Improving three-dimensional visualisation of cellular porosity in
human dentin using two photon microscopy

Seunghwan G. Lee<sup>a,b</sup>, Elsa Vennat<sup>c</sup>, Kwangseuk Kyhm<sup>b*</sup>, Aurélien Gourrier<sup>a*</sup>

<sup>a</sup>Univ. Grenoble Alpes, CNRS, LIPhy, 140 rue de la Physique, Grenoble, France; <sup>b</sup>Pusan National University, Department of Opto & Cogno Mechatronics Engineering, 57, Oryundae-ro, Geumjeonggu, Busan, Republic of Korea 4624; <sup>c</sup>Université Paris-Saclay, CentraleSupélec, ENS Paris-Saclay, CNRS, LMPS – Laboratoire de Mécanique Paris-Saclay, 91190 Gif-sur-Yvette, France

ABSTRACT

Three-dimensional visualisation of dentin cellular porosity is essential to understand the mechanosensing response of human teeth. However, the inherent light scattering nature of dentin and its refractive index mismatch limit the imaging depth of standard confocal laser scanning microscopy (CLSM) to approximately 20 µm, which is insufficient for structural analysis. This study presents a methodology that enhances the imaging depth of dentin porosity through two approaches. First, we optimised sample preparation to improve in-depth image quality by polishing surfaces down to sub-wavelength roughness and employing a mounting medium to minimise refractive index differences. Second, we implemented two-photon microscopy (TPM), which is advantageous for deep-tissue imaging compared to standard CLSM. The imaging depth was evaluated using standard image quality metrics and expert visual perception. Our results demonstrate improved imaging depth exceeding 90 µm, representing more than a four-fold enhancement over standard CLSM.

**Keywords:** Dentin porosity, Two-photon microscopy, mineralised tissue, bioimaging, dentin

*kskyhm@pusan.ac.kr

*aurelien.gourrier@univ-grenoble-alpes.fr

# 1. INTRODUCTION

The structural analysis of dental tissues provides fundamental insight for dental research. Dentinal porosity remains poorly characterised and could be important to understand the biological and mechanical properties of human teeth better. Dentin porosity consists of micro-scale tubules (1-2 µm in diameter) and sub-micron branches (0.2-0.5 µm in diameter) [1-3]. We previously demonstrated that the presence of branches can cause local stress concentration[4]. Furthermore, dentin porosity is believed to play an essential role in mechanosensing in human teeth. According to the hydrodynamic theory, dentin sensitivity is at least partly determined by fluid flow within the porosity in response to external stimuli[5]. Since the fluid dynamics depend on geometric characteristics of porosity such as diameter, length, and connectivity, a three-dimensional porosity visualisation provides fundamental information for mechanical and biological studies of human teeth.

However, the three-dimensional visualisation of dentin porosity presents specific challenges. Dentin exhibits strong scattering of visible light, thus reducing the collected signal intensity[6-7]. In addition, the refractive index mismatch between the sample and mounting medium further degrades image quality by disrupting the coherent focusing of light at the intended focal plane. Because the refractive index of dentin depends on mineral density, it varies from 1.43 to 1.57[8]. Degradation of image quality due to scattering and refractive index mismatch becomes more pronounced with depth, limiting the imaging depth of standard CLSM to 20 µm.
To resolve these issues, we consider two distinct lines of improvement: sample preparation and imaging. For sample preparation, embedding, sectioning, surface polishing, and staining are sequentially performed. The tooth sample is embedded in resin and sectioned to expose the dentin surface, and then the dentin surface is polished sequentially to the sub-wavelength level. Initially, large-size grains (~10 µm) are used to remove major surface irregularities and cutting marks, and then smaller-size grains (a few µm) are applied to reduce surface roughness progressively. In the final stage, nanometer-level abrasives (200 nm) are used to achieve sub-wavelength-level polishing. This surface polishing process is designed to minimise surface scattering effects. During the staining process, we diffuse a mounting medium containing fluorescent molecules into the dentin porosity. The fluorescence signal can therefore be optimised by minimising the refractive index mismatch using a mounting medium with a refractive index similar to dentin.

To improve imaging depth, we exploit two-photon microscopy (TPM). The near-infrared excitation wavelength used in TPM corresponds to an optical window where scattering and absorption are significantly reduced for biological tissue. TPM utilises nonlinear absorption, which confines light-matter interactions to the focal point, thus reducing background noise [9]. Therefore, TPM is expected to achieve significantly deeper imaging depths in scattering samples compared to CLSM.

In this study, we investigate the improvement in imaging depth achieved by the methodology described above. To quantitatively evaluate the improvement effect of surface polishing, we prepared two groups of specimens with contrasting surface conditions. We sequentially polished the first group to 200 nm, sub-wavelength level, while the second group was degraded down to 5 µm roughness. To analyse the effect of refractive index matching, samples from each group were stained with PBS, glycerol, and Vectashield containing Rhodamine B. Correlative 3D imaging using CLSM and TPM was performed on each sample to compare the imaging depth between the two imaging modalities. Improvements in imaging depth were evaluated using standard image quality metrics and expert visual perception.

## 2. METHODS

### Sample preparation for improved dentin porosity visualisation

Extracted human teeth were initially cleaned using an ultrasonic bath to remove food debris and then subjected to disinfection with a Chloramine-T solution (0.5% - 5g/L). Following disinfection, specimens were fixed in 70% ethanol for a minimum of 48 hours, then secured to sample holders using beeswax and air-dried at room temperature for 24 hours (Figure 1 (a)). The dried samples were subsequently embedded in athermal curing epoxy resin (EPOFIX, Struers) (Figure 1 (b)). Sectioning was performed in the mesiodistal direction using a diamond-blade saw (Struers E0D15 mounted on a Presi Mecatome T210) under continuous water cooling (Figure 1 (c,d)). The sectioned surfaces were thoroughly cleaned in distilled water and dried between glass slides to prevent bending from drying under ambient conditions for 24 hours. The section was then mounted onto a metallic sample holder using cyanoacrylate adhesive to fix the surrounding embedding resin for subsequent polishing (Figure 1 (d)).

Primary polishing was conducted to remove cutting marks resulting from sectioning. This process was performed manually using 2000-4000 grit SiC abrasive paper (EU grade, Struers) with corresponding grain sizes ranging from 10.3 µm to 5 µm (Figure 1 (f)). Secondary polishing was achieved using diamond polishing spray (DP-spray P, Struers) combined with aqueous lubricant oil (DP-lubricant red, Struers) on a Presi Minitech 233 polishing machine, progressively reducing grain sizes from 3 µm to 0.2 µm (Figure 1(g)). Surface polishing effectiveness was assessed at each grain size stage through light microscopy examination (Aristomet, Leitz) using a 20× objective lens (NA 0.45) to evaluate polishing mark removal (Figure 1(e-g)). Sample thickness uniformity was maintained by monitoring height variations relative to the metallic plate using a digital gauge (Ostartools, 824-343) at four designated corner positions, with a precision of ±10 µm. Upon completion of polishing, all sections underwent a final cleaning in distilled water, followed by 48 hours of drying between glass slides at ambient temperature to ensure the complete removal of residual polishing particles.
<figure><img src="image_1.png" /><figcaption>Figure 1. Sample preparation workflow. (a) The extracted tooth is secured to the sample holder with beeswax for drying, and subsequently (b) embedded in epoxy resin and (c,d) sectioned using a diamond saw. (e) Bright-field microscopy image showing cutting marks on the unpolished surface after sectioning. Surface image after polishing with (f) 5 µm and (g) 0.2 µm grain sizes, demonstrating progressive removal of polishing marks. (h) Final porosity staining process using a mounting medium containing Rhodamine B.</figcaption></figure>

The polished samples were stained for 72 hours at room temperature in a mounting medium containing 0.02 wt% Rhodamine B (Figure 1(h)). Following staining, all samples were mounted between slide glasses using the same mounting medium. Following staining, all samples were mounted between slide glasses using the same mounting medium.

## Sample preparation for comparative analysis

Six sections prepared according to our sample preparation protocol were divided into two groups: polished and degraded. The polished group underwent sequential surface polishing down to 0.2 µm grain size to achieve sub-wavelength surface polishing. The degraded group was subjected to surface roughening using 360-grade (EU) SiC abrasive paper with 40 µm particle size while maintaining the original sample thickness to simulate rough surface conditions.

Following surface treatment, samples from both groups were stained with one of three mounting media: glycerol, phosphate-buffered saline (PBS), or Vectashield, each containing 0.02 wt% Rhodamine B. All specimens underwent staining for 72 hours at room temperature under controlled conditions. Upon completion of staining, samples were mounted between slide glasses using the identical mounting medium employed during the staining process.

The naming of samples corresponds to the mounting medium and surface conditions. (e.g., glycerol polished)

## Correlative imaging

Fluorescence images were obtained using CLSM (SP8, Leica) and TPM (custom-built system).

CLSM imaging employed a 561 nm laser with an oil immersion (Type F immersion liquid, Leica, n=1.5180) objective lens (40×, 1.3 NA). The theoretical lateral and axial resolutions were 220 nm and 800 nm, respectively. Fluorescence detection was performed using a photomultiplier tube (PMT) with a spectral range of 569-696 nm. Images were captured with a field of view (FOV) of 354.6 × 354.6 µm², pixel resolution of 172 nm, and z-step intervals of 500 nm in the dentin-enamel junction (DEJ) region.

TPM imaging employed an 800 nm infrared laser with an oil immersion (IMMOIL-F30CC, Olympus, n=1.518) objective lens (40×, 1.3 NA). The theoretical lateral and axial resolutions were 330 nm and 1000 nm, respectively. Fluorescence detection was achieved using a PMT with a 510-670 nm wavelength detection range. Images were acquired with an FOV of 228 × 228 µm², pixel resolution of 172 nm, and z-steps of 500 nm in the same region with CLSM.
PBS-stained samples were also imaged using an oil immersion objective. This was to minimise hardware variables such as numerical aperture and transmission of the objective lens. Furthermore, to demonstrate image quality degradation due to refractive index mismatch, samples mounted in PBS (n = 1.33), a medium exhibiting a significant refractive index difference compared to glycerol (n = 1.43) and Vectashield (n = 1.45), were imaged.

### 3. RESULTS AND DISCUSSION

Dentin porosity contains cell processes, organic substances and inorganic minerals, but a significant portion of its volume previously occupied by cell processes is found to be vacant after the extraction and preparation. Therefore, to achieve a comprehensive three-dimensional visualisation, the porosity needs to be fully filled with a fluorescent medium. This approach relies on diffusion rather than chemical binding, posing the risk of staining medium diffusing out from the stained porosity when mounting in an unstained medium.

Figure 2(a) shows time-lapse images of a porosity stained with glycerol containing 0.02 wt% Rhodamine B and mounted without any additional stain in the mounting medium. Four minutes after staining, the staining medium begins to escape from the porosity. After 12 minutes, a tubule can be observed in which most of the staining medium has escaped. The weak signals at the two edges of the tubule could also be mistaken for distinct signals from two smaller tubules or branches, which poses a significant challenge in image processing.

Figure 2(b) shows time-lapse images of the porosity after staining and mounting with the same staining medium, following our sample preparation method. The concentration equilibrium is established between the internal porosity and the surrounding medium, thereby preventing diffusion-induced signal loss. This demonstrates that our method effectively maintains signal consistency during long-term fluorescence imaging.

<figure><img src="image_2.png" /><figcaption>Figure 2. Time-lapse image of dentin porosity. Images were acquired using TPM over 12 minutes with a temporal resolution of 8 seconds. (a) Image of the sample stained with glycerol containing 0.02 wt% Rhodamine B and sealed with a cover glass without additional stain in the mounting medium. The loss of fluorescence over time is observed (arrows). (b) Image of the sample after the same staining process as (a), but mounted with the stained medium.</figcaption></figure>

Figure 3 (a-c) shows CLSM images of polished samples stained with PBS, glycerol, and Vectashield, respectively, at a depth of 25 µm. Dentinal tubules (yellow arrows), branches (green arrows), and optical aberrations (purple arrows) can be observed in Figure 3(a). The optical aberrations appear as elongated signal tails extending 9 µm from the end of the dentinal tubules, which can pose a significant obstacle for image processing. In particular, the intensity of the branches is low, making it difficult to separate them from optical aberrations. Tubules appear with a significantly higher contrast than branches. This suggests that branches are more sensitive to image quality degradation with depth than tubules. Therefore, the imaging depth limit for 3D porosity visualisation should be defined as the maximum depth at which the signal intensity of branches is similar to the optical aberration or background. From this perspective, when assessing
images of samples stained with glycerol (Figure 3(b)) and Vectashield (Figure 3(c)), it can be qualitatively confirmed that these two mounting media can provide an imaging depth of 25 µm or more. In particular, compared to PBS, the contrast of branches is significantly higher. Figure 3(d) shows an image of glycerol-degraded samples obtained by CLSM at a depth of 20 µm. It exhibits distinct optical aberrations, high background signals, and low branch contrast compared to polished samples (Figure 3(b)). This demonstrates that surface polishing provides a significant improvement in the porosity visualisation.

Figure 3(e) and (f) show CLSM and TPM images of glycerol polished samples obtained at a depth of 90 µm in the same region of interest (ROI). Compared to CLSM, TPM exhibits a significantly lower background signal and much higher branch contrast relative to optical aberrations. In contrast, in CLSM, the signal from the dentinal tubules is still well preserved, but the intensity of the branches is similar to, or below, the level of optical aberrations. These results demonstrate that TPM can provide imaging depths of 90 µm or more in glycerol polished samples, a significant improvement of more than four times compared to standard CLSM.

<figure><img src="image_3.png" /><figcaption>Figure 3. Fluorescence image comparison across different samples and imaging modalities. Images acquired using CLSM at 25 µm depth from (a) PBS polished, (b) glycerol polished and (c) Vectashield polished samples. Green arrows indicate branches, yellow arrows indicate dentinal tubules, and purple arrows indicate optical aberrations. (d) CLSM image of glycerol degraded sample at 20 µm depth demonstrating reduced image quality due to surface roughness. Correlative imaging of (e) CLSM and (f) TPM at 90 µm depth of the glycerol polished sample. Mean fluorescence intensity degradation with depth comparing (g) CLSM and TPM for Vectashield polished sample, (h) Vectashield polished and degraded sample with TPM and (i) Vectashield, glycerol, and PBS polished sample with TPM.</figcaption></figure>

Figure 3(g-i) shows examples of mean signal intensity degradation with depth according to imaging modality, surface polishing, and mounting medium. TPM shows improved mean intensity stability compared to CLSM. This difference
becomes more pronounced as depth increases. The mean signal intensity of TPM remains 33% higher than that of CLSM from 10 µm (Figure 3(g)). The mean signal intensity of polished samples is consistently equal to or higher than that of degraded samples at all depths (Figure 3(h)). In the mounting medium comparison, results differed from those observed by expert visual perception (Figure 3(i)). Vectashield and glycerol show similar trends, with both media exhibiting lower mean signal intensities than PBS up to 25 µm. Analysis of the total decrease in mean signal intensity showed that glycerol and Vectashield showed a 50% decrease up to 25 µm, while PBS showed a decrease of more than 66%. At 20 µm depth, PBS, glycerol, and Vectashield exhibited similar mean fluorescence intensities. However, a qualitative examination of the images revealed substantially lower branch contrast in PBS (Figure 3(a)) compared to both glycerol (Figure 3(b)) and Vectashield (Figure 3(c)). This discrepancy occurs because the analysis performed on the entire image includes background noise and tubule signals, thereby obscuring the assessment of branch image quality. This suggests the need for the development of advanced image processing methods that can selectively analyse the porosity.

## 4. CONCLUSION

This study demonstrated that sub-wavelength polishing of the sample surface, refractive index-matched mounting, and TPM application provide a significant improvement in imaging depth for three-dimensional porosity visualisation. Sample surface polishing played a key role in reducing background noise and optical aberrations. Refractive index matching using glycerol and Vectashield mounting media showed superior performance compared to PBS. Notably, TPM exhibited lower background signals and high branch contrast compared to CLSM. Under optimised conditions with glycerol polished, TPM imaging achieved an imaging depth of over 90 µm, representing a fourfold improvement over standard CLSM. This study presents a methodology that enables improved three-dimensional analysis of dentin porosity and provides a standardised protocol that can be widely applied to the study of the biological and mechanical properties of human teeth.

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