8
2.0
2.2
Remin- 2 Days
Remin- 5 Days
Remin- 10 Days
Remin- 20 Days
Remin- 28 Days
Am
plit
ude R
atio (
V/V
0)
Frequency (Hz)
1 10 100 1000
-9
-6
-3
0
3
6
9
12
PTR Amplitude
PTR Phase
Phase D
iffe
rence ( -
0)
Frequency (Hz)
1 10 100 1000
1.2
1.4
1.6
1.8
2.0
2.2
Remin- 2 Days
Remin- 5 Days
Remin- 10 Days
Remin- 20 Days
Remin- 28 Days
Am
plit
ude R
atio (
V/V
0)
Frequency (Hz)
1 10 100 1000
-9
-6
-3
0
3
6
9
12
PTR Amplitude
PTR Phase
Phase D
iffe
rence ()
Frequency (Hz)
100 µm
117
Figure A.1.3. PTR amplitude ratio and phase difference with respect to the final
demineralization state for a sample in the low fluoride (1 ppm) treatment group, under 830-nm
laser excitation. Corresponding microradiograph and mineral volume profile are shown in the
adjacent figures. Error bars, when not visible, are of the size of the symbols.
1 10 100 1000
1.1
1.2
1.3
1.4
1.5
1.6
Remin- 2 Days
Remin- 5 Days
Remin- 10 Days
Remin- 20 Days
Remin- 28 DaysA
mplit
ude R
atio (
V/V
)
Frequency (Hz)
1 10 100 1000
-6
-4
-2
0
2
4
6
8
PTR Amplitude
PTR Phase
Phase D
iffe
rence ( -
)
Frequency (Hz)
1 10 100 1000
1.1
1.2
1.3
1.4
1.5
1.6
Remin- 2 Days
Remin- 5 Days
Remin- 10 Days
Remin- 20 Days
Remin- 28 Days
Am
plit
ude R
atio (
V/V
0)
Frequency (Hz)
1 10 100 1000
-4
-2
0
2
4
6
8
PTR Amplitude
PTR Phase
Phase D
iffe
rence (-
0)
Frequency (Hz)
100 µm
118
Figure A.1.4. PTR amplitude ratio and phase differences with respect to the final
demineralization state for a sample in the high fluoride (1000 ppm) treatment group, under 830-
nm laser excitation. Corresponding microradiograph and mineral volume profile are shown in the
adjacent figures. Error bars, when not visible, are of the size of the symbols.
1 10 100 1000
1.0
1.5
2.0
Am
plit
ude R
atio (
V/V
0)
Frequency (Hz)
1 10 100 1000
-12
-10
-8
-6
-4
-2
0
2
4
6
8
10
12
14
Remin- 2 Days
Remin- 5 Days
Remin- 10 Days
Remin- 20 Days
Remin- 28 Days
PTR Amplitude
PTR PhasePhase D
iffe
rence (-
0)
Frequency (Hz)
100 µm
1 10 100 1000
1.0
1.2
1.4
1.6
1.8
2.0
2.2
Am
plit
ude R
atio (
V/V
0)
Frequency (Hz)
1 10 100 1000
-12
-10
-8
-6
-4
-2
0
2
4
6
8
10
12
14
Remin- 2 Days
Remin- 5 Days
Remin- 10 Days
Remin- 20 Days
Remin- 28 Days
PTR Amplitude
PTR Phase
Phase D
iffe
rence (-
0)
Frequency (Hz)
119
13.2 Appendix 2
The following figures present the theoretical fits of sound (Fig. A.2.1) demineralized (A.2.2a-d)
and remineralized enamel (Figs. A.2.3 – A.2.5) superposed on the experimental data. In all cases,
a good fit between theoretical curves and experimental data points was observed. From the
theoretical fitting curves to experimental data, opto-thermophysical depth profiles for multi-
layered enamel were reconstructed.
Figure A.2.1. Multi-parameter fitting of amplitude and phase curves of a sound tooth.
Experimental data are represented by symbols (*). Calculated theory is shown as solid lines.
101
102
10-1
100
Frequency (Hz)
PTR
Am
plit
ud
e (a
.u.)
101
102
-85
-80
-75
-70
-65
Frequency (Hz)
PTR
ph
ase,
deg
120
Figure A.2.2. PTR amplitude and phase experimental and 3-layer theory plots for the (a) 10-day
demineralized sample and (b) the 40-day demineralized sample. Experimental data are
represented by symbols and calculated theory is shown as solid lines.
101
102
-100
-90
-80
-70
-60
Frequency (Hz)
PTR
pha
se, d
eg
Sound enamel
Demin-5day
Demin-10day
101
102
10-2
10-1
100
101
Frequency (Hz)
PTR
Am
plit
ude
(a.u
.)
Sound enamel
Demin-5day
Demin-10day
(A)
101
102
-75
-70
-65
-60
-55
Frequency (Hz)
PTR
ph
ase,
deg
Sound enamel
Demin-5day
Demin-10day
Demin-15day
Demin-20day
Demin-30day
Demin-40day
101
102
10-1
100
Frequency (Hz)
PTR
Am
plit
ud
e (a
.u.)
Sound enamel
Demin-5day
Demin-10day
Demin-15day
Demin-20day
Demin-30day
Demin-40day
(B)
121
Figure A.2.3. PTR amplitude and phase experimental and 3-layer theory plots for the fluoride-
free sample. Experimental data are represented by symbols and calculated theory is shown as
solid lines.
Figure A.2.4. PTR amplitude and phase experimental and 3-layer theory plots for the low
fluoride sample. Experimental data are represented by symbols and calculated theory is shown as
solid lines.
101
102
10-1
100
Frequency (Hz)
P
TR A
mp
litu
de
(a.u
.)
Demin-10day
Remin-2day
Remin-5day
Remin-10day
Remin-20day
Remin-28day
101
102
-70
-68
-66
-64
-62
-60
Frequency (Hz)
PTR
ph
ase,
deg
Demin-10day
Remin-2day
Remin-5day
Remin-10day
Remin-20day
Remin-28day
101
102
10-1
100
Frequency (Hz)
PTR
Am
plit
ud
e (a
.u.)
Demin-10day
Remin-2day
Remin-5day
Remin-10day
Remin-20day
Remin-28day
101
102
-70
-68
-66
-64
-62
Frequency (Hz)
PTR
ph
ase,
deg
Demin-10day
Remin-2day
Remin-5day
Remin-10day
Remin-20day
Remin-28day
122
Figure A.2.5. PTR amplitude and phase experimental and 3-layer theory plots for the high
fluoride group. Experimental data are represented by symbols and calculated theory is shown as
solid lines.
101
102
10-1
100
Frequency (Hz)
PTR
Am
plit
ud
e (a
.u.)
Demin-10day
Remin-2day
Remin-5day
Remin-10day
Remin-20day
Remin-28day
101
102
-80
-75
-70
-65
Frequency (Hz)
PTR
ph
ase,
deg
Demin-10day
Remin-2day
Remin-5day
Remin-10day
Remin-20day
Remin-28day
123
13.3 Appendix 3
13.3.1 Additional optothermal parameters
The following section presents the auxiliary parameters derived from the theoretical fitting
program outlined in Table 6 and not described in detail within the text. These parameters
include: the cosine of the scattering angle (g), the infrared absorption coefficient (μIR), the optical
reflection coefficients from the L1-L2 boundary (R2) and L2-L3 boundary (R3) and lastly the non-
radiative energy conversion efficiency within each layer (ηNR).
Supplementary optothermal depth profiles for the 10-day demineralized sample and the 40-day
demineralized are shown in Figs. A.3.1 and A.3.2, respectively. In addition, the auxiliary
parameters from the samples in the fluoride-free (Fig. A.3.3), low fluoride (Fig. A.3.4) and high
fluoride group (Fig. A.3.5) are shown.
Figure A.3.1. The change in optothermal parameters as a function of time for the 10-day
demineralized sample. (a) Scattering anisotropy, (b) IR absorption coefficient, (c) Reflection
coefficients and (d) Non-radiative efficiency, are presented for each layer over the
demineralization period. Layer 1 = surface layer; Layer 2 = lesion body.
0 5 10
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Co
s Sc
atte
rin
g A
ngl
e (
g)
Treatment Time (days)
Layer 1
Layer 2
0 5 10
60000
80000
100000
120000
140000
160000
180000
200000
IR A
bso
rpti
on
Co
eff
icie
nt
(m-1
)
Treatment Time (days)
0 5 10
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Re
fle
ctio
n C
oe
ffic
ien
t (R
)
Treatment Time (days)
R2
R3
0 5 10
30
40
50
60
70
80
90
No
n-R
adia
tive
Eff
icie
ncy
(%
)
Treatment Time (days)
Layer 1
Layer 2
(b)(a)
(d)(c)
124
Figure A.3.2. The change in optothermal parameters as a function of time for the 40-day
demineralized sample. (a) Scattering anisotropy, (b) IR absorption coefficient, (c) Reflection
coefficients and (d) Non-radiative efficiency, are presented for each layer over the
demineralization period. Layer 1 = surface layer; Layer 2 = lesion body.
0 10 20 30 40
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Co
s Sc
atte
rin
g A
ngl
e (g
)
Treatment Time (days)
Layer 1
Layer 2
0 10 20 30 4050000
75000
100000
125000
150000
175000
IR A
bso
rpti
on
Co
eff
icie
nt
(m-1
)
Treatment Time (days)
0 10 20 30 40
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
Re
fle
ctio
n C
oe
ffic
ien
t (R
)
Treatment Time (days)
R2
R3
0 10 20 30 400
10
20
30
40
50
60
No
n-R
adia
tive
Eff
icie
ncy
(%
)
Treatment Time (days)
Layer 1
Layer 2
(b)(a)
(d)(c)
125
Figure A.3.3. The change in optothermal parameters as a function of time for the fluoride-free
sample. (a) Scattering anisotropy, (b) IR absorption coefficient, (c) Reflection coefficients and
(d) Non-radiative efficiency, are presented for each layer over the de- and remineralization
periods. Layer 1 = surface layer; Layer 2 = lesion body.
0 10 20 30 40
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
ReminDemin
Co
s Sc
atte
rin
g A
ngl
e (g
)
Treatment Time (days)
Layer 1
Layer 2
0 10 20 30 4020000
30000
40000
50000
60000
70000
80000
90000
100000
110000
ReminDemin
IR A
bso
rpti
on
Co
eff
icie
nt
(m-1
)
Treatment Time (days)
0 10 20 30 40
0.0
0.2
0.4
0.6
0.8
1.0
ReminDemin
Ref
lect
ion
Co
effi
cien
t (R
)
Treatment Time (days)
R2
R3
0 10 20 30 400
10
20
30
40
50
60
70
80
ReminDemin
No
n-R
adia
tive
Eff
icie
ncy
(%
)
Treatment Time (days)
Layer 1
Layer 2
(b)
(d)(c)
(a)
126
Figure A.3.4. The change in optothermal parameters as a function of time for the low fluoride
sample. (a) Scattering anisotropy, (b) IR absorption coefficient, (c) Reflection coefficients and
(d) Non-radiative efficiency, are presented for each layer over the de- and remineralization
periods. Layer 1 = surface layer; Layer 2 = lesion body.
0 10 20 30 40
0.3
0.4
0.5
0.6
0.7
0.8
0.9
ReminDeminCo
s Sc
atte
rin
g A
ngl
e(g)
Treatment Time (days)
Layer 1
Layer 2
0 10 20 30 4060000
80000
100000
120000
140000
160000
180000
200000Demin Remin
IR A
bso
rpti
on
Co
eff
icie
nt
(m-1
)
Treatment Time (days)
0 10 20 30 40
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
ReminDemin
Ref
lect
ion
Co
effi
cien
t (R
)
Treatment Time (days)
R2
R3
0 10 20 30 40
10
20
30
40
50
60
70
80
ReminDemin
No
n-R
adia
tive
Eff
icie
ncy
(%
)
Treatment Time (days)
Layer 1
Layer 2
(b)
(d)(c)
(a)
127
Figure A.3.5. The change in optothermal parameters as a function of time for the high fluoride
sample. (a) Scattering anisotropy, (b) IR absorption coefficient, (c) Reflection coefficients and
(d) Non-radiative efficiency, are presented for each layer over the de- and remineralization
periods. Layer 1 = surface layer; Layer 2 = lesion body.
0 10 20 30 40
0.2
0.4
0.6
0.8
1.0
ReminDeminCo
s Sc
atte
rin
g A
ngl
e (g
)
Treatment Time (days)
Layer 1
Layer 2
0 10 20 30 40
20000
40000
60000
80000
100000
120000
140000
160000
180000
200000Demin Remin
IR A
bso
rpti
on
Co
eff
icie
nt
(m-1
)
Treatment Time (days)
0 10 20 30 40
0.0
0.2
0.4
0.6
0.8
1.0
Remin
Demin
Re
fle
ctio
n C
oe
ffic
ien
t (R
)
Treatment Time (days)
R2
R3
0 10 20 30 40
10
20
30
40
50
60
70
80
90
ReminDeminNo
n-R
adia
tive
Eff
icie
ncy
(%
)
Treatment Time (days)
Layer 1
Layer 2
(b)
(d)(c)
(a)
128
13.4 Appendix 4
The following section presents an assessment of the error in experimental data on the theoretical
derivation of optical and thermophysical parameters. In the first test, 3 individual fits were
performed and compared in order to evaluate sources of error from experimental data. The first
fit involved the experimental raw PTR amplitude and phase data to generate a set of opto-
thermophysical parameters. In the second and third fits, the maximum and minimum PTR
amplitude and phase ranges were determined by adding and subtracting experimental standard
deviations from the averaged PTR signals, respectively (Fig. A.4.1). The percentage difference
was then calculated between the resultant parameters derived from the first fit and the average of
the minimum and maximum range in order to assess the deviation in parameter as a function of
the standard deviation of the PTR measurements (Table A.4.1).
Figure A.4.1. The influence of PTR raw data standard deviation on the outcome of theoretical
fitting. Error max and Error min refer to PTR amplitude and phase signals plus and minus
standard deviation, respectively. Experimental data are represented by symbols and calculated
theory is shown by solid or dotted lines.
A good agreement between the averaged PTR data and the average PTRmax and PTRmin can be
seen. This illustrates that large error bars in the PTR raw data measurement can have a
significant influence on the generated set of opto-thermophysical parameters, however,
differences were mainly seen in the optical properties and less so in the thermal properties and
thickness values. Therefore, for future applications of the theoretical algorithm it is important to
maximize the SNR of the experimental data and/or fit the raw data to a polynomial function in
101
102
100
Frequency (Hz)
PT
R A
mp
litu
de
(a
.u.)
3-L Theory- Error MIN
3-L Theory- Error MAX
Experimental- Error MIN
Experimental- Error MAX
101
102
-72
-70
-68
-66
-64
-62
Frequency (Hz)
PT
R P
ha
se
(d
eg
)
3-L Theory- Error MIN
3-L Theory- Error MAX
Experimental- Error MIN
Experimental- Error MAX
129
order to attain a smooth curve for the fitting procedure, as extraneous data points can add
significant deviation in the generated parameters. Adhering to these guidelines would
significantly reduce the overall calculation time of the theoretical algorithm, which at present is
the time-limiting factor, and augment the validity of the derived parameters.
Table A.4.1. Percentage difference attributed to the standard deviation of experimental PTR
measurements.
The results of a second test on the validity of the computational algorithm are presented in Table
A.4.2. In this test the PTR curves from frequency scans at the final treatment point were fitted
with ‗open‘ and ‗closed‘ thickness limits. Closed thickness limits refer to the fitting procedure
outlined in Fig. 13 and described in detail in sub-section 1.7 of chapter 1. This involved fitting
the final PTR treatment curve based on the maximum and minimum thicknesses determined
from the TMR mineral content depth profiles. The closed limits refer to the situation where the
final thicknesses are known values. Open thickness limits refers to the situation where it is
assumed that the final thicknesses are unknown values. The limits that were defined for layer 1
and layer 2 for the ‗open‘ fit were determined from the minimum and maximum range of
thicknesses for the surface layer and lesion body from all TMR mineral content profiles in the
study. The results of this satellite experiment demonstrated large variation in the generated
optical properties, while less deviation was noted for the thermal properties. Most importantly,
the thickness values showed great convergence to more or less the same values. This indicates
that layer thicknesses could be predicted within ≈20% error which strengthens the overall power
and legitimacy of the derived theoretical formalism in non-destructively quantifying layer
Parameters PTR Amp and phase + S.D.
(PTRMax)
PTR Amp and phase – S.D.
(PTRMIN)
Average (PTRMax) and
(PTRMIN)
Fitted PTR average
Percent difference
(%)
µa1 114 104 109 141 26
µa2 22 51 37 43 16
µs1 151156 105971 128564 117427 9
µs2 100530 144472 122501 144369 16
κ1 0.76 0.35 0.56 0.53 5
κ2 0.45 0.48 0.47 0.45 3
α1 7.1 x 10-7 6.2 x 10-7 6.7 x 10-7 7.3 x 10-7 9
α2 2.5 x 10-7 2.8 x 10-7 2.7 x 10-7 2.5 x 10-7 6
L1 20.2 14.0 17.1 18.7 9
L2 79.8 93.1 86.5 92.1 6
130
thicknesses. The fact that the ‗open‘ thickness values converged to more or the same values as
the ‗closed‘ is an important finding, since the former situation may occur clinically where the
thicknesses are clearly unknown.
Table A.4.2. The percent differences for fitting the final demineralized and final remineralized
PTR curves with open thickness limits (DOPEN and ROPEN) and closed thickness limits (DLIMIT and
RLIMIT). D and R refer to demineralized and remineralized, respectively. For an explanation of
‗open‘ and ‗closed‘ limits see text body. Subscript numbers refer to the layer, where 1 is the
intact surface layer (layer 1) and 2 is the lesion body (layer 2).
Parameters DLIMIT DOPEN Percent
difference (%)
RLIMIT ROPEN Percent
difference (%)
µa1 141 78 58 141 36 118
µa2 43 40 7 40 31 24
µs1 117427 134711 14 226 210 69
µs2 144369 111874 25 149920 58982 87
κ1 0.53 0.37 35 0.68 0.76 11
κ2 0.45 0.53 17 0.43 0.44 1
α1 7.3 x 10-7 6.8 x 10-7 7 6.7 x 10-7 6.4 x 10-7 4
α2 2.5 x 10-7 3.0 x 10-7 18 2.2 x 10-7 2.1 x 10-7 4
L1 18.7 18.7 0 15.1 12.1 22
L2 92.1 102.1 10 39.7 39.3 1
131
13.5 Appendix 5
13.5.1 The effect of incubation in the humid chamber on backscatter PTR-LUM
signals
At the end of each designated treatment period, individual samples were removed from their
treatment solutions, washed, dried and placed in a thermodynamic chamber overnight before
PTR-LUM measurements were executed. In order to examine the effects of PTR-LUM signal
drift due to changes in humidity over time of exposure inside the humid chamber, the following
satellite measurement was performed. A sound enamel sample was scanned at day 0 and after 2
and 4 days of incubation inside the humid chamber. Prior to each measurement the sample was
removed from the chamber, allowed to dry in the air for 40 min, followed by a further 20 min
under direct laser radiation for thermal stabilization purposes. From the results presented in Fig.
A.5.1, it is apparent that incubation in the humid box overnight or over a weekend did not
significantly affect PTR signal generation. On the other hand, LUM after 2 days also did not
exhibit any change in signal however, after 4 days there was slight decrease in amplitude and
small scale changes in phase. Thus, small changes in LUM signals may be influenced by sample
hydration levels over time rather than signal being solely dependent on individual treatments,
consistent with recent PTR-LUM studies (Jeon et al. 2007, 2008).
132
Figure A.5.1. The effect of incubation time in the humidity chamber on PTR - LUM signals.
13.5.2 The effect of the treatment solutions on transmission-mode
PTR-LUM
Using a thin sheet of aluminum foil in place of enamel section, frequency scans were performed
before and immediately following the addition of the acid gel medium to the treatment container.
After gel was decanted, PTR amplitude decreased with a marked behaviour at low modulation
frequencies and PTR phase lag decreased with enhanced phase curvature in the low modulation
frequency range (Fig. A.5.2a). Frequency scans following the replacement of the
demineralization gel with the remineralizing solution are shown in Fig. A.5.2b. A switch from
the demineralizing to remineralizing solution produced a slight decrease in amplitude across the
1 10 100 1000
1E-4
1E-3
0.01
Am
plit
ude (
a.u
.)
Frequency (Hz)1 10 100 1000
0.12
0.14
0.16
0.18
0.20
0.22PTR Amplitude LUM Amplitude
Am
plit
ude (
a.u
.)
Frequency (Hz)
1 10 100 1000
-90
-85
-80
-75
-70
-65 PTR Phase
Phase (
Deg)
Frequency (Hz)1 10 100 1000
-18
-16
-14
-12
-10
-8
-6
-4
-2
Humid Box- Day 0
Humid Box- Day 2
Humid Box- Day 4
LUM phase
Phase (
Deg)
Frequency (Hz)
133
entire modulation frequency range. PTR phase of the remineralizing solution curve converged to
the same values of the demineralization gel curve, apart from a small decrease in phase lag at 1 –
2 Hz.
Figure A.5.2. Transmission-mode PTR frequency response of an aluminum foil sample
illustrating the effect of demineralization gel (a) and demineralizing and remineralizing solutions
(b) on signal generation.
The same experiment as detailed above using the demineralization gel was performed with an
enamel section and is shown in Fig. A.5.3. After the addition of the demineralization gel, PTR
amplitude decreased very slightly across the entire modulation frequency range. PTR phase
showed no effect at frequencies above 4 Hz. At 1 Hz, PTR phase lag decreased whereas an
134
increase in phase lag was observed at 2 - 3 Hz. A large decrease in both LUM amplitude and
phase was noted after gel was added.
Figure A.5.3. Transmission-mode PTR-LUM signals with an enamel section under empty
treatment container and demineralization gel filled container conditions.
Following all demineralization treatments, the accumulation of out-diffused mineral and organic
factions from the treated enamel section sated the demineralizing gel media. The effect of PTR-
LUM signal generation in the particle-laden used demineralization gel vs. fresh particulate-free
gel was tested and shown in Fig. A.5.4. After the replacement of the undisturbed, used gel with
fresh medium, PTR amplitude slightly increased across the entire modulation frequency range
with a concomitant small decrease in phase lag at 1 - 3 Hz with minimal change in the phase
frequency response at frequencies greater than 3 Hz. A comparable increase in LUM amplitude
1 10 100
1E-4
1E-3
0.01
Am
plit
ude (
a.u
.)
Frequency (Hz)1 10 100 1000
0.30
0.35
0.40
0.45
0.50
0.55
Empty Container
Gel - filled Container
PTR Amplitude LUM Amplitude
Am
plit
ude (
a.u
.)Frequency (Hz)
1 10 100
-120
-115
-110
-105
-100
-95
-90
-85
-80
PTR Phase
Phase (
Deg)
Frequency (Hz)1 10 100 1000
-14
-12
-10
-8
-6
-4
-2
LUM phase
Phase (
Deg)
Frequency (Hz)
135
was also noted with a slight increase in phase minimum. As the incident radiation must
propagate through the demineralizing and remineralizing media to reach the tooth surface, the
presence of significant scatters within the bulk media can significantly affect the amount of laser
energy deposited inside the tooth, as evidenced from the change in PTR-LUM signals with the
fresh vs. used demineralizing gel. Thus, particulates within the demineralizing medium cannot be
ruled out as a source of signal generation in transmission measurements as small contributions to
PTR-LUM signals are noted.
Figure A.5.4. The effect of new and used demineralization gel on transmission-mode PTR-LUM
signal generation.
1 10 100 1000
1E-4
1E-3
0.01
Am
plit
ude (
a.u
.)
Frequency (Hz)1 10 100 1000
0.18
0.24
0.30
PTR Amplitude LUM Amplitude
Am
plit
ude (
a.u
.)
Frequency (Hz)
1 10 100 1000
-150
-140
-130
-120
-110
-100
-90
-80
-70
-60
-50 PTR Phase
Phase (
Deg)
Frequency (Hz)1 10 100 1000
-16
-14
-12
-10
-8
-6
-4
-2
Undisturbed Demin Gel
New Demin Gel Added
LUM phase
Phase (
Deg)
Frequency (Hz)
136
14 References
Al-Khateeb S, Exterkate R, Angmar-Månsson B and ten Cate B. (2000). Effect of acid-etching
on remineralization of enamel white spot lesions. Acta Odont Scand 58(1):31-36.
Al-Khateeb S, Exterkate RAM, de Josselin de Jong E, Angmar-Månsson B and ten Cate JM.
(2002). Light-Induced Fluorescence Studies on Dehydration of Incipient Enamel Lesions.
Caries Res.36: 25–30.
Almond D and Patel P. (1996). Photothermal science and techniques. Kluwer Academic
Publishers, Chapman and Hall, London.
Amaechi BT, Higham SM and Edgar WM. (1998). Factors affecting the development of carious
lesions in bovine teeth in vitro. Arch Oral Biol 43: 619–628.
Amaechi BT, Higham SM, Edgar WM. (1999). Effect of sterilization methods on the structural
integrity of artificial enamel caries for intra-oral cariogenicity tests. J Dent 27: 313-316.
Amaechi BT and Higham SM. (2001). Eroded enamel lesion remineralization by saliva as a
possible factor in the site-specificity of human dental erosion. Archs Oral Biol 46(8): 697-
703.
Amaechi BT and Higham SM. (2002). Quantitative light-induced fluorescence: A potential tool
for general dental assessment. J Biomed Opt 7: 7-13.
Amaechi BT. (2009). Emerging technologies for diagnosis of dental caries: The road so far. J
Appl Phys 105: 102047-1 – 102047-9.
Amjad Z and Nancollas GH. (1979). Effect of Fluoride on the Growth of Hydroxyapatite and
Human Dental Enamel. Caries Res 13:250-258.
Amjad Z, Koutsoukos PG and Nancollas GH (1981). The mineralization of enamel surface. J
Dent Res 60: 1783-1792.
Anderson P and Elliott JC. (1985). Scanning x-ray microradiographic study of the formation of
caries-like lesions in synthetic apatite aggregates. Caries Res 19: 403-406.
137
Anderson P and Elliott JC. (1987). Coupled diffusion as basis for subsurface demineralization in
dental caries. Caries Res 21:522- 525.
Anderson P and Elliott JC. (1992).Subsurface Demineralization in dental enamel and other
permeable solids during acid dissolution. J Dent Res 71(8): 1473-1481.
Anderson P, Levinkind M and Elliott JC (1998). Scanning microradiographic studies of rates of
in vitro demineralization in human and bovine dental enamel. Arch Oral Biol 43: 649-656.
Anderson P, Bollet-Quivogne FRG, Dowker SEP and Elliott JC. (2004). Demineralization in
enamel and hydroxyapatite aggregates at increasing ionic strengths. dissolution. Arch Oral
Biol 49: 199-207.
Ando M, Gonzalez-Cabezas C, Isaacs RL, Eckert GJ and Stookey GK. (2004). Evaluation of
Several Techniques for the Detection of Secondary Caries Adjacent to Amalgam
Restorations. Caries Res 38: 350–356.
Angmar-Månsson B and ten Bosch JJ. (1987) Optical methods for the detection and
quantification of caries. Adv Dent Res 1(1): 14 - 20.
Angmar-Månsson B and ten Bosch JJ. (1993). Advances in methods for diagnosing coronal
caries--a review. Adv Dent Res 7(2): 70-9.
Angmar-Mänsson B, Al-Khateeb S and Tranaeus S. (2000). Quantitative light-induced
fluorescence: current research. In: Proceedings of 4th Annual Indiana Conference on Early
Detection of Dental Caries II, May 19-22, 1999. Stookey GK, editor. Indianapolis, IN:
Indiana University School of Dentistry, pp. 203-217
Angmar-Månsson B and ten Bosch JJ. (2001) Quantitative light-induced Fluorescence (QLF): a
method for assessment of incipient caries lesions. Dento maxillo Radiol 30: 298 – 307.
Aoba T, Okazaki M, Takahashi J and Moriwaki Y. (1978). X-ray diffraction study on
remineralization using synthetic hydroxyapatite pellets. Caries Res 12: 223-230.
Aoba T. (1997). The effect of fluoride on apatite structure and growth. Crit Rev Oral Biol Med
8(2): 136-153.
Aoba T. (2004). Solubility properties of human tooth mineral and pathogenesis of dental caries.
Oral Diseases 10: 249-257.
138
Aoba T and Fejerskov O. (2002). Dental Fluorosis: Chemistry and Biology. Crit Rev Oral Biol
Med 13(2): 155-170.
Areas EPG and Galembeck F. (1991). Adsorption of carboxymethyl cellulose onto
hydroxyapatite. J Coll Interf Sci 147:371-377
Arends J and Jongebloed WL. (1979). Ultrastructural Studies of Synthetic Apatite Crystals. J
Dent Res 58(2): 837-843.
Arends J and ten Cate JM (1981). Tooth enamel remineralisation. J Cryst Growth 53:135-147.
Arends J, Christoffersen J. (1986). The nature of early caries lesions in enamel. J Dent Res 65: 2-
11.
Arends J and ten Bosch JJ. (1992). Demineralization and Remineralization Evaluation
Techniques. J Dent Res (Spec Iss) 71: 924-928.
Arends J, Ruben JL and Inaba D. (1997). Major Topics in Quantitative Microradiography of
Enamel and Dentin: R Parameter, Mineral Distribution Visualization, and Hyper-
Remineralization. Adv Dent Res 11: 403-414.
Armstrong WG. (1963). Fluorescence characteristics of sound and carious human dentine
preparations. Arch Oral Biol 8: 79–90.
Astvaldsdottir A, Tranaeus S, Karlsson L and Holbrook WP. (2010). DIAGNOdent
measurements of cultures of selected oral bacteria and demineralized enamel. Acta Odontol
Scand Early Online: 1 - 6.
Bader JD, Shugars DA, Bonito AJ. (2001). Systematic reviews of selected dental caries
diagnostic and management methods. J Dent Educ 65: 960-968.
Baelum V. (2010). What is an appropriate caries diagnosis? Acta Odontol Scand 68: 65–79.
Balageas DL, Krapez JC and Cielo P. (1986). Pulsed photothermal evaluation of layered
materials. J Appl Phys 59: 348-357.
Barker RE, Rafoth RF and Ward RW. (1972). Thermally Induced Stresses and Rapid
Temperature Changes in teeth. J Biomed Mater Res 6: 305-325.
139
Baumgartner A, Dichtl S, Hitzenberger CK, Sattmann H, Robl B, Moritz A, Fercher AF and
Sperr W. (2000). Polarization-sensitive optical coherence tomography of dental structures.
Caries Res 34(1): 59-69.
Benn DK. (1994). Radiographic caries diagnosis and monitoring. Dentomaxillofac Radiol 23:
69–72.
Borsboom PCF and ten Bosch J.J. (1983). A Fibre-Optic Scattering Monitor for Application on
Bulk Biological Tissue, Paper and Plastic. In: Proc. of the Max Born Cent. Conf., SPIE, Vol
369. Bellingham, WA, USA: SPIE pp. 417-421.
Boyle EL, Higham SM, Edgar WM. (1998). The Production of Subsurface Artificial Caries
Lesions on Third Molar Teeth. Caries Res 32:154-158.
Braden M. Heat conduction in normal human teeth (1964). Arch Oral Biol 9: 479–486.
Braden M. (1985). Physics in Dentistry. Phys Technol 16: 58-62.
Brown WS, Dewey WA and Jacob HR. (1970). Thermal properties of teeth. J Dent Res 49: 752–
755.
Brudevold F, Mccann HG and Gron P. (1968). An enamel biopsy method for determination of
fluoride in human teeth. Arch Oral Biol. 13(8): 877-885.
Can AM, Darling CL and Fried D. (2009). High-resolution PS-OCT of enamel remineralization.
Proc. SPIE 6843(1): 68430T.
Craig RG, and Peyton FA. (1961). Thermal Conductivity of Teeth Structures, Dentin Cements,
and Amalgam. J Dent Res 40: 411.
ten Cate JM. (1983). The effect of fluoride on enamel de- and remineralization in vitro and in
vivo. Cariology Today. Int. Congr., Zürich: 231-236
ten Cate JM. (1990). In vitro studies on the effects of fluoride on de- and remineralization. J
Dent Res 69:614-619.
ten Cate JM. (1997). Review on fluoride, with special emphasis on calcium fluoride mechanisms
in caries prevention. Eur J Oral Sci 105(5): 461-465.
140
ten Cate JM. (2001).Remineralization of caries lesions extending into dentin. J Dent Res 80:
1407-1411.
ten Cate JM. (2006). Biofilms, a new approach to the microbiology of dental plaque. Odontology
94: 1-9.
ten Cate JM and Arends J. (1977). Remineralization of artificial enamel lesions in vitro. Caries
Res 11:277-286.
ten Cate JM and Arends J. (1980). Remineralization of artificial enamel lesions. III. A study of
the deposition mechanism. Caries Res 14:351-358.
ten Cate JM, Jongebloed WL and Arends J. (1981). Remineralization of artificial enamel lesions
in vitro. IV. Influence of fluorides and diphosphonates on short- and long-term
remineralization. Caries Res 15:60–69.
ten Cate JM and Featherstone JDB. (1991). Mechanistic Aspects of the Interactions between
Fluoride and Dental Enamel. Crit Rev Oral Biol Med 2(2): 283-296.
ten Cate JM, Lagerweij MD, Wefel JS, Angmar-Månsson B, Hall AF, Ferreira Zandona AG, et
al. (2000). In vitro validation studies of Quantitative Light-induced Fluorescence. Early
detection of dental caries II. Proceedings of the 4th Annual Indiana Conference, Indiana
University Press; 230–246.
CDC. (2001). Recommendations and Reports: Recommendations for using fluoride to prevent
and control dental caries in the United States. MMWR 50(RR-14): 1-42
Chebotareva GP, Nikitin AP, Zubov BV and Chebotarev AP. (1993). Investigation of teeth
absorption in the IR range by the pulsed photothermal radiometry. Proc SPIE;2080:117-128.
Cheong WF, Prahl SA and Welch AJ. (1990). A review of optical properties of biological media.
IEEE J Quan Electron 26: 2166-2185.
Chow LC and Vogel GL (2001). Enhancing Remineralization. J Op Dent (Suppl.) 6:27-38.
Choo-Smith LP, Dong CS, Cleghorn B and Hewko M. (2008). Shedding New Light on Early
Caries Detection. JCDA 74(10): 913-918.
141
Christoffersen J and Arends J.(1982). Progress of artificial carious lesions in enamel. Caries Res
16:433-439.
Craig RG and Peyton FA. (1961). Thermal Conductivity of Tooth Structure, Dental Cements,
and Amalgam. J Dent Res 40: 411-418.
Curzon MEJ and Featherstone JDB. (1983). Chemical composition of enamel, in Lazzari EP
(ed): Handbook of Experimental Aspects of Oral Biochemistry. Boca Raton, CRC Press, 123
– 135.
Damen JJM, Exterkate RAM and ten Cate JM. (1997). Reproducibility of TMR for the
Determination of Longitudinal Mineral Changes in Dental Hard Tissues. Adv Dent Res 11(4):
415-419.
Darling AI and Crabb HSM. (1956). X-ray absorption studies of human dental enamel. Oral
Surg 9: 995-1009.
Darling C, Huynh G and Fried D. (2006). Light scattering properties of natural and artificially
demineralized dental enamel at 1310-nm. J Biomed Opt 11(3):034023(1-11).
Dawes C. (2003). What is the critical pH and why does a tooth dissolve in acid? J Can Dent
Assoc 69(11):722-724.
de Josselin de Jong E, Linden AHIM, ten Bosch JJ. (1987). Longitudinal microradiography: a
non-destructive automated quantitative method to follow mineral changes in mineralised
tissue slices. Phys Med Biol 32: 1209-1220.
de Josselin de Jong E and van der Veen MH. (2007). TMR- Automated Analysis and Mineral
Assessment of Tooth Tissue Sections with Intact Natural Surfaces. Caries Res. 41: 323.
de Josselin de Jong E, Higham S, Smith PW, van Daelen CJ and van der Veen MH. (2009).
Quantified light-induced fluorescence, review of a diagnostic tool in prevention of oral
disease. J Appl Phys. 105: 102031.
de Rooij JF and Nancollas GH. (1984). The formation and remineralization of artificial white
spot lesions: A constant composition approach. J Dent Res 63: 864-867.
Dowker SEP, Anderson P, Elliott CJ and Gao XJ. (1999). Crystal chemistry and dissolution of
calcium phosphate in dental enamel. Mineral Mag 63(6): 791-800.
142
El-Brolossy TA, Abdalla S, Hassanein OE, Negm S, and Talaat H. (2005). Photoacoustic and
electron microscopic studies of remineralized artificially carious enamel and dentin. J Phys IV
125: 685-688.
Elliott JC, Dowker SEP, Davis GR, Walker CS, Wassif HS and Anderson P. (2008). Is the Rate
of Demineralisation in a Caries Lesion Diffusion or Surface Reaction Controlled? Caries Res
42: 203–204.
Faller RV. (1995). The application of in situ models for evaluation of new fluoride-containing
systems Adv Dent Res 9(3): 290-299.
Featherstone JDB, Duncan JF and Cutress TW (1978). Surface layer phenomena in in vitro early
caries-like lesions of human tooth enamel. Arch Oral Biol 23: 397-404.
Featherstone JDB, Duncan JF and Cutress TW. (1979). A mechanism for dental caries based on
chemical processes and diffusion phenomena during in vitro caries simulation on human tooth
enamel. Arch Oral Biol 24: 101–112.
Featherstone JDB and Mellberg JR (1981). Relative rates of progress of artificial carious lesions
in bovine, ovine and human enamel. Caries Res 15: 109–114.
Featherstone JDB, ten Cate JM, Shariati M and Arends J. (1983). Comparison of Artificial
Caries-Like Lesions by Quantitative Microradiography and Microhardness Profiles. Caries
Res17: 385-391.
Featherstone JDB and Zero DT. (1992). An in situ model for the simultaneous assessment of
inhibition of demineralization and enhancement of remineralization. J Dent Res 71: 804–810.
Featherstone JDB. (1999). Prevention and reversal of dental caries: role of low level fluoride.
Community Dent Oral Epidemiol 27: 31–4
Featherstone JD. (2000). The science and practice of caries prevention. J Am Dent Assoc 131:
887-899.
Featherstone JDB and Fried D. (2001). Fundamental interactions of lasers with dental hard
tissues. Med Laser Appl 16: 181-194.
Featherstone JDB. (2008). Dental caries: a dynamic disease process. Aust Dent J 53(3): 286-
291.
143
Featherstone JDB. (2009). Remineralization, the natural caries repair process— the need for
new approaches. Adv Dent Res 21:4-7.
Fejerskov O, Larsen MJ, Richards A and Baelum V. (1994). Dental tissue effects of fluoride.
Adv Dent Res 8(1): 15-31.
Ferreira Zandoná A and Zero DT. (2006). Diagnostic tools for early caries detection. J Am Dent
Assoc 137: 1675-1684.
Francescut P, Zimmerli B, and Lussi A. (2006). Influence of Different Storage Methods on Laser
Fluorescence Values: A Two-Year Study. Caries Res 40:181–185.
Fried D, Featherstone JDB, Glena RE, Bordyn B and Seka W. ( 1993). The light scattering
properties of dentin and enamel at 543, 632, and 1053 nm. In: Lasers in orthopedic, dental,
and veterinary medicine II. Gal D, O'Brien S, Vangsness CT, White J, Wigdor H, editors.
Bellingham, WA: SPIE: 240-245.
Fried D, Glena RE, Featherstone JDB, and Seka W. (1995). Light scattering properties of natural
and artificially demineralized dental enamel at 1310 nm. Appl Opt 34: 1278-1285.
Fried D, Glena RE, Featherstone JDB, Seka W. (1995). Nature of light scattering in dental
enamel and dentin at visible and near-infrared wavelengths. Appl Opt 34: 1278–1285.
Fried D, Xie J, Shafi S, Featherstone JDB, Breunig T and Lee CQ. (2002). Early detection of
dental caries and lesion progression with polarization sensitive optical coherence tomography.
J Biomed Opt 7(4): 618–27.
Fujikawa H, Matsuyama K, Uchiyama A, Nakashima S and Ujiie T. (2008). Influence of salivary
macromolecules and fluoride on enamel lesion remineralization in vitro. Caries Res 42: 37-
45.
Gao XJ, Elliott JC and Anderson P (1991). Scanning and contact microradiographic study of the
effect of degree of saturation on the rate of enamel demineralization. J Dent Res 70:1332-
1337.
Gao XJ, Elliott JC, Anderson P and Davis GR. (1993). Scanning microradiographic and
microtomographic studies of remineralization of subsurface enamel lesions. J Chem Soc
Faraday Trans 89: 2907-2912.
144
Gao XJ, Elliott JC and Anderson P (1993a). Scanning microradiographic study of the kinetics of
subsurface demineralization in tooth sections under constant-composition and small constant-
volume conditions. J Dent Res 72:923-930.
Girkin JM, Hall AF and Creanor SL. (2000). Multi-photon imaging of intact dental tissue. Early
detection of dental caries II. Proceedings of the 4th Annual Indiana Conference, Indiana
University Press; 155–168.
Gmur R, Giertsen E, van der Veen MH, de Josselin de Jong E, ten Cate JM and Guggenheim B.
(2006). In vitro quantitative light-induced fluorescence to measure changes in enamel
mineralization. Clin Oral Invest 10:187-195.
Gonzalez-Cabezas C, Dunn E, Ando M, Eggertsson H, Eckert G, Fontana M and Stookey G.
(2001). Detection of small carious lesions on root surfaces of extracted teeth. Caries Res 35:
282.
Gray JA. (1962). Kinetics of the dissolution of human dental enamel in acid. J Dent Res 41: 633-
645.
Gray JA and Francis MD. (1963). Physical chemistry of enamel dissolution. In: Sognnaes RF,
Editor. Mechanisms of hard tissue destruction. Washington (DC): American Association for
the Advancement of Science, 213-260.
Groeneveld A, Purdell-Lewis DJ, and Arends J. (1975). Influence of the mineral content of
enamel on caries-like lesions produced in hydroxyethylcellulose buffer solutions. Caries Res
9: 127-138.
Groeneveld A, Theuns HM and Kalter PGE. (1975a). Microradiography of developing artificial
dental caries-like lesions in man. Arch Oral Biol 23: 75-83.
Groeneveld A and Arends J. (1975). Influence of pH and demineralization time on mineral
content, thickness of surface layer and depth of artificial caries lesions. Caries Res 9: 36–44.
Groenhuis RAJ, Jongebloed WL and ten Bosch JJ.(1980). Surface roughness measurement of
acid-etched and demineralized bovine enamel measured by a laser speckle method. Caries
Res 14: 333-340.
145
Groenhuis RAJ, ten Bosch JJ and Ferwerda HA. (1981). Scattering of light by dental enamel:
theoretical model compared with experiments. In: Scattering and absorption of light in turbid
materials, especially dental enamel, thesis University of Groningen, pp. 25-42.
Gupta PK, Ghosh N and Patel HS. (2007). Chapter 5: Lasers and laser tissue interaction. In:
Fundamentals and applications of biophotonics in dentistry. Eds: Kishen, A. and A. Asundi,
Imperial College Press. pp 123-151.
Gwinnett AJ. (1967). The ultrastructure of the ―prismless‖ enamel of permanent human teeth.
Archs oral Biol 12: 381-387.
Hafstrom-Bjorkman U, Sundstrom F, de Josselin de Jong E, Oliveby A and Angmar-Mansson B.
(1992). Comparison of laser fluorescence and longitudinal microradiography for quantitative
assessment of in vitro enamel caries. Caries Res 26: 241–7.
Hall A and Girkin JM. (2004). A review of potential new diagnostic modalities for caries lesions.
J Dent Res 83 (Spec Iss C): C89-C94.
Hibst R and Gall R. (1998). Development of a diode laser-based fluorescence caries detector.
Caries Res 32: 294.
Hibst R and Paulus R. (1999). Caries detection by red excited fluorescence: investigations on
fluorophores. Caries Res 33: 295.
Hicks J, Garcia-Godoy F and Flaitz C. (2003). Biological factors in dental caries: role of saliva
and dental plaque in the dynamic process of demineralization and remineralization (part 1). J
Clin Pediatr Dent 28(1): 45- 52.
Higuchi WI, Gray JA, Hefferren JJ and Patel PR. (1965). Mechanisms of Enamel Dissolution in
Acid Buffers. J Dent Res 44: 330-341.
Iijima Y, Takagi O, Ruben J and Arends J. (1999). In vitro Remineralization of in vivo and in
vitro Formed Enamel Lesions. Caries Res 33:206-213.
Ingram GS and Edgar WM. (1994). Interactions of Fluoride and non-Fluoride agents with the
caries process. Adv Dent Res 8:158-6.
146
Issa AI, Preston KP, Preston AJ, Toumba KJ and Duggal MS. (2003). A study investigating the
formation of artificial subsurface enamel caries-like lesions in deciduous and permanent teeth
in the presence and absence of fluoride. Arch Oral Biol 48(8): 567-571.
Jackson D. (1971). Genetic endowment and dental caries. Archs oral Biol. 16: 1433-1441.
Jeon RJ, Hellen A, Matvienko A, Mandelis A, Abrams SH, Amaechi BT. (2008). In vitro
Detection and Quantification of Enamel and Root Caries Using Infrared Photothermal
Radiometry and Modulated Luminescence. J Biomed Opt 13: 034025.
Jeon RJ, Matvienko A, Mandelis A, Abrams SH, Amaechi BT and Kulkarni G. (2007). Detection
of Interproximal Demineralized Lesions on Human Teeth in vitro Using Frequency-Domain
Infrared Photothermal Radiometry and Modulated Luminescence. J Biomed Opt 12: 034028.
Jeon RJ, Han C, Mandelis A, Sanchez V and Abrams SH. (2004). Diagnosis of Pit and Fissure
Caries Using Frequency-Domain Infrared Photothermal Radiometry and Modulated Laser
Luminescence. Caries Res 38:497-513.
Jeon RJ, Mandelis A, Sanchez V and Abrams SH. (2004a). Non-intrusive, non-contacting
frequency-domain photothermal radiometry and luminescence depth profilometry of natural
carious and artificial sub-surface lesions in human teeth. J Biomed Opt 9(4): 804 – 819.
Johansson B. (1965).Remineralization of Slightly Etched Enamel. J Dent Res 44:64-70.
Johnson NW (1967). Some aspects of the ultrastructure of early human caries seen with the
electron microscope. Arch Oral Biol 12: 1505-1521.
Jones FH. (2001). Teeth and bones: applications of surface science to dental materials and
related biomaterials Surf Sci Rep 42: 75-205
Jones RS and Fried D. (2006). Quantifying the Remineralization of Artificial Caries Lesions
using PS-OCT. Proc. SPIE 6137: 613780-1-613780-7.
Kakaboura A and Papagiannoulis L. (2005). Bonding of resinous materials on primary enamel,
in dental hard tissues and bonding,‖ in Interfacial Phenomena and Related Properties, T.
Eliades and C. Watts, eds. Springer pp. 35–51.
Kidd EAM and Joyston-Bechal S. (1980). Histopathological appearance of caries-like lesions of
enamel created artificially in vitro in acidified gels containing fluoride. Caries Res 14: 40-44
147
Kidd EAM and Joyston-Bechal S. (1997). Essentials of Dental Caries. 2nd
edition. New York:
Oxford University Press.
Klinger HG and Weideman W (1986). Enhancement of in vivo remineralization of approximal
initial caries in man by an organic and inorganic remineralization agent. Arch Oral
Biol;31:269-272.
Ko CC, Tantbirojn D, Wang T and Douglas WH. (2000). Optical scattering power for
characterisation of mineral loss. J Dent Res. 79: 1584–1589.
Kodaka T, Kuroiwa M and Higashi S. (1991). Structural and distribution patterns of surface
‗prismless‘ enamel in human permanent teeth. Caries Res 25:7-20.
Kodaka T. (2003). Scanning electron microscopic observations of surface prismless enamel
formed by minute crystals in some human permanent teeth. Anatomical Science International
78(2): 79-84.
Koulourides T, Phantumvanit P, Munksgaard EC and Housch T. (1974). An intraoral model used
for studies of fluoride incorporation in enamel. J Oral Pathol 3:185 - 196.
Krutchkoff DJ, and Rowe NH. (1971). Chemical Nature of Remineralized Flattened Enamel
Surfaces. J Dent Res 50(6): 1621-1625.
Lagerweij M, van der Veen MH, Ando M, Lukantsova L and Stookey G. (1999). The validity
and repeatability of three light induced fluorescence systems: An in-vitro study. Caries Res
33: 220-226.
Lagerweij MD and ten Cate JM. (2006). Acid susceptibility at various depths of pH-cycled
enamel and dentine specimens Caries Res 40: 33-37
Lammers PC, Borggreven JMPM, Driessens FCM, van Dijk JWE. (1991). Influence of fluoride
and carbonate on in vitro remineralization of bovine enamel. J Dent Res 70: 970–974.
Larsen MJ and Fejerskov O. (1978). Structural Studies on Calcium Fluoride Formation and
Uptake of Fluoride in Surface Enamel in vitro. Scand J Dent Res 86: 337-345.
Larsen MJ and Fejerskov O. (1989). Chemical and structural challenges in remineralization of
dental enamel lesions Scand J Dent Res 97(4): 285-96.
148
Larsen MJ and Richards A. (2001). The influence of saliva on the formation of calcium fluoride-
like material on human dental enamel. Caries Res 35: 57-60.
Limeback H. (1994).Enamel formation and the effects of fluoride. Community Dent Oral
Epidemiol 22:144-7.
Lussi A, Imwinkelried S, Pitts N, Longbottom C, Reich E. (1999). Performance and
reproducibility of a laser fluorescence system for detection of occlusal caries in vitro. Caries
Res 33: 261-266.
Lussi A and Reich E. (2005). The influence of toothpastes and prophylaxis pastes on
fluorescence measurements for caries detection in vitro. Eur J Oral Sci 113: 141–144
Lynch RJM, Mony U and ten Cate JM. (2007). Effect of lesion characteristics and mineralizing
solution type on enamel remineralization in vitro. Caries Res 41: 257-262.
Macho GA and Berner MA. (1993). Enamel thickness of human maxillary molars reconsidered.
Am. J. Phys. Anthropol. 92: 189-200
Magalhães AC, Moron BM, Comar LP, Wiegand A, Buchalla W and Buzalaf MAR. (2009).
Comparison of Cross-Sectional Hardness and Transverse Microradiography of Artificial
Carious Enamel Lesions Induced by Different Demineralising Solutions and Gels. Caries Res
43:474-483.
Mandelis A. (1994). Signal-to-noise ratios in lock-in amplifier synchronous detection: A
generalized communications system approach with application to frequency-, time-, and
hybrid (rate-window) photothermal measurements. Rev Sci Instrum 65: 3309 – 3323.
Mandelis A. (2001). Diffusion Wave Fields: Mathematical Methods and Green Functions.
Springer, New York.
Mandelis A, Nicolaides L, Feng C and Abrams SH. (2000). Novel dental depth profilometric
imaging using simultaneous frequency-domain infrared photothermal radiometry and laser
luminescence. Proc. SPIE 3916: 130–137.
Mandelis A and Feng C. (2002). Frequency-domain theory of laser infrared photothermal
radiometric detection of thermal waves generated by diffuse-photon-density wave field in
turbid media. Phys. Rev. E 65: 021909 (1-19).
149
Manesh SK, Darling C and Fried D. (2008). Imaging natural and artificial demineralization on
dentin surfaces with polarization sensitive optical coherence tomography. Lasers in Dentistry
XIV Proc. SPIE, vol. 6843: 68430M–7M.
Manesh SK, Darling CL and Fried D. (2009). Polarization-sensitive optical coherence
tomography for the nondestructive assessment of the remineralization of dentin. J Biomed Opt
14(4): 044002.
Margolis HC and Moreno EC (1985). Kinetic and thermodynamic aspects of enamel
demineralisation. Caries Res 19:22-35.
Margolis HC and Moreno EC (1992). Kinetics of Hydroxyapatite Dissolution in Acetic, Lactic,
and Phosphoric Acid Solutions. Calcif Tissue Int 50: 137-1435.
Margolis HC, Zhang YP, Lee CY, Kent RL and Moreno EC (1999). Kinetics of enamel
demineralization in vitro. J Dent Res 78:1326-1335.
Marshall GW, Marhsall SJ, Kinney JH and Balooch M. (1997). The dentin substrate: structure
and properties related to bonding. J Dent 25 (6): 441-458.
Matvienko A, Mandelis A and Abrams SH. (2009a). Robust multiparameter method of
evaluating the optical and thermal properties of a layered tissue structure using photothermal
radiometry‖ Appl Opt 48(17): 3193-3204.
Matvienko A, Mandelis A, Hellen A, Jeon RJ, Abrams SH and Amaechi BT. (2009b).
Quantitative analysis of incipient mineral loss in hard tissues. Proc. SPIE 7166: 71660C
Matvienko A, Mandelis A, Jeon RJ and Abrams SH. (2009c). Theoretical analysis of coupled
diffuse-photon-density and thermal-wave field depth profiles photothermally generated in
layered turbid dental structures. J App Phys 105: 102022.
Mellberg JR and Mallon DE (1984): Acceleration of Remineralization in vitro by Sodium
Monofluorophosphate and Sodium Fluoride. J Dent Res 63:1130-1135
Miake Y, Saeki Y, Takahashi M, and Yanagisawa T. (2003). Remineralization effects of xylitol
on demineralized enamel. J Electron Microsc. 52: 471–476.
Minesaki Y. (1990). Thermal properties of human teeth and dental cements. Shika Zairyo Kikai
9: 633—46.
150
Minet O, Dörschel K, and Muller G. (2006). Lasers in biology and medicine. Laser Applications.
Landolt–Börnstein 8: 279 - 310.
Mobley J and Vo-Dinh T. (2003). ―Optical properties of tissue‖, in Biomedical Photonics
Handbook, Tuan Vo-Dinh, Ed., Chap. 2, pp. 1–76, CRC Press, New York.
Moreno EC, and Zahradnik RT. (1974). Chemistry of Enamel Subsurface Demineralization In
vitro. J Dent Res (Suppl. 2) 53: 226–235.
Mount GJ and Hume WR. (2005). Preservation and restoration of tooth structure. 2nd ed.
Queensland: Knowledge Books and Software.
Mujat C, van der Veen MH, Ruben JL, ten Bosch JJ, Dogariu A. (2003). Optical pathlength
spectroscopy of incipient caries lesions in relation to quantitative light fluorescence and lesion
characteristics. Appl Opt 42:2979-2986
Murdoch-Kinch CA and McLean ME. (2003). Minimally invasive dentistry. J Am Dent Assoc
134: 87-95.
Nanci A. (2003). Enamel: composition, formation, and structure; in Nanci, A. (ed): Ten Cate‘s
Oral Histology: Development, Structure, and Function. St. Louis, Mosby, (6th
ed) pp 3-15,
145–191.
Nancollas GH. (1979). Enamel apatite nucleation and crystal growth. J Dent Res 58(8): 861-869.
Nicolaides L, Mandelis A and Abrams SH. (2000). Novel dental dynamic depth profilometric
imaging using simultaneous frequency-domain infrared photothermal radiometry and laser
luminescence. J Biomed Opt 5(1): 31-39.
Nicolaides L, Chen Y, Mandelis A and Vitkin IA. (2001). Theoretical, experimental, and
computational aspects of optical property determination of turbid media by using frequency-
domain laser infrared photothermal radiometry. J Opt Soc Am A 18 (10): 2548 – 2556.
Nicolaides L, Feng C, Mandelis A and Abrams SH. (2002). Quantitative dental measurements by
use of simultaneous frequency-domain laser infrared photothermal radiometry and
luminescence. Appl Opt 41(4): 768-777.
O‘Brien WJ. (1997). Dental Materials and Their selection. Chicago, IL: Quintesssence
Publishing Co, Inc. pp 331-404.
151
Odor TM, Watson TF, Pitt Ford TR and McDonald F.( 1996). Pattern of transmission of laser
light in teeth. Int Endod J 29: 228-234.
Ogaard B. (2001). CaF2 Formation: Cariostatic Properties and Factors of Enhancing the Effect.
Caries Res 35 (Suppl. 1): 40-44.
Palamara J, Phakey PP, Rachinger WA and Orams HJ. (1986). Ultrastructure of the intact
surface zone of white spot and brown spot carious lesions in human enamel. J Oral Pathol 15:
28–35.
Panas AJ, Żmuda S, Terpiłowski J, and Preiskorn M. (2003). Investigation of the thermal
diffusivity of human tooth hard tissue, Int J Thermophys 24 (3):837–848
Panas AJ, Preiskorn M Dabrowski M and Żmuda S.(2007). Validation of hard tooth tissue
thermal diffusivity measurements applying an infrared camera. Infrared Phys Technol 49(3):
302-305.
Pearce EIF, Coote GE and Larsen MJ. (1995). The distribution of Fluoride in carious human
enamel. J Dent Res 74: 1775-82.
Pinelli C, Campos Serra M and de Castro Monteiro Loffredo L. (2002). Validity and
reproducibility of a laser fluorescence system for detecting the activity of white-spot lesions
on free smooth surfaces in vivo. Caries Res. 36(1): 19-24.
Poole DFG, Shellis RP and Tyler JE. (1981). Rates of formation in vitro of dental caries-like
enamel lesions in man and some non-human primates. Archs oral Biol 26: 413-417.
Pretty IA, Hall AF, Smith PW, Edgar WM and Higham SM. (2002). The intra- and inter-
examiner reliability of quantitative-light induced fluorescence (QLF) analyses. Br Dent J.
193(2): 105-9.
Pretty IA, Edgar WM and Higham SM. (2002a). Detection of in vitro demineralization of
primary teeth using quantitative light-induced fluorescence (QLF). Int J Paediatr Dent 12:
158-167.
Pretty IA and Maupomé G. (2004). A Closer Look at Diagnosis in Clinical Dental Practice: Part
5. Emerging Technologies for Caries Detection and Diagnosis. J Can Dent Assoc 70(8): 540a-
540i.
152
Pretty IA. (2006). Caries Detection and Diagnosis: Novel Technologies. J Dent 34 (10): 727-
739.
Ripa LW, Gwinnett AJ and Buonocore MG. (1966). The ―prismless‖ outer layer of deciduous
and permanent enamel. Arch Oral Biol 11: 41-48.
Ricketts DNJ, Kidd EAM and Wilson RF. (1997). The effect of airflow on site specific electrical
conductance measurements used in the diagnosis of pit and fissure caries in vitro. Caries Res
31:111–118.
Robinson C, Shore RC, Brookes SJ, Strafford S, Wood SR and Kirkham J. (2000) The chemistry
of enamel caries. Crit Rev Oral Biol Med. 11 (4): 481-495.
Ruben J, Arends J, Christoffersen J. (1999). The Effect of Window Width on the
Demineralization of Human Dentine and Enamel. Caries Res 33:214-219.
Saitoh M, Shiota Y, Kaneko K, Hayashi J, Moro H, Koizumi H, Igarashi T and Nishiyama M.
(2000).Thermal Properties of Dental Materials: Part 10 Crown and Bridge Resins Containing
High Concentration of Filler. J J Dent Mate 19(5): 441-447.
Selwitz RH, Ismail AI and Pitts NB. (2007). Dental Caries. Lancet 369: 51–59.
Shi XQ, Welander U, Angmar-Månsson B. (2000). Occlusal caries detection with KaVo
DIAGNOdent and radiography: an in vitro comparison. Caries Res 34(2):151–8.
Shi XQ, Tranaeus S and Angmar-Månsson B. (2001). Comparison of QLF and DIAGNOdent for
quantification of smooth surface caries. Caries Res 35(1): 21–6.
Silverstone LM, Poole DF.(1969).Histologic and ultrastructural features of remineralized carious
enamel. J Dent Res 48(5): 766-770.
Silverstone LM. (1972). Remineralization of human enamel in vitro. Proc Roy Soc Med 65:32.
Silverstone LM. (1977). Remineralization phenomena. Caries Res 11 (Suppl. 1): 59-84.
Silverstone LM, Wefel JS, Zimmerman BF, Clarkson BH, and Featherstone MJ. (1981).
Remineralization of Natural and Artificial Lesions in Human Dental Enamel in vitro. Caries
Res 15: 138-157.
153
Silverstone LM, Wefel JS (1981). The effect of remineralization on artificial caries-like
lesions and their crystal content. J Cryst Growth 53:148-159
Silverstone LM. (1983). Remineralization and Enamel Caries: New Concepts. Dent Update
10:261-273.
Smith CE. (1998). Cellular and chemical events during enamel maturation. Crit Rev Oral Biol
Med 9(2): 128-161.
Soyenkoff BC and Okun JH. (1958). Thermal Conductivity Measurements of Dental Tissues
with the Aid of Thermistors. JADA 57: 23.
Spitzer D and ten Bosch JJ. (1975). The absorption and scattering of light in bovine and human
dental enamel. Calcif Tissue Res 17:129-137.
Spitzer D and ten Bosch JJ. (1976). The total luminescence of bovine and human dental enamel.
Calcif Tissue Res 20: 201-208.
Spitzer D and ten Bosch JJ. (1977). Luminescence quantum yields of sound and carious dental
enamel. Calcif Tissue Res 24:249-251.
Stookey GK. (2005). Quantitative Light Fluorescence: A Technology for Early Monitoring of the
Caries Process. Dent Clin N Am 49: 753-770.
Tam A C. (1985).Pulsed photothermal radiometry for noncontact spectroscopy, material testing
and inspection measurements. Infrared Phys 25: 305-313.
Tanaka R, Shibata Y, Manabe A, Miyazaki T. (2009). Mineralization Potential of Polarized
Dental Enamel. PLoS ONE 4(6): e5986.
Thuy TT, Nakagaki H, Kato K, Phan AH, Inukai J, Tsuboi S and Nakagaki H et al. (2008).
Effect of strontium in combination with fluoride on enamel remineralization in vitro, Arch
Oral Biol 53: 1017–1022.
Tohda H, Yanagisawa T, Tanaka N and Takuma S. (1990). Growth and fusion of apatite crystals
in the remineralized enamel. J. Electron Microsc. 39: 238–244.
154
Tranæus S, Shi X-Q, Angmar-Månsson B. (2005). Caries risk assessment: methods available to
clinicians for caries detection. Community Dent Oral Epidemiol 33: 265–73.
Tschoppe P, Meyer-Lueckel H and Kielbassa AM. (2008). Effect of carboxymethylcellulose-
based saliva substitutes on predemineralised dentin evaluated by microradiography, Arch Oral
Biol 53: 250–256
Tuchin VV and Altshuler GB. (2007). Chapter 9: Dental and Oral Tissue Optics. In:
Fundamentals and applications of biophotonics in dentistry. Eds: Kishen, A. and A. Asundi,
Imperial College Press. pp 245-296.
Tucker K, Adams M, Shaw L and Smith AJ. (1998). Human Enamel as a Substrate for in vitro
Acid Dissolution Studies: Influence of Tooth Surface and Morphology. Caries Res 32:135-
140.
van der Veen MH and de Josselin de Jong E. (2000). Caries activity detection by dehydration
with QLF.In: Stookey GK, editor. Proceedings of the 4th Annual Indiana Conference, May
19–22, 1999, Indianapolis, Indiana. Early detection of dental caries II. Cincinnati (OH):
SpringDot Publishing: 251 – 259.
Vargaftik NB, Filippov LP, Tarzimanov AA and Totskii EE. (1994). Handbook of thermal
conductivity of liquids and gases; CRC Press: Boca Raton, FL, p. 68 - 70.
Varughese K and Moreno EC (1981). Crystal growth of calcium apatites in dilute solutions
containing fluoride. Calcif Tissue Int 33: 431–439.
Vissink A, `s-Gravenmade EJ, Gelhard TBFM, Panders AK and Franken MH. (1985).
Rehardening properties of mucin- or CMC-containing saliva substitutes on softened human
enamel. Caries Res 19:212-8.
Weatherell J, Robinson C and Hallsworth AS. (1974). Variation in the chemical composition of
human enamel. J Dent Rest 53: 180-192.
Whittaker DK.(1982). Structural variations in the surface zone of human tooth enamel observed
by scanning electron microscopy. Arch Oral Biol 27: 383–392.
Winston AE and Bhaskar SN. (1998). Caries prevention in the 21st century. JADA 129: 1579-
1587.
155
Wong L, Cutress TW and Duncan JF. (1987). The influence of incorporated and adsorbed
fluoride on the dissolution of powdered and pelletized hydroxyapatite in fluoridated and non-
fluoridated acid buffers. J Dent Res 66: 1735-1741.
Wu MS, Higuchi WI, Fox JL and Friedman M. (1976). Kinetics and Mechanism of
Hydroxyapatite Crystal Dissolution in Weak Acid Buffers Using the Rotating Disk Method. J
Dent Res 55: 496-505.
Xue J, Li W and Swain MV. (2009). In vitro demineralization of human enamel natural and
abraded surfaces: A micromechanical and SEM investigation. J Dent 37(4): 264-272.
Yamazaki H, Litman A and Margolis HC. (2007). Effect of fluoride on artificial caries lesion
progression and repair in human enamel: Regulation of mineral deposition and dissolution
under in vivo-like conditions. Arch Oral Biol 52: 110-120.
Yanagisawa T and Miake Y. (2003). High-resolution electron microscopy of enamel-crystal
demineralization and remineralization in carious lesions J Electron Microsc 52(6): 605–613.
Yonese M, Fox JL, Nambu N, Hefferren JJ and Higuchi WI. (1981). Fluoride remineralization of
demineralized bovine tooth enamel and hydroxyapatite pellets. J Pharm Sci 70(8): 904 - 907.
Zahradnik R. (1979). Modification by Salivary Pellicles of In vitro Enamel Remineralization. J
Dent Res. 58: 2066-2073 .
Zhang XZ, Anderson P, Dowker SEP and Elliott JC. (2000). Optical Profilometric Study of
Changes in Surface Roughness of Enamel during in vitro Demineralization. Caries Res 34:
164–174.
Zhang YP, Kent Jr RL, Margolis HC. (2000a). Enamel demineralization under driving forces
found in dental plaque fluid. Eur J Oral Sci 108:207-213.
Zijp JR and ten Bosch JJ (1991). Angular dependence of HeNe-laser light scattering by bovine
and human dentine. Arch Oral Biol 36:283-289.
Zijp JR and ten Bosch JJ. (1993). Theoretical model for the scattering of light by dentin and
comparison with measurements. Appl Opt 32(4): 411-415.
Zijp JR, ten Bosch JJ and Groenhuis RAJ. (1995). HeNe-laser light scattering by human dental
enamel. J Dent Res 74: 1891-1898.
156
Zijp JR. (2001). Optical properties of dental hard tissues. University of Groningen, The
Netherlands.
Zimmerman B, Silverstone LM, Wefel J and Clarkson BH. (1978). The Effect of Calcium
Concentration on Remineralization of Enamel. J Dent Res. 57: 182.
Zuerlein MJ, Fried D, Featherstone J D B and Seka W. (1999). Optical properties of dental
enamel in the mid-IR determined by pulsed photothermal radiometry IEEE J. Sel. Top.
Quantum Electron. 5: 1083–9.
Zuerlein MJ, Fried D, Seka W and Featherstone JDB. (1998). Modeling thermal emission in
dental enamel induced by 9-11 µm laser light Appl Surf. Sci. 127-129: 863-868.