Unveiling enamel demineralization mechanisms by sensitive dielectric differentiation based on terahertz nanospectroscopy

The early stage of dental caries, i.e. demineralization, has always been a topic of concern to dentists. Understanding the essential mechanism of its occurrence is of great significance for the prevention and treatment of dental caries. However, owing to limitations in resolution and the detection capabilities of diagnostic tools, the study of enamel demineralization has always been a challenge. Terahertz (THz) technology, especially the combination of scanning near-field optical microscopy (s-SNOM) and THz time-domain spectroscopy (TDS), due to its nanoscale resolution, has shown great advantages in the field of biological imaging. Here, a THz s-SNOM system is used to perform near-field imaging of enamel before and after demineralization at the nanoscale. It can be found that near-field signals decrease significantly after demineralization. This is due to the changes of the crystal lattice and the transfer of mineral ions during demineralization, which leads to a decrease in the permittivity of the enamel. The novel approach in this study reveals the essence of demineralization and lays the groundwork for additional research and potential interventions.


Sample preparation
The bovine tooth samples used in this experiment were provided by West China Hospital of Stomatology, Sichuan University.Research regarding the appropriateness of replacing bovine for human teeth has shown that subtle morphological differences do exist between the two substrates, as both tissues behave similarly but not necessarily identically [1,2].Bovine enamel was found to be more porous, and have higher carbonate [3] but lower fluoride contents [4].However, the two behave similar enough to provide an acceptable alternative with the advantage of reduced variability of the hard tissue substrate [5].Freshly extracted bovine permanent incisors without cracks and lesions were cut with a low-speeded saw cutting machine and stored in deionized water containing 0.5% thymol.The samples were ground and polished using a diamond-coated band saw with continuous water cooling (Struers Minitom, Struers, Copenhagen, Denmark) to produce flat, parallel dentin and enamel surfaces.The dentin side of the specimen was ground to a uniform thickness with 200 grades of silicon carbide papers.The samples were embedded with epoxy resin, and the enamel surface was continuously ground with water-cooled carborundum discs of waterproof silicon carbide paper (1000, 2000, and 5000grit; Matador), polished with 1μm diamond polishing suspension ( DiaDuo-2, Struers, Copenhagen, Denmark)on the polishing cloth (MD-Nap, Struers, Copenhagen, Denmark) until the enamel side had at least 4×6 mm highly polished surface, resulting in the removal of the about 200-300μm depth of enamel surface.The surface of each sample except for the polished enamel was covered with a double layer of acid-resistant varnish and then stored at 4°C and 100% relative humidity till use.Sound enamel samples were immersed in the demineralization buffer, containing 50 mM acetic acid, 2.2 mM CaCl 2 , 2.2 mM KH 2 PO 4 •2H 2 O, 5.0 mM NaN 3 and 1.0 ppm F, pH adjusted to 4.5, placed in a shaker (37℃, 100 rpm/min) for 5 days.The demineralized samples were cut vertically by a low-speed, water-cooled diamond saw (Minitom, Struers, Copenhagen, Denmark) to the enamel window area to obtain slices of two different surfaces.

Other near-field results
In addition to 0.8 THz, we also select several other frequencies for near-field imaging, and the results are shown in the Figure S1a.It can be found that the normal and demineralization area still have a clear division.In order to minimize the effect of height fluctuation, we select demineralized and normal enamel regions with height fluctuation in the range of 650-750 µm.As can be seen from Figure S1b, the near-field signals in the demineralized region at each frequency point are significantly lower than those in the normal region.In addition, we also analyze the phase information of the near-field signal of these points.The spectra of points 1-4 in Figure 2c are shown in Figure S1c.It can be seen that the spectra of the normal enamel region is significantly larger than that of the demineralized region, and with the increase of frequency, the spectrum also increases.

Element distribution before and after demineralization
As shown in the Table S1, the minerals after demineralization, mainly Ca and P ions, have some losses, but the overall change is little.

TDS system
The TDS system setup is shown in Figure S2.TDS THz source (TeraSmart, menlosystems TERA15-TX-FC) is a THz time-domain spectrometer based on a photoconductive antenna (PCA)with a laser repetition rate of 100 MHz and a frequency of 0.2-2 THz.The system has a dynamic range of over 80 dB.The laser central wavelength is 1560 nm, the pulse width is 100 fs, and the average power is more than 10 mW.A femtosecond laser interacts with the photoconductive antenna to generate terahertz waves, which reach the detector after passing through the sample.Another femtosecond laser beam also reaches the detection end after passing through the delay line, sampling the terahertz pulses returning to the detector at different times.The amplitude and phase information of the terahertz pulses returning to the detector are obtained, and the corresponding waveform is plotted to calculate the permittivity of the enamel before and after demineralization.

Method for calculating the permittivity of tooth enamel
We take the signal when the sample is not put in as the reference signal, and measure the Terahertz wave through the normal and demineralized enamel as the sample signal.The complex refractive index of the sample is a function of frequency (ω) and can be determined by Eq.S1, where the real part n (ω) and imaginary part κ (ω) of the complex refractive index are calculated by Eq.S2 and Eq.S3 respectively, where c represents the speed of light in vacuum, d represents the thickness of the sample, and n air represents the refractive index of air.H (ω) represents the transfer response function of the system, which is obtained by dividing the Fourier transform result of the sample signal (Gs(ω)) with the Fourier transform result of the reference signal (Gr(ω)) (Eq.S4). ) From this, we can further calculate the real part ε 1 (ω) and the imaginary part ε 2 (ω) of the permittivity according to the samples n (ω) and κ(ω) (Eq.S5-S7).

Fig. S1 .
Fig. S1.(a) Other frequency imaging results.(b) Normal and demineralized near-field signal amplitudes between 650 and 700μm at several frequencies (c) The normalized 2 nd order nearfield spectra of points 1-4.(d) Normalized 2 nd order near-field phase of normal and demineralized area between 650 and 700 µm.

Table . S1
. Element distribution before and after demineralization.