Abstract
BackgroundConfocal 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.MethodsIn 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.ResultsThree-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.ConclusionsThree/four-dimensional (3D/4D) imaging and processing in confocal microscopy is of great interest and benefit to clinical dental researchers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12903-016-0282-0) contains supplementary material, which is available to authorized users.
Highlights
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
Three-dimensional reconstruction for thick carious lesions Specimens used in this study were soft pulp tissues connected with hard dentin tissues (Fig. 1a). 2D images with an overlay of the differential interference contrast (DIC) channel image of a healthy control tooth were displayed scant expression of Glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein B (S100B) (Fig. 1b)
Three-dimensional reconstruction for detection of an oral pathogen in periodontitis tissues Using 3D reconstruction to analyse oral microbial biofilm structure, we found one of the specific pathogens P. gingivalis to be widely localised inside the periodontal pocket epithelial tissues from a series of 2D-stack images from periodontal disease tissue samples (Fig. 3b) compared to clinically healthy gingiva where the pathogens were only located on the gingival surface (Fig. 3a)
Summary
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. Confocal laser scanning microscope (CLSM) has been developed and improved enormously over the past 10 years This powerful technology has several advantages over conventional epi-fluorescence microscopy, including improved performance in contrast and free out-of-focus blur for thin or thick specimens [1], The technology enables to capture thin optical sections from thick specimens with controllable depth of field to produce 2D z-stack images through a three-dimensional. Most microscopic samples are essentially transparent, and the depths of fields of focused samples are exceptionally narrow Another advantage of confocal laser scanning microscope (CLSM) is the capacity to distinguish between different depths of a sample, which is free of out-of-focus blur for thin or thick specimens [1]. This process can provide good quality high resolution images for publishing
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