Three/four-dimensional (3D/4D) microscopic imaging and processing in clinical dental research

Ping Ye, Hong Yu, Mojgan Houshmandi

Confocal laser scanning microscope (CLSM) has been widely employed in our laboratory for structural and functional analysis of clinical dental specimens and live cell imaging of cultured oral epithelial cells. In this vitro study, a Fluoview 1000 (Olympus) confocal system was utilised to study thick sections of carious lesions (40-100 μm) and periodontal disease tissue samples (20-40 μm) by 2D Z stacking imaging and 3-dimentional (3D) reconstruction. Four-dimensional (4D) imaging when including time or position points was used for live cells to assess penetration/localisation/co-localization of oral pathogen proteins and therapeutic drugs. Three-dimensional (3D) reconstruction revealed latent features of carious hard tissues (strongly expressed amelogenin proteins in dentin tubules), and soft tissues (increased glial markers GFAP and S100B in pulp components). We also found the oral microbial specific pathogens, Porphyromonas gingivalis to be widely localised inside the periodontal pocket epithelial tissues as detected by 3D reconstruction from a series of 2D sections from periodontal disease tissue samples. 4D live cell imaging showed the diffusion patterns of fluorescent molecules in response to a bacterial virulence factor, the pathogen (gingipain haemagglutinin) domain that attacked epithelial integrity. This technology also showed uptake of a novel porphyrin-linked metronidazole antibiotic into epithelial cells to kill intracellular oral pathogen, P. gingivalis. Three/four-dimensional (3D/4D) imaging and processing in confocal microscopy is of great interest and benefit to clinical dental researchers.