We report the implementation of a unique multimodal nonlinear optical microscopy (i.e., coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), third harmonic generation (THG) and two photon excitation fluorescence (TPEF)) platform for label-free imaging of dentin. A picosecond tunable laser together with an OPO is used as the excitation source for simultaneously multimodal imaging. CARS shows similar information as TPEF in dentin, but it has a higher sectioning performance than TPEF and thus it is a good alternative for TPEF. Microtubule structure is revealed nearby dentin enamel junction (DEJ) from the multimodal images. This work demonstrates that combining different nonlinear optical imaging modalities can provide new insights into the understanding of morphological structures and biochemical/biomolecular distributions of the dentine without the need of labeling.
In this study, we are using two-photon (2-p) excited autofluorescence and second harmonic (SH) as imaging modalities
to investigate dental sections that contains the enamel and the dentin. The use of near-infrared wavelengths for multiphoton
excitation greatly facilitates the observation of these sections due to the hard tissue's larger index of refraction
and highly scattering nature. Clear imaging can be achieved without feature altering preparation procedures of the
samples. Specifically, we perform polarization resolving on SH and lifetime analysis on autofluorescence. Polarization
resolved SH reflects the preferred orientation of collagen while very different autofluorescence lifetimes are observed
from the dentin and the enamel. The origin of 2-p autofluorescence and SH signals are attributed to hydroxyapatite
crystals and collagen fibrils, respectively. Hydroxyapatite is found to be present throughout the sections while collagen
fibrils exist only in the dentin and dentinoenamel junctions.
Recently, it was documented that the combined treatment of fluoride and laser can induce an even greater increase in caries resistance than laser or fluoride treatment alone. However, the real mechanisms remained unclear. The aim of our present pilot study is to characterize the crystallographic changes in the human enamel treated with fluoride (F), laser (L), and combined fluoride-laser (LF) therapies using Micro-XRD and Micro-FTIR, so as to elucidate the true mechanisms of the combined effects of fluoride and Er:YAG laser on human enamel. Three sound human teeth were selected and 3 windows were created on each tooth surface. The 3 windows were later subjected to the F or L or LF treatment, respectively. The Micro-XRD patterns for the 9 windows were recorded before and after the treatments. Three sections from another 3 sound human teeth were selected for Micro-FTIR investigation and 3 windows were created on the cut surface of each section. The 3 windows were later subjected to the F or L or LF treatment, respectively. The FTIR patterns for the 9 windows were recorded before and after the treatments. The results revealed that both the LF and L treatments caused the contraction in the a-aixs and the improvement in the enamel crystallinity. Though the difference in the a-axis contraction between the LF and L-treated windows was not very significant (0.006Å), this crystallographic change might suggest more than 34% decrease in the enamel solubility. In conclusion, both the L and LF therapies may improve the crystalline stability and thus acid resistance of human enamel.
Ultrafast lasers have found increasing use in scanning optical microscopy due to their very high peak power in generating multiphoton excitations. A mode-locked Ti:sapphire laser is often employed for such purposes. Together with a synchronously pumped optical parametric oscillator (OPO), the spectral range available can be extended to 1050-1300 nm. This broader range available greatly facilitates the excitation of second harmonic generation (SHG) and third harmonic generation (THG) due to better satisfaction of phase matching condition that is achieved with a longer excitation wavelength. Dental sections are then investigated with the contrasts from harmonic generation.
In the past decade several studies have demonstrated the increased acid resistance in enamel demineralization after laser irradiation. However, the exact mechanism of action to this effect still remains a speculation. Recently, the role of organic matrix was revealed to be significant in the laser-induced inhibition of enamel demineralization. The aim of the present study was to characterize the lipid component of organic matrix in mature lazed enamel and unlazed enamel histochemically using a hydrophobic fluorescent probe with Confocal Laser Scanning Microscope (CLSM). Partial decalcification of thin enamel sections was carried out using 0.5 M of EDTA in a stainless steel grid for 5 hours, following fixation with 3.5% paraformaldehyde. Thereafter the sections were stained with Nile red coupled with CLSM. The intensity of the light reflection was analyzed under the same conditions for all specimens, ruling out the autofluorescence in the control sections. Confocal imaging revealed a diffuse and increased fluorescence of the lipid stain in the lazed areas suggesting that the swelling and coating of organic matrix on the surface of enamel crystals in the peri and interprismatic spaces is rendering the increased acid resistance. These findings will substantiate the proposed organic blocking theory in partially explaining the laser-induced prevention of enamel demineralization.
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