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

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.