Raman confocal microscopy atlas of human tooth

P.-Y. Collart-Dutilleul, S. Piglionico, R. Younes, Y. Messat, S. Garrabé, H.

Salehi, J.-C. Durand, F. Cuisinier, A. Desoutter

► To cite this version:

P.-Y. Collart-Dutilleul, S. Piglionico, R. Younes, Y. Messat, S. Garrabé, et al., Raman confocal microscopy atlas of human tooth. Archives of Oral Biology, 2025, 173, pp.106189. 10.1016/j.archoralbio.2025.106189. hal-05368493

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# Raman confocal microscopy atlas of human tooth

P.-Y. Collart-Dutilleul<sup>a,b</sup>, S. Piglionico<sup>a,c</sup>, R. Younes<sup>a</sup>, Y. Messat<sup>a</sup>, S. Garrabé<sup>a,b</sup>, H. Salehi<sup>d</sup>, J.-C. Durand<sup>a,b</sup>, F. Cuisinier<sup>a,b</sup>, A. Desoutter<sup>a,*</sup>

## ARTICLE INFO

**Keywords:**

Tooth

Enamel

Dentin

Cementum

Hydroxyapatite

Raman microscopy

## ABSTRACT

**Objective:** Dental histology is a field that has been studied since the early 19th century. Most of the techniques used have been based on white-light microscopy or histological staining, or more recently immunohistochemical staining. With the advent of lasers coupled to confocal microscopy, Raman spectra can be measured in large numbers to create a detailed chemical atlas of the human tooth, offering new insights into its composition and structure.

**Design:** A total of twenty teeth, with 5 teeth from each type premolar, molar, incisor and canine were selected. Five teeth of different types pre-molar and molar incisor canine (total of 20 teeth) These teeth were sectioned and polished, and pulps extracted and cut into thin layers, to perform chemical mapping of all these tissues and components, including secondary structures, based on Raman scattering.

**Results:** We obtained images reconstructed from the intensities of the various characteristic peaks, enabling us to create an atlas of the tooth. A part of the result confirm previous study, but some structures have been revealed for the first time by chemical cartography: cementum dentin junction, dental pulp, Retzius striae, scallop pattern, Hunter-Schreger bands, sheat enamel prism content and dentin branches.

**Conclusion:** The present study thus provides the dental research and practice community with a complete chemical mapping of the fundamental and secondary constituents of the dental organ, with optical resolution.

## 1. Introduction

The tooth is made up of the hardest tissues in the human body and function as the first organ of digestion. Hydroxyapatite (HAP) constitutes the mineral phase present in dentin, enamel dans cementum teeth. It has the chemical formula Ca₅(PO₄)₃(OH). To distinguish between the two types of Ca²⁺ ions, it can be written as Ca(I)₄Ca(II)₆(PO₄)₆(OH)₂. Ca(I) is linked to 9 oxygen ions, which are bonded to 6PO₄ groups, while Ca(II) is connected to 7 oxygen ions: 6 from PO₄ and one from hydroxyl (OH). The HAP crystal mesh belongs to C₆ class, with a cyclic group of type Cₙ = 2π/n with n = 6, and space group P6₃/m. This mineral phase provides significant mechanical resistance to the tooth (Zheng et al., 2013).

Whether an incisor, canine, premolar or molar, each contains a central chamber where dental pulp resides. Its rich in various tissues

such as nerves blood vessels, lymphatic vessels and stem cells. This chamber is encapsulated in dentin, which is the majority constituent of the tooth. It is composed of 70 % mineral phase, 20 % organic matter and 10 % water. It also includes tubules that connect the pulp chamber to the junction with the enamel. This constitution allows dentin to be distinguished chemically and morphologically from enamel. The organic matrix of dentin is mainly composed of type I collagen (85 %) with less amount of type V and III collagens and proteins (Van Hassel, 1971).

In dentin we distinguish between the primary dentin (created during development of the organ up to the apex), secondary dentin (formed after the apex formation) and tertiary dentin (produced as scarring after a trauma). In contrast, enamel is essentially mineral (96 %) with a little amount of water (3 %) and a residue of organic matter (1 %) (Williams et al., 1989). This can be divided into several zones: the junction

<footnote><sup>a</sup> LBN, Univ. Montpellier, Montpellier, France</footnote>

<footnote><sup>b</sup> Dental Clinic, CHU Montpellier, Montpellier, France</footnote>

<footnote><sup>c</sup> National University, Cuyo, Argentina</footnote>

<footnote><sup>d</sup> University of Strasbourg, CNRS, Inserm, ICube, UMR, Strasbourg 7357, France</footnote>

<footnote>* Correspondence to: LBN, Univ. Montpellier, 545 avenue du professeur Jean Louis Viala, Cedex 5, 34193, France.</footnote>

<footnote>E-mail addresses: pierre-yves.collart-dutilleul@umontpellier.fr (P.-Y. Collart-Dutilleul), sofiapiglionico@hotmail.com (S. Piglionico), richard.younes@umontpellier.fr (R. Younes), yacine.messat@umontpellier.fr (Y. Messat), sarah.garrabe@umontpellier.fr (S. Garrabé), s_hamideh@yahoo.com (H. Salehi), jed-cedric.durand@umontpellier.fr (J.-C. Durand), frederic.cuisinier@umontpellier.fr (F. Cuisinier), alban.desoutter@umontpellier.fr (A. Desoutter).</footnote>
between enamel and dentin (DEJ), which includes secondary structures such as the bushes and the spindles. Furthermore, Dental enamel is organized into sheaths, with the interprismatic substance between these sheaths. The streaks of Retzius mark the progression of secreted cells the odontoblasts during crown formation, with their extension into per-kyemata on the surface of the enamel. The circadian rhythm of enamel formation is inscribed within the enamel sheaths through variations in hydroxyapatite concentrations, contributing in daily striations. The enamel is organized in a prism, where the inter-prismatic substance is inserted in between. Both are composed of carbonated apatite crystal-lites, which are themselves made up poly-substituted hydroxyapatite. Dental enamel has a complex micro-structure, with several levels of organization. The basic structure is made of HAP crystals with a section of approximately 25 × 70 nm (Daculsi & Kerebel, 1978) and 100 µm length (Daculsi et al., 1984). The length of these crystals in the direction of the c-axis of the enamel prism is directly related to the rigidity of the enamel (Eimar et al., 2012). These crystals are themselves organized into fibers, fibers and finally enamel prisms (Cui & Ge, 2007).

Many techniques have been made to understand the tooth's arrangement, concerning its internal composition, or its relationships with its surrounding structures: ligament and alveolar bone (Nanci, 2013). First, optical microscopy techniques were used, then followed by higher resolution methods such as the transmission electron microscopy (Gerould, 1944). A large number of techniques have been developed to understand the organ's architecture, chemical composition (Weatherell et al., 1974), microstructure (Orilisi et al., 2021) and even the mechanical properties of its constituents (Gutiérrez-Salazar & Reyes-Gasga, 2003; Y.-R. Zhang et al., 2014). Modern microscopy techniques have enabled to obtain detailed images of enamel prisms (Radlanski et al., 2001), as well as variations in orientation of HAP crystal (Beniash et al., 2019; Habelitz et al., 2001). Classical histology, based on staining or microscopy, has been widely used, and all macro- and microstructures are thoroughly understood and published (Johnson, 1986).

However, this organ histology is still studied today, both for understanding of healthy teeth and for investigating teeth with pathologies such as caries and amelogenesis imperfecta (Desoutter et al., 2022). Confocal Raman microscopy is a chemical analysis tool which is the subject of numerous developments in biology and medicine (Ozaki, 1988; C. Zhang et al., 2015; Ember et al., 2017). it provides several advantages: the possibility of producing spectra or images in aqueous solutions (unlike IR spectroscopy); require less sample preparation for a single spectrum; and provides non-destructive, contactless analysis. Moreover, the confocal properties allows to carry out multilevel, and three-dimensional reconstructions (Majzner et al., 2013).

Although numerous studies have examined several elements of the dental organ separately, a complete chemical mapping of the tooth still lacking. Firstly, the main mineral constituent, HAP was studied by Raman spectroscopy, and the relationship between the intensities of the constituent peaks and the polarization of the incident light was established (Leroy et al., 2002; Tsuda et al., 1996a). Phosphate stretching modes as carbonate (A and B types) and OH peaks in enamel have intensities that are related to the angle between polarization and c-axis of hexagonal crystal. OH peak, being very weak, could be quasi invisible if this angle is 90° (Tsuda & Arends, 1994).

Studies employing spectral measurement have revealed the internal composition of enamel including the dentin, the enamel and the junction between them, as well as the cementum (Timchenko et al., 2016), the surface enamel (Ko et al., 2005; Mihály et al., 2009), and the dentin beside the dentin enamel junction (Alebrahim et al., 2015; Desoutter et al., 2023; Slimani et al., 2017). Likewise, certain secondary structures could be studied or presented by Raman microscopy such as tufts and spindles (Desoutter et al., 2021a) or daily cross striation of enamel [28]. In these studies, Raman scattering was used to accumulate thousands of spectra in a region of interest, and use certain peaks in the spectrum, as 960 cm⁻¹/PO₄³⁻ to reconstruct images where each point represents an intensity of selected peak. See supplementary table 1 for summary for

previous works on sound human teeth with Raman microscope.

In the present study, we used confocal Raman microscopy to generate representative chemical mapping of all the structures of a healthy human dental organ, including the three mineralized components (dentin, enamel and cementum), the different junctions, and the secondary structures (enamel drapes, spindles, Hunter-Schreger bands, striae of Retzius, dentin branches), as well as the dental pulp. The samples come from permanent adult teeth and teeth from children wether deciduous or permanent are not included. Images of tooth structures and microstructures based on the variation in Raman mode's intensity corresponding to certain molecular bonds, such as phosphate or CH bonds were realized. The presented results reveal a comprehensive and homogeneous analysis (with the same wavelength and intensity), for the study of different part of tooth. This study represents an advanced step in the understanding of both the chemical properties of this organ and the structural arrangements between the different materials that compose it. Although the present study is limited by the absence of aged and decidual teeth, and therefore age-related pathologies have not been investigated, the results presented provide a reference for chemical mapping, under controlled conditions (laser wavelength, resolution...) of all dental structures at microscopic level. Moreover, variations of chemical composition in structures could exist between individuals, as well as between tooth types and intra-dental positions.

## 2. Material and methods

### 2.1. Sample preparation

In this study, samples destined to the study of hard tissues were prepared without any fixation, embedding or decalcifying solutions, to not affect result due to changes of chemical composition of diffusing volume. However, these processes cannot be avoided to study soft tissue as dental pulp. A recent study proves that for Raman spectroscopy of cells from the periodontum, formaldehyde and methanol derivatives, used in our study, are a viable option. Besides, after the use of each chemical product, a thorough washing protocol is carried out to eliminate the remains of these solutions, which reduce the presence of chemicals inside the analysed tissue

### 2.2. Dental hard tissue

Human incisor, canine, pre moral and molar teeth (5 each), extracted for orthodontic reasons with patient consent - process No. 2014-2198 - local ethical research committee "Montpellier Sud-Méditerranée IV", were collected and stored in distilled water. All donors were between 32 and 47 years old. Each tooth was sectioned longitudinally using a diamond saw (Isomet 2000, Buehler, USA). The slices thicknesses were 0.5 mm. All slices were polished both side, with an abrasive disc from 1200 and 2400 grit and a velvet disc with diamond pastes of 0.25 and 0.1 micrometre particle sizes to smooth the surface, using a polishing table (Escil, France). Then, samples were cleaned with an ultrasonic bath to remove particles and polishing past, two times 180 seconds and three times rinced with distilled water. Samples were keeping hydrated in 4 degrees frigo.

### 2.3. Dental pulp

The human dental pulp tissue was isolated from extracted wisdom teeth from young healthy patients (from 23 to 40 years old). Informed consent was obtained from the patients after receiving approval by the local ethics committee (Comité de protection des Personnes Sud Méditerranée IV, Centre Hospitalier de Montpellier). We used a previously established protocol to recover pulp cells (Collart-Dutilleul et al., 2015). Briefly, the teeth were cleaned with 2 % chlorhexidine, then the teeth were broken into two pieces in the Cement-Enamel junction. An initial
cut was made with a diamond disc on a rotary handpiece without touching the pulp, then the parts were separated with a scalpel. Finally, the pulp tissue was recovered from its cavity by using a tweezer. The pulp tissue was included in histogel (Epredia, Thermo Fisher Scientific, Massachusetts, USA) and fixed in 4% Paraformaldehyde (PFA) solution overnight. The fixed sample were then cut longitudinally using a vibratome Vt1200, (Leica, Welzlar, Germany) with a thickness of 80 µm and kept in Dulbecco's phosphate buffered saline (DPBS, Sigma-Aldrich, Saint Louis, USA) until the scans.

Human healthy and freshly extracted premolars for ortodontic reasons were obtained from patients after giving informed consent approved by the local ethics committee (Comité de protection des Personnes Sud Mediterrannée IV, Centre Hospitalier de Montpellier). Teeth were fixed in Paraformaldehyde 4 % during 48 h. Later samples were rinsed in DPBS and placed in a rapid decalcifying solution (Shandon TBD-2 Decalcifer, 12607926, Fisher Scientific, Pittsburgh, USA), around 20 times the volume of the sample. The decalcifying product was renewed every day during 4 days. The samples were then rinsed with DPBS, treated in an impregnation automaton Leica ASP300 and included in paraffin in a MYR EC350 paraffin embedding station. The paraffin blocks were cut with a Microm HM340E microtome with Niagara system to obtain 3 µm thick sections that were placed in coated glass slides (Superfrost plus, Thermo Fisher Scientific, Massachusetts, USA).The samples were then dewaxed with xylene, rehydrated with ethanol baths in decreasing concentrations and conserved under immersion in DPBS.

## 2.4. Raman scattering

Raman scattering phenomenon occurs when a monochromatic electromagnetic wave interacts with a molecule. This interaction induces a dipole moment within the molecule, influenced by its electronic structure and orientation relative to the polarization of the light. The molecule's polarizability, a measure of how its electronic cloud deforms under the electromagnetic field, plays a central role in this process. Vibrational movements of the molecule cause variations in polarizability, leading to different frequency components in the scattered light. These components include, Rayleigh Scattering, with the same frequency as the incident light, Stokes and Anti-Stokes Scattering where the scattered light loses and wins energy, resulting in a lower and higher frequency, respectively. The Stokes and Anti-Stokes shifts reflect the molecular vibrations and provide insight into the molecule's structural and vibrational properties. This is the basis of Raman spectroscopy, a technique used to analyze molecular composition and behavior.

## 2.5. Confocal Raman microscope

Alpha300R confocal Raman Microscopy (Witec, Ulm, Germany) has been used for single spectra recording and scanning area. The laser wavelength is 532 nm, produced by a frequency doubled Nd:YAG laser (Newport, Evry, France). Polarized light from the laser is transported to the microscope via optical fiber. The microscope is equipped with an air objective × 20 with numerical aperture (NA) of 0.46 (Nikon, Otawara, Japan). Spatial resolution is -using the formula $0.61λ/NA$- around 700 nm for the x20 objective. Spot size is 1.3 micrometer radius. Power at the focal plane is 10 mW, so power density is $7.5 mW/µm^2$. This, for all the samples expect for cementum, where power is 4 mW and power density 3 mW/µm$^2$.

Slices of teeth were placed on the piezoelectric table. In scanning mode, integration time of a single spectrum was 0.2–0.5 second (depend on number of points) and 2 seconds for single spectrum, with an average of 5 accumulated spectra. Sometimes, integration time of scan was longer, to see more clearly variation of weak peak, as shown in results section. Image size was determined by the region of interest (ROI) chosen by the user. In this study, image size is approximatively a square of 100 µm side. The signal is collected on the electron charge coupled device (EMCCD) camera (DU 970N-BV353, Andor, Hartford, USA),

equipped with a 1600 × 200 pixel chip. UHTS 300 spectroscopy system with a 70 % throughput transmission and a grating of 600 lines per mm provides a spectral resolution of 3–5 cm$^{-1}$. System control is performed with Witec control 1.5. Data acquisition was performed using Image Plus 5.01 software from Witec.

## 2.6. Spectra analysis

For the analysis of a single spectrum, the Spectragraph software v1.2.16 (Friedrich Menges, Germany) was used. Two treatments were carried out on the spectra to present them correctly: subtraction of the background and normalization by the highest intensity peak. For hard tissues, the peak of highest intensity is always that corresponding to the phosphate bond, PO$^3$.

## 2.7. Reconstruction of chemical mapping

The interest of confocal Raman microscopy lies in the possibility of recording a large number of scans over a defined area, each point corresponding to a spectrum. From there, we can reconstruct the image of the intensity of a certain chemical bond, depending on what we are trying to observe. However, it turns out that the most intense peak is that of the phosphate or more precisely of the $v_1$ PO$_4$ stretching mode. It is therefore this peak which will be favored for the structures of tooth, whose composition is essentially composed of HAP. PO$_4$$^3-$ is the peak that the signal-to-noise ratio is best.

To make a filter can be applied to a portion of the spectral range: here, at the limits of the phosphate peak which is located at $960 cm^{-1}$. "Sum filter" integrate the space occupied by the peak. Thus, each point will be transformed into a value, and this value, by application of a color table (look up table), into hue. The choice of look up table was guide by the dynamics of peak values in chemical cartography and structures represented. Thus, we will have an "image" which will reflect the intensity of the phosphate peak, the patterns of which we will be able to appreciate, and see whether or not they correspond to this something and allow information to be drawn from it. We can see the different stages of reconstructing an image from the selection of a peak of interest in Fig. 2-C. Binarization and analyze of some images was performed with Fiji software (v1.51, National Institutes of Health, Washington, USA).

# 3. Results and discussion

As will be presented in this section, we first succeeded in recording representative spectra in all the constituent elements of the tooth: enamel, dentin, cementum, but also the dental pulp.

Concerning enamel, we must distinguish between interior enamel, inaccessible without cutting the tooth, and surface enamel. Indeed, as we can see in Fig. 2, the shape of the peaks whose peaks are located at 1071 and 1103 $cm^{-1}$ are not identical, and represent a biomarker of the interior enamel/surface enamel distinction, as previously reported (Ko et al., 2005).

Likewise, all the materials constituting the tooth could be scanned and give rise to reconstructions. The variability of biological samples sometimes required numerous acquisitions before being able to present maps allowing the structures of interest to be visualized. Also, the power of the laser sometimes has to be adapted to the material. Indeed, if enamel is very resistant, cementum or dentin are materials which can heat up during the scan and distort the results or destroy part of the surface layer of the sample. Each scanned area has its particularities, analyzes are presented here based on the relevant elements for each structure analyzed. Du to the geometry of laser beam focalized on the sample, the depth of surface probed is around 2/3 micrometers.

## 3.1. Representative spectra

In biological samples, spectra have significant variability depending
<figure><img src="image_4.png" /><figcaption>Fig. 1. Scheme of tooth structures.</figcaption></figure>

on location. Indeed, samples are rarely homogeneous, including in dental enamel. Also, the spectra presented in Fig. 3 are only indicative. In particular, the ratio between the intensity of the different peaks can vary greatly. Thus an enamel spectrum recorded at the level of the dentin enamel junction will have different ratios, and even sometimes different peaks from a spectrum recorded below the surface of the enamel. Also, as indicated, they were normalized to clearly distinguish each peak. Assignment of Raman peak for each material was done with previous measurement published in literature. Due to the spectral resolution of spectroscope (around more than 3 cm⁻¹), there may be variations in the corresponding wave numbers between published studies (Matousek and Morris, 2010; Timchenko et al., 2021; Tsuda et al., 1996b) (see Table 1).

In this table are presented the frequency shifts in Raman spectroscopy, which can be related to molecular vibrational modes. However, another relevant information is the intensity of peaks, which is related to the concentration of the compound in the diffusing volume (Favors et al., 2005). Since the absolute intensity depends on the sampling conditions and the measuring instrument (illumination intensity, alignment, temperature, etc.), it is useful to normalize the peaks using the v1 phosphate mode intensity in order to compare peak ratios between them, giving an indication of the change in the relative composition of the diffusing volume. The normalization results are presented in Supplementary Table 2.

These results must be taken with caution due to sensibility of Raman spectra. Indeed, the spectrum obtained by Raman scattering is sensitive to several factors, as explained above. In particular, the angle formed between the orientation of the hydroxyapatite mesh and the polarization

of the laser can significantly influence the intensity of the peaks (Tsuda & Arends, 1994). Thus, the OH peak, located at 3580 cm⁻¹, is particularly dependent on the polarization of the laser, and can be almost non-existent if the orientation of the c axis of the HAP mesh is perpendicular to the polarization of the laser.

The Raman characteristic of the materials constituting the tooth, in terms of a unique characteristic and representative spectrum, has already been reported. The wave number for the same chemical signature can however vary from one experiment to another, or from one publication to another. Among the phenomena that can be studied, one comes from the instrumentation: it is sometimes necessary to check that the Rayleigh peak corresponds to zero on the abscissa. If this is not the case, a correction must be applied to the frequency shift of the analyzed photos.

The spectra presented in Fig. 3 have all already been listed (Tsuda et al., 1996a), with known peaks and their relationship with chemical modes. Thus, it is possible to then reconstruct the different intensity maps for the most characteristic connections. It should be noted that not all spectra are recorded under the same conditions. Indeed, cementum, for example, has a spectrum with a lower signal-to-noise ratio than enamel or dentin, which explains its noisier appearance.

The spectra presented are representative. However, relative intensity ratios may vary. Indeed, each scanned area has its own characteristics, and each point of the scan can see the concentrations of chemical constituents vary.

For the following images we use some of the peaks presented above to calculate images whose hues represent intensity levels. We choose images that are representative of the studied area and we briefly discuss
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Microscopy based on Raman scattering. A) Energy diagram of Rayleigh, Stokes and anti-Stokes scattering; B) Scheme of Raman spectra; C) different step of chemical mapping of a surface of interest, by selection of peak intensity.</figcaption></figure>

their significance. It should be pointed out that each of the structures described is common to all teeth, maxillary and mandibular, although there may be variations between each type of tooth. To understand what is new in present results, and what is just a confirmation of previous work, see Table 3.

To better compare the studied structures with previous findings from conventional histology staining, scanning electron microscopy (SEM), or other imaging techniques used to examine dental structures, see Supplementary Table 3. These reference images provide helpful context for interpreting the mappings created with confocal Raman microscopy.

## 3.2. Enamel prism

Fig. 4 shows reconstructed longitudinal sections of tooth enamel, in which the prisms are clearly identified.

Moreover, we highlight the presence of organic residue in the zone corresponding to the enamel sheath is shown in Fig. 4A. This image is the first chemical cartography revealed the presence such presence of a CH peak for those recorded at the junction between two prisms. The relatively long acquisition time of each spectrum (1.5 seconds in this case). This about 1 µm zone separates enamel prism. On this image interprismatic enamel is not visible. The use of the peak corresponding to the bonding of the phosphate PO₄³⁻ provides very good contrast (Fig. 4B). In this zone, enamel rods are around 5 µm width. Fig. 4C shows the combination of intensities of interest (mineral and organic) from Figs. 4A and 4B. Representative spectra for each of the zones prismatic and interprismatic zones are shown in Fig. 4D.

Note also that the periodic striations are not systematically observed, even with long integration times. This indicates that depending on the cutting plane these daily variations in the HAP level are visible or not. In Fig. 4, these variations are barely visible. Note also that as in all the images, the presence of the OH peak at 3580 cm⁻¹ is very strongly dependent on the angle formed between the c axis of the enamel prism and the polarization of the laser, as was demonstrated (Tsuda & Arends, 1994). The polarization of the laser used is horizontal. In this scan, prism are also horizontally oriented. This explains the presence of the OH

peak, clearly visible (Fig. 4D).

## 3.3. Dentine

Longitudinal and cross-sectional reconstruction of dentin is presented. Cartography reconstructed are the first with optical resolution on sound dentin; this allows to measure the diameters of the canaliculi, and the accumulation of spectra allows to observe the chemical composition of dentin and its variations. It is possible to measure width of canaliculi and their density in dentine.

The image in Fig. 5C, reconstructed from the intensity of peak 960 (PO₄³⁻), shows an increased concentration of HAP around tubuli.

Although there are many studies using Raman spectroscopy, there is little description of dentin from a histological point of view by Raman microscopy. In Fig. 5, we can visualize the difference between a longitudinal section, along the dentin tubules, and a transverse section, perpendicular to the tubules. We thus see that the concentration of HAP is not homogeneous outside the tubule. Indeed, the peritubular zone has a greater intensity of the phosphate peak. Conversely, the pixels corresponding to the intensity of the organic peak (2800 cm⁻¹) visible in Fig. 5D are weak in the area close to the tubule, giving the impression of enlargement of the diameter of these structures. This accumulation of HAP and the lower level of organic matter in the peritubular zone is compatible with the results of mechanical tests, by AFM or by nanoindentation, showing significantly greater precision of the peritubular zone. In a study published in 2010 it was shown a hardness of 4.7 (SD 0.8) GPa in the peritubular zone while measurements carried out in the intertubular material showed a hardness of only 1.8 (SD 0.4) GPa (Cohen et al., 2008). It is therefore consistent with these results to note a higher HAP rate around the day of the tubuli.

## 3.4. Surface enamel

To scan surface enamel, the tooth must be locally relatively flat, parallel to the focal plane, to allow the scattering volume of the laser to contain the same quantity of material, in orders of magnitude of similar
<figure><img src="image_6.png" /><figcaption>Fig. 3. A: Representative mean Raman spectrum of dentin, enamel, surface enamel and cementum. B: Raman spectra of coronal and radicular. Each spectra is mean of 10 spectra: 2 spectra from 5 samples, each spectra represent mean of 5 spectra integrated during 2 seconds, collected in representative zone of each sample type.</figcaption></figure>

level. Fig. 6 shows reveal such surface enamel with Raman microscopy. As presented above, surface enamel has a particular spectrum, with a shift in peaks located between 1007 and 1103 cm⁻¹ (see Table 1). As shown in Supplementary Figure 1, the characteristic spectra of internal enamel and surface enamel are clearly distinguishable due to difference in this region. This makes it possible to distinguish the two types of email.

The peak of the phosphate is similar to the interior enamel, and allows for good image color dynamics, as shown in Fig. 6A. It makes possible to obtain good contrast dynamics and to be able to observe structures such as enamel prisms or perikymata. The enamel prisms are visible and it is also possible to evaluate their density and characteristic size. Likewise, in all the scans carried out on the surface of the enamel, we were able to note the presence of peaks which do not correspond to HAP. This is the case of a peak at 2800–3000 cm⁻¹ (CH bond), characteristic of organic elements which could be nutrient residues inserted in the interstices, as shown in Fig. 6B.

Prisms perpendicular to surface are not always visible, and depend

on the surface condition. When prism is not visible there is a layer of aprismatic enamel. The arrangement of prism at the surface is showing the alignment of ameloblast during amelogenesis. SEM imaging has already shown this type of structure on the surface (Kodaka et al., 1991). These images also make it possible to measure the size of the enamel prisms and their surface density. The peak located at 2800 cm⁻¹ corresponds to organic elements which could be accommodated, like food or biofilm. Likewise, the image presented in Fig. 6B presents the surface structures, the perikymata, which are linked to the variation in ameloblast secretions. Indeed, this rhythm of secretion produces the striae of Retzius in the enamel, and the perikymata on the surface and their number and periodicity can be a marker of the duration of enamel formation (McFarlane et al., 2014). Raman microscopy allows analyze surface organization and chemical composition nondestructively.

### 3.5. Dentine enamel junction

The junction between dentin and enamel is shown in Fig. 7 and
<caption>**Table 1**</caption>

Chemical bond and vibrational mode, correlated to Raman shift (in cm⁻¹) of dental hard tissues. Bold characters indicate modes used in this study to produce images.

<table><thead><tr><th></th><th>Surface<br>enamel<br>(cm<sup>-1</sup>)</th><th>Inner<br>enamel<br>(cm<sup>-1</sup>)</th><th>Dentine<br>(cm<sup>-1</sup>)</th><th>Cementum<br>(cm<sup>-1</sup>)</th></tr></thead><tbody><tr><td>ν2 phosphate (PO<sub>4</sub>3-)</td><td>433</td><td>433</td><td>433</td><td>433</td></tr><tr><td>ν4 phosphate (PO<sub>4</sub>3-)</td><td>448</td><td>448</td><td>448</td><td>448</td></tr><tr><td></td><td>584</td><td>593</td><td>583</td><td>583</td></tr><tr><td></td><td>613</td><td>613</td><td>593</td><td>593</td></tr><tr><td>ν1 phosphate (PO<sub>4</sub>3-)</td><td>960</td><td>960</td><td>960</td><td>960</td></tr><tr><td>Collagen (Phe)</td><td>-</td><td>-</td><td>1007</td><td>1007</td></tr><tr><td>ν3 phosphate (PO<sub>4</sub>3-)</td><td>1027</td><td>1027</td><td>1047</td><td>1047</td></tr><tr><td></td><td>1056</td><td>1047</td><td></td><td></td></tr><tr><td>B type v1 Carbonate<br>(CO32-)</td><td>1071</td><td>1071</td><td>1071</td><td>1071</td></tr><tr><td>A type v2 Carbonate<br>(CO32-)</td><td>1104</td><td>1107</td><td>1107</td><td>1107</td></tr><tr><td>(NH) Amide III</td><td>-</td><td>-</td><td>1260</td><td>1260</td></tr><tr><td>(NH) Amide III non-<br>polar triple helix of<br>collagen</td><td>-</td><td>-</td><td>1275</td><td>1275</td></tr><tr><td>CH2 (wagging)</td><td>-</td><td>-</td><td>1460</td><td>1460</td></tr><tr><td>Advanced glycation end<br>products (AGEs)-<br>Pentosidine</td><td>-</td><td>-</td><td>1550</td><td>1550</td></tr><tr><td>Amide I (C=O)</td><td>-</td><td>-</td><td>1665</td><td>1665</td></tr><tr><td>Organic peaks -<br>protein groups: C-H,<br>CH2</td><td>-</td><td>-</td><td>2800/<br>3000</td><td>2800/<br>3000</td></tr><tr><td>OH</td><td>3580</td><td>3580</td><td>3580</td><td>3580</td></tr></tbody></table>

confirms previous study about this zone. The junction is represented by two distinct materials, and 4 identifiable zones: tubular dentin, the dentin coat, the junction and finally the prismatic enamel. The junction is characterized by a transition between dentin and enamel. Fig. 7A represents the variation in intensity of the CH peak located between 2800 and 3000 cm⁻¹. The high intensity in yellow corresponds to dentin, while black corresponds to low intensity, or even zero intensity. Thus, we see that the intensity of the CH peak does not decrease suddenly, but on the contrary, gradually fades over around ten micrometers in the enamel, above the dentin/enamel separation.

This correlates with Figs. 7B and 7D. Fig. 7B represents the variation in intensity of the peak of the phosphate PO₄³⁻, and we see an inverse gradient in the junction zone: a low relative intensity in the dentin, which however mainly comprises HAP, and a strong intensity in the enamel, with a gradual increase over an area of 10 micrometers. Thus, the intensity profiles presented in Fig. 7D along the green line in Figs. 7A and 7B represent this translation zone.

The power of Raman makes it possible to compare the dynamics of various chemical bonds with high sensitivity. Thus, the DEJ is defined as a transition zone in which the HAP level increases from dentin to prismatic enamel, while the organic phase, measured with the CH peak at 2800 cm⁻¹, has an opposite dynamic: the rate of loss of dentin towards enamel. An example of such dynamic is illustrated by the intensity profiles in Fig. 7D.

### 3.6. Dentin cementum junction

Dentin and cementum have a similar chemical composition, which is reflected in the spectra shown in Fig. 2. The reconstruction of the intensities of the peaks of the phosphate PO₄³⁻ at 960 cm⁻¹ and CH at 2800/3000 cm⁻¹ of the dentin-cementum junction are shown respectively in Figs. 8A and 8B. This is the first image of dentine cementum

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Enamel prism Raman images. A) 2800–3000 cm⁻¹ peaks intensity corresponds to organic phase (CH, CH₂, CH₃) reconstructed image. B) 960 cm⁻¹ ν₁ PO₄³⁻ stretching mode intensity peaks reconstructed image. C) 2800–3000 cm⁻¹ peaks intensity corresponds to organic phase (CH, CH₂, CH₃) and 960 cm⁻¹ phosphate Raman peak intensity image combined; red: high intensity of phosphate; blue: high intensity of interprismatic material. D) representative singles spectra in enamel prism and interprismatic material.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Human dentine. A and C) $960 \text{ cm}^{-1}$ $v_1 \text{PO}_4^{3-}$ stretching mode intensity peaks reconstructed image B) $2800–3000 \text{ cm}^{-1}$ organic bounds (CH, CH$_2$, CH$_3$) intensity peaks reconstructed image C) dentine transverval section 960 D) image of ratio between 960 and 2800 modes. White arrows: tubuli branches. Black arrow: increased concentration of phosphate around tubuli.</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Two different surface crown teeth: with and without perikymata. A) $960 \text{ cm}^{-1}$ $v_1 \text{PO}_4^{3-}$ stretching mode reconstructed image; B) $2800–3000 \text{ cm}^{-1}$ peak intensity reconstructed image correspond to organic material (CH, CH$_2$, CH$_3$).</figcaption></figure>

junction with Raman microscopy. The intensity profiles along the green line are shown in Figure D, where it is possible to see two junctions represented by a drop in intensity of phosphate and CH. The intensity variation corresponding to the phenylalanine peak at $1110 \text{ cm}^{-1}$ is shown in Fig. 8C. The intensity collapse zones of the phosphate and CH peaks correspond to intensity maxima of the $1110 \text{ cm}^{-1}$ phenylalanine peaks. Three parts are visible: dentine, acellular cementum and cellular cementum, from left to right. Cementum is a term covering three variants of the tissue surrounding the dental roots up to the neck. The cementum can then be attached to the enamel, or partially cover it by a

few tens or hundreds of micrometers, or even leave a space.

Cementum can be acellular, or on the contrary cellular. The latter type contains traces of osteocement. We thus distinguish acellular cementum with extrinsic fiber (AEFC), cellular cementum with intrinsic fiber (CIFC) and mixed stratified cellular cementum (CMSC). Finally, cementum is the attachment site of the ligament, the more external part is the Sharpey's fibers insertion site (blue arrows in Fig. 8A).

Concerning the composition, cementum is highly mineralized, containing mainly HAP. It is structured in layers, which in some cases, can reflect annual apposition cycles. Several studies have attempted to study
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Human dentine enamel junction Raman images. A) 2800–3000 cm⁻¹ (CH, CH₂, CH₃) intensity peaks reconstructed image. B) 960 cm⁻¹ v₁ PO₄³⁻ stretching mode intensity peaks reconstructed image. C) CH and phosphate Raman peak intensity image combined; red: high intensity of phosphate; blue: high intensity of interprismatic material. D) v₁ symmetric stretching phosphate mode and 2800–3000 cm⁻¹ CH intensity profile plotted along green line in A; De: dentine; En: enamel; DEJ: dentin enamel junction.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Dentin cementum junction. A) 960 cm⁻¹ v₁ phosphate stretching mode intensity peaks reconstructed image. B) 2800–3000 cm⁻¹ organic bounds (CH, CH₂, CH₃) intensity peaks reconstructed image C) 1007 phenylalanine. Black arrow: cementocyte. Blue arrows: Sharpey's fibers insertion site. De: dentine; GIT: granular layer of Tomes; Ac: acellular cementum; Cc: cellular cementum.</figcaption></figure>
these cementum layers to estimate the age at tooth extraction, or the age of death in the case of a tooth removed after a death (Pinto et al., 2022). The structure and content of cementum make it a material that is more difficult to scan in Raman microscopy. Indeed, it is necessary to reduce the intensity of the laser or to reduce the integration time, to avoid the phenomena of combustion of the surface of the sample, as explained above. Also, during the experiments, it was noted on several occasions areas that had undergone destruction by heating (dark characteristic area). Although the spectra are, as a result, noisier, it is quite possible to reconstruct chemical maps, including to perceive the different types of cementum. Indeed, chemically, acellular and cellular cementum are very close, as can be seen in Fig. 8. However, we can clearly visualize the marks of cementocytes, as well as the insertion sites of Sharpey's fibers. The intensity profiles of the phosphate (PO₄³⁻) peaks at 960 cm⁻¹ and CH at 2800–3000 cm⁻¹, illustrated in Fig. 8D, show a strong correlation between the mineralization rates and organic elements such as lipids and proteins. Also, it can be seen that hypomineralized areas, particularly at the junctions between the dentin and the cementum layer, can

have a chemical signature revealing traces of phenylalanine, which may reasonably suggest the presence of collagen in the spaces left vacant. This collagen may be the result of the presence of fibrils from neighboring connective tissue, rich in collagen I and III.

### 3.7. Dental pulp

Fig. 9 shows for the first time two pulp chemical maps based on Raman scattering, radicular and coronal. Dental pulp tissue is an organized soft tissue placed in the empty space in the middle of the tooth. This tissue is well organized, composed by a fibrous extracellular matrix containing cells, blood vessels, nerves and lymphatic elements. The histology of the dental pulp corresponds to a loose connective tissue containing the components common to all connective tissues but with a great variability depending on the area studied, whether at the root or crown level, the marginal zone, the Weill layer or the central zone, but also due to the high variety of histological components: blood vessels, nerves, connective fibers, mucopolysaccharides, odontoblasts,

<figure><img src="image_12.png" /><figcaption>Fig. 9. Dental pulp. Up: dentine pulp junction. Middle: coronal. Down: radicular. A) lipids/proteins 2854 cm⁻¹ Raman peak intensity image of dentine pulp junction. B) proteins 2930 cm⁻¹ Raman peak intensity image of dentine pulp junction. C) CH 2800–3000 cm⁻¹ Raman peak intensity image of coronal dental pulp. D) methyl/phospholipids/lipids 1450 cm⁻¹ Raman peak intensity image of coronal dental pulp. E) CH 2800–3000 cm⁻¹ Raman peak intensity image of radicular dental pulp. F) methyl/phospholipids/lipids 1450 cm⁻¹ Raman peak intensity image of radicular dental pulp.</figcaption></figure>
fibroblasts, Höhl cells, type I and III collagen, etc (Nanci, 2013).

Besides histology in human dental pulp could presents variations between coronal and radicular level due to anatomical contouring of hard tissue that exerts mechanical signs affecting the organization of soft tissue. As a result, the recorded spectra in coronal or radicular portion are both quite variable, but also include many peaks, as shown in Fig. 3 and Table 2. The results show that pulp tissue is organized in accordance with its known histology (Nanci, 2017).

In the peripheral part of the dental pulp, the cellular layer of odontoblast adjacent to dentin could be clearly observed in Fig. 9 (A-B). Below this odontogenic layer it could be observed the zone of Weil or cell-free zone. The architecture of this region is determined by the extracellular matrix composed mainly by reticular fibers (Korff's fibers), organized in bundles oriented parallel to the root long axis and passing from the central area. It could be observed on the images obtained by chemical mapping corresponding to matrix, an organization comprising globular clusters of around 10 micrometers, with a high concentration of CH bonds corresponding to groupings of cells, a collagenous matrix. This zone also is known by containing numerous capillaries and nerves, in accordance with the image of the coronary pulp, where it could be seen a space of low intensity density of all the peaks, suggesting the location of a nerve or a blood vessel oriented longitudinally (Fig. 9 C-D). Beneath the cell-free zone is the cell-rich zone observed in Fig. 9 (E-F). This region contains fibroblasts capable of degrading and synthesizing collagen and also undifferentiated mesenchymal cells that can differentiate into odontoblasts, fibroblasts, or macrophages.

### 3.8. Hunter-Schreger bands (HSB)

Fig. 10 presents a result obtained in an area corresponding to a HSB. This area was chosen in white light, by the appearance of a characteristic light band. The reconstruction with the $v_1$ stretching mode phosphate peak located at $960 cm^{-1}$ makes it possible to observe the density of the enamel sheaths involved in the phenomenon, as well as the orientation, as can be seen in Fig. 10A. Also, by thresholding the maps in an identical manner to obtain binary images, it is possible to measure the relationship between the center of the sheath and the interprismatic material. Thus, we find here a lower occupancy rate for the prisms occupying the dark part (41.3 %) compared to the prisms in the light areas (54.5 %).

HSBs result from variations in the orientation of prism groups in dental enamel. These variations can be visualized in white light, with the prism groups forming alternating light and dark bands. In Raman microscopy, the dynamics of the intensity of the phosphate images clearly show the orientation of the prisms, particularly the groups oriented parallel to the cutting plane of the sample, as seen in Fig. 10. In this figure, we can also see the intensity variations along the prisms, resulting from the circadian activity of the ameloblasts. Using the

<caption>**Table 2**</caption>

Chemical bond and vibrational mode, correlated to Raman shift (in cm-1) of dental soft tissue (dental pulp). Bold characters indicate modes used in this study to produce dental pulp images.

<table><caption>**Table 3**</caption><thead><tr><th>Chemical bond</th><th>Raman shift (cm-1)</th><th>Description</th></tr></thead><tbody><tr><td>Cytosine</td><td>690</td><td>Cytosine</td></tr><tr><td>C-C</td><td>824</td><td>Collagen backbone</td></tr><tr><td>C-O-H ring</td><td>860</td><td>Glycogen</td></tr><tr><td>P-O</td><td>950</td><td>DNA backbone</td></tr><tr><td>Phenyl group</td><td>1002</td><td>Phenylalanine</td></tr><tr><td>C-H in-plane bending vibrations</td><td>1175</td><td>Phe and Tyr</td></tr><tr><td>CH and NH</td><td>1260</td><td>Amide III, peptide backbone</td></tr><tr><td>CH2 and CH3</td><td>1450</td><td>Methyl and methylene, peptide side chains, phospholipids, lipids</td></tr><tr><td>C=O</td><td>1650</td><td>Amide I</td></tr><tr><td>C-H</td><td>2800-3000</td><td>CH</td></tr></tbody></table>

Summary table of results.

<table><thead><tr><th>Structure</th><th>Previous results confirmed</th><th>New results</th></tr></thead><tbody><tr><td>Enamel Prism</td><td>Enamel prisms were identified with high phosphate concentration (Pezzotti et al., 2017)</td><td>Separation between prism by lamellae containing organic residues</td></tr><tr><td>Dentine</td><td>Reconstructions showed tubules size, orientation and density. Chemical variations and structural details (Desoutter et al., 2022)</td><td>Chemical cartography on sound tissue with optical resolution</td></tr><tr><td>Surface Enamel</td><td></td><td>Surface scans revealed pattern of surface prisms and perikymata. Organic residues were identified, possibly from biofilms or nutrients.</td></tr><tr><td>Dentine-Enamel Junction (DEJ)</td><td>A gradual transition zone with increasing HAP concentration from dentin to enamel was mapped as previously published (Desoutter et al., 2022)</td><td></td></tr><tr><td>Dentine-Cementum Junction</td><td></td><td>Variations in phosphate and organic bonds revealed distinct zones, including dentine, acellular, and cellular cementum. Sharpey's fibers insertion sites were also mapped.</td></tr><tr><td>Dental Pulp</td><td></td><td>Chemical maps of coronal and radicular pulp highlighted variations in lipid and protein content, reflecting tissue organization and presence of collagen.</td></tr><tr><td>Hunter-Schreger Bands (HSBs)</td><td></td><td>The alternating color bands reflect the combination of variations in enamel prism orientation and PAH concentration.</td></tr><tr><td>Striae of Retzius and Lamination</td><td></td><td>Periodic variations in HAP concentration reveal striae connected to the rhythmic activity of ameloblasts secretion during enamel formation.</td></tr><tr><td>Enamel Cross Striation</td><td>Variation in phosphate intensity concentration highlights periodic changes in HAP linked to circadian secretion patterns of ameloblasts (Desoutter et al., 2021a)</td><td></td></tr><tr><td>Scallop Patterns at DEJ</td><td></td><td>Large and small scallops with organic material penetration from dentine in enamel</td></tr><tr><td>Enamel Spindles</td><td>Hypomineralized spindle structures extending from dentin tubules were mapped (Desoutter et al., 2021a)</td><td></td></tr><tr><td>Enamel Tufts</td><td>Weakly mineralized structures (Desoutter et al., 2021a)</td><td>Orientation of tufts along enamel prism groups</td></tr><tr><td>Dentine Branches</td><td></td><td>A network of tubule branches within dentin was characterized, with variations in angles and spacing.</td></tr><tr><td>Interglobular Dentin (IGD)</td><td></td><td>Cartography of mineralization defects with organic material residues were observed.</td></tr></tbody></table>
<figure><img src="image_13.png" /><figcaption>**Fig. 10.** HSB diazones parazones. A) $960 \text{ cm}^{-1}$ $v_1 \text{PO}_4^3-$ stretching mode intensity peaks reconstructed image. B) up: grey shade image of the two characterized zone of HSB, cropped from A; down: binarized images, same zone.</figcaption></figure>

binarization of the phosphate peak intensity image, we can clearly see the orientation of the prisms. Since only the orientation varies, it is obvious that it is this parameter that absorbs or scatters the light. This variation demonstrates the strong anisotropy of dental enamel. It is also noteworthy that the sensitivity of the OH peak intensity ($3850 \text{ cm}^{-1}$) to the relative orientation between the laser polarization and the c axis of the HAP mesh allows the reconstruction of the HSB (images not shown).

## 3.9. Striae of Retzius and lamination

Fig. 11 shows the Retzius striations obtained by scanning the enamel outside the aprismatic enamel zone above the enamel-dentin junction. The relative variations in intensity of the $\text{PO}_4^3-$ phosphate peak in certain areas are characteristic, and quantifiable by an intensity profile as shown in Fig. 11B. In the scanned area, the distance between two dark areas measures between 20 and 30 µm, and secondary lines of intensity variations are spaced of 7/8 µm.

Lamination and Retzius striae are only observed in cross-sections of teeth, and form parallel lines between them, spaced variably from one to the other, this has been correctly documented by histology works (Nanci, 2013) although recent literature continues to discuss the biorhythm significance of these structures (McFarlane et al., 2021). One of these lines is particularly studied: the neonatal line, which corresponds to the depression of the enamel at the time of birth, and corresponding to a particularly marked striae of Retzius (Weber & Eisenmann, 1971). The Raman results are well correlated with what is known about Retzius streaks in classical histology. The variations in the intensity profile make it possible to precisely measure its rhythmicity. Indeed, in chemical mapping, a variation in intensity is observed, which suggests that in addition to the circadian variation in the secretion of enamel prism material, the secretion activity experiences other longer-period cycles, with phases of lower mineralization, visible by the

distances between two grooves in the intensity profile.

It is thus possible to measure the number of days which correspond to a decrease in the secretory activity of ameloblasts, or on the contrary, the number of days which correspond to a strong secretory activity.

## 3.10. Enamel cross striation

The variations in intensity of the $v_1$ symmetric stretching Raman mode of the phosphate, seen in the image in Fig. 11, are clearly identifiable at higher magnification in Fig. 12. The prims appeared as rods of highly mineralized nodules. Those nodules are periodically spaced with around 4/5 µm along the prism longitudinal axis. They form the so-called cross striation. The distance between adjacent enamel prims is round 6 µm. The intensity profiles presented in Fig. 12B corresponding to the two green lines in Fig. 12A allow comparing phosphate intensity variation. As well demonstrated in Fig. 12, chemical mapping of enamel, which allows visualizing variations in HAP concentration along an enamel prism, is a powerful tool for measuring the spatial frequencies of cross striations (illustrated here by relatively spherical formations), but also for observing the alignment of these structures perpendicular to the orientation of the enamel prism. Raman microscopy has an advantage here over techniques that only visualize the surface of the sample. Indeed, probing the sample in a volume diffusing over a few micrometers of depth, by recording the intensity of the peak corresponding to phosphate, makes it possible to distinguish variations in the concentration of the material, which electron microscopy fails to illustrate (Desoutter et al., 2019). The cross striation of enamel is correlated with the circadian regulation of ameloblasts with variation expression transcription factors Per2 and Bmal1 in synchronized murine ameloblasts (Lacruz et al., 2012).

The ratio between the total width of the enamel and the spatial distance between cross striations can indicate the duration of dental

<figure><img src="image_14.png" /><figcaption>**Fig. 11.** Striae of Retzius. A) $960 \text{ cm}^{-1}$ $v_1 \text{PO}_4^3-$ stretching mode peak intensity reconstructed image. B) $960 \text{ cm}^{-1}$ $v_1 \text{PO}_4^3-$ stretching mode peak intensity profile along green line shown in A. Blue arrows: striae of Retzius. Black arrows: lamination.</figcaption></figure>
<figure><img src="image_15.png" /><figcaption>**Fig. 12.** Cross striation. A) 960 cm⁻¹ ν₁ PO₄³⁻ stretching mode intensity reconstructed images B) intensity profile along green (up) and blue (down) lines shown in A. Blue arrow: cross striations. Ep: enamel prism; Ip: interprism enamel.</figcaption></figure>

enamel formation. However, the spatial frequency of cross striations is dependent on the location: 2.6 ± 0.3 for the internal enamel, close to the DEJ, and 5.5 ± 0.8 µm for the external enamel, close to the surface (Shellis, 1983) demonstrating variation in enamel deposition rate during amelogenesis.

### 3.11. Scallop pattern

The email-dentin junction is shown in Fig. 13. This image is representative of a specific area in which a large scallop pattern (80 µm) is combined with smaller scallops (10/15 µm), as shown in Fig. 13A (double arrows). These variations in size are dependent on the area in which the scan is performed and the type of tooth. The presence of scallops of different sizes (2/3 µm and ~100 µm) connected to tooth type has been described previously (Brauer et al., 2010). However, the origin and role of scallops are currently unclear. The transition zone

between enamel and dentine (DEJ) shows a certain penetration of organic material within the dental enamel, over an area of about ten micrometers. This penetration revealed by the intensity mapping of the peak located at 2800/3000 cm⁻¹ also shows a network of tangled structure, suggesting the presence of a high concentration of organized protein, for example collagen (zoom in Fig. 13B). We can also see the convergence of the tubules at the tip of the scallop-type structure. The structures located at the junction have already been visualized and presented, once the enamel has been removed from the tooth, in particular by scanning electron microscopy (Marshall et al., 2003a).

### 3.12. Enamel spindle

The image in Fig. 14 shows intensity reconstructions in a part of the enamel-dentin junction where a spindle can be seen. This structure appears to be an extension of a canal, rather rectilinear in shape and

<figure><img src="image_16.png" /><figcaption>**Fig. 13.** Scallop pattern at DEJ. A) 960 cm⁻¹ ν₁ PO₄³⁻ stretching mode intensity reconstructed image. E: Enamel, D: dentine B) 2800–3000 cm⁻¹ organic bounds (CH, CH₂, CH₃) intensity peaks reconstructed image. C) CH 2800/3000 cm⁻¹ intensity peak profile, along red line shown in B) Blue arrow: organic penetration in enamel. D: dentine; E: Enamel; DEJ: dentine enamel junction. Double green arrows: large scallop pattern; double dark blue arrows: small scallop pattern.</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>**Fig. 14.** Spindle. A) and C) 960 cm⁻¹ ν₁ PO₄³⁻ stretching mode intensity reconstructed image B) and D) 2800–3000 cm⁻¹ organic bounds (CH, CH₂, CH₃) intensity peaks reconstructed image.</figcaption></figure>

hypomineralized, whose contents do not appear to be invested with protein. Indeed, image 14B shows the variation in intensity of the CH peak in the area, and the absence of organic material appears fairly

homogeneous, with no demarcation at the level of the structure described. Enamel spindles have two characteristics reported in the literature: they are hypomineralized, and are extensions of tubules

<figure><img src="image_18.png" /><figcaption>**Fig. 15.** Drapes. Enamel drapes. A) and C) 960 cm⁻¹ ν₁ PO₄³⁻ stretching mode intensity reconstructed image. B) and D) 2800–3000 cm⁻¹ organic bounds (CH, CH₂, CH₃) intensity peaks reconstructed images.</figcaption></figure>
present in dentin (Palamara et al., 1989). Raman microscopy results confirm both these elements. Two types of spindles can be seen in the images in Fig. 14: on the one hand, completed structures between 15 and 20 micrometers (Fig. 14A), and on the other, smaller structures of less than 10 micrometers (Fig. 14C). Their density along the DEJ is highly dependent on location, and the smaller spindles are densely pack.

In Fig. 14D, we can distinguish an intensity of the CH 2800/3000 cm⁻¹ peak at the spindles appearing in Fig. 14C. This is consistent with the presence of protein residues inside these structures. This observation is not always clearly visible (see Fig. 14B), which can be explained by the preparation method of the simple, based on polishing and ultrasound baths.

### 3.13. Enamel drapes

Fig. 15 shows images of enamel drapes. The images on the left, 15 A and 15 C, are related to the intensity of the peak corresponding to the phosphate elongation, while images 15B and 15D are related to the intensity of the peak corresponding to the CH bond. In both cases, we can see a structure that tends to widen from base to apex in an undulating fashion, and which, as with the spindle, is weakly mineralized and without organic content at the time of scanning.

The reconstruction of the chemical maps, both for the phosphate (960 cm⁻¹) and for the CH bonds (2800–3000 cm⁻¹), clearly shows hypomineralization, but also a low rate of organic matter. However, it is possible that the cleaning actions of the samples could have removed elements. Indeed, the presence of proteins in the enamel drapes has already been well documented (Robinson & Hudson, 2011).

It can be seen that the drapes are not connected to the tips of the scallop patterns, but that their base seems to randomly start from the junction without any determined pattern. They also seem to be often oriented in the direction of the prism groups, which could suggest a link between the drapes and the Hunter-Shreger bands, as has already been suggested in the literature (Risnes, 1987).

Raman spectra of enamel drapes and spindles are similar to those of enamel (Fig. 3), but with much lower peak intensities. This is due to the fact that the laser's scattering volume necessarily includes HAP located below the focal plane itself.

### 3.14. Branches in dentine

In Fig. 16, where dentine is cutting in a plane parallel to dentin axe, the branches are scanned from the tubule to the inner part of inter-tubular material. Indeed, usual network of tubules originating in the pulp chamber and extending towards the enamel junction (Nanci, 2013) is combine to another one, formed by branches. Connected to localization in the dentin, three types of branches were identified: a Y-shaped branch 0.5–1 µm in diameter, within 250 µm of the peripheral dentin and close to the DEJ; another type located at root level. The branches and the dentine central axe are forming an angle, comprise between 25

and 45 degrees, spaced around 3–5 µm (Mjör & Nordahl, 1996).

The network of tubules and smaller-diameter branches embedded in the intertubular material plays a mechanical role that was the subject of a finite-element study (Vennat et al., 2021). Because the microscope scatters a section of diameter ~350 nm, the resolving power of the instrument does not allow us to visualize a difference in composition between the tubuli sheathing and the rest of the material. The same applies to the branches. However, the nanoindentation studies mentioned above indicate a higher hardness in the peritubular zone, and SEM studies show a higher density of PAHs. It is therefore possible that such an organization (sheath denser than the intertubular material) could be found in the branches. Further investigation will highlight this point.

### 3.15. Interglobular dentin (IGD)

IGD is not a structure as such, but a defect in the dentin, in which there are residues of cells or organic material. However, its presence is very common in the dentin, it is rare for a tooth to be completely devoid of it. As a result, IGD appears as an area of the order of ten micrometers, of irregular shape, grouped in the dentin sometimes in the form of a circle around the pulp chamber, below the dental mantle (Nanci, 2013).

IGD is also located in the cervical areas as well as at the level of the coronary third (Jayawardena et al., 2009a). IGD has been associated in several studies as a sign of vitamin C deficiency (D'Ortenzio et al., 2018; Veselka and Snoeck, 2021; Veselka et al., 2019) or overexposure to fluoride during dentin formation (Nanci, 2013).

Fig. 17 A thus shows a clear mineralization defect, while Fig. 17B shows the trace of organic residues. As previously described, the pattern of tubules is not interrupted through defect, and continues from on side of IGD to the other.

The two spectra in Figure D, representative of dentin and the interior of the IGD. Thus, the spectrum plotted in black shows an inversion of the peak of highest intensity between the phosphate and CH peak, compared to the spectrum of dentin plotted in red.

Fig. 17 C is the mapping of the intensity ratio of the CH peak located at 2800 cm⁻¹ and the intensity of the Phosphate peak at 960 cm⁻¹. This last figure clearly illustrates the presence of organic residues within the IGD.

## 4. Conclusion

For the first time, a study makes it possible to obtain images in the form of chemical maps of all the components of the healthy tooth with optical resolution Confocal Raman microscopy demonstrates its potential in dental histology. The present study serves as a basis in this field, presenting results obtained under homogeneous conditions in terms of resolution, laser power and wavelength. Chemical mapping could show the global structure as classical microscopy does, but with molecular bound signature. The resolution of the optical system, depending on the numerical aperture of the objective used, makes it possible to map sub-

<figure><img src="image_19.png" /><figcaption>Fig. 16. Branches in dentine. A) 960 cm⁻¹ ν₁ PO₄³⁻ stretching mode intensity reconstructed image. B) zoom on blue rectangle of A). in this zone alpha is 26 degrees.</figcaption></figure>
<figure><img src="image_20.png" /><figcaption>**Fig. 17.** Interglobular dentine. A) 960 cm⁻¹ v₁ PO₄³⁻ stretching mode intensity reconstructed image. B) 2800–3000 cm⁻¹ organic bounds (CH, CH₂, CH₃) intensity peaks reconstructed images. C) cartography of ratio intensity between CH bound dans Phosphate peaks. D) representative Raman spectra of IGD and dentin, red and black spectrum correspond to red and black dots shown in A and B.</figcaption></figure>

micrometric structures and observe their composition.

If certain results have already been shown in the literature, in particular concerning the dentin enamel junction or daily variations of enamel, others are shown for the first time by Raman microscopy. This is the case for the branches of dentin, the concentration of HAP in the peritubular zone or the maps of the dental pulp. Thus, this study made it possible for the first time to visualize a high concentration rate of HAP in the peritubular zone, the possible presence of organic material at the junction of the enamel prisms, the variations of HAP in the different bands of the HSBs allowing the filling rates to be compared between the two orientations of the bands. We were also able to measure the variation in secretion of material in the enamel constituting the striae of Retzius.

These results can also be compared with other studies on mechanical properties. Conversely, variations in HAP indicate a modification of the mechanical properties in the organs, and allow us to better understand the relative share of each element.

Chemical mapping therefore makes it possible to observe variations in mineral and organic components within the organ, and can therefore be a tool for measuring variations between individuals, but also between organs of different species. Teeth being the most mineralized organ, it is also the one that is best preserved. Analysis by chemical imaging would therefore make it possible to compare dental organs, including in a non-destructive manner, between different species, including ancient ones.

## Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

Study has been realized in EDMOS platform created with the financial support of the Region Occitanie (France) and the European Regional

Development Fund (ERDF).

## Statement of author contributions

Pierre-Yves Collart-Dutilleul: analysis and interpretation of data drafting the article it critically for important intellectual content; final approval of the version to be submitted;

S. Piglionico: contributed to conception and design, data acquisition, analysis, and interpretation, drafting the article it critically for important intellectual content; final approval of the version to be submitted

R. Younes: contributed to conception and design, data interpretation, drafting the article it critically for important intellectual content; final approval of the version to be submitted

H. Salehi: contributed to conception and design, data interpretation, drafting the article it critically for important intellectual content; final approval of the version to be submitted

Y. Messat: contributed to conception and design, data acquisition, analysis, and interpretation, drafting the article it critically for important intellectual content; final approval of the version to be submitted

S. Garrabe: contributed to conception and design, data interpretation, drafting the article it critically for important intellectual content; final approval of the version to be submitted

J-C Durand: contributed to conception and design, data interpretation, drafting the article it critically for important intellectual content; final approval of the version to be submitted

F. Cuisinier: contributed to conception and design, data acquisition, analysis, and interpretation, drafting the article it critically for important intellectual content; final approval of the version to be submitted

Alban Desouter: contributed to conception and design, data acquisition, analysis, and interpretation, drafting the article it critically for important intellectual content; final approval of the version to be submitted
# Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.archoralbio.2025.106189.

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