Application of Focused Ion Beam to Root Canal Biofilms Research

Objectives: Many studies have been shown micro-organisms in the root canal or outside root canal system by scanning electron microscopic (SEM) observation, that make possible for visualizing micro-organism in their natural state. It is possible that FIB etches (or mills) the SEM sample surface locally with sub-micron precision and allows non-destructive deposition at the sample surface. Recently it has been applied to biological tissues to expose their ultrastructure for examination. In this study, we observed the cross-sectional SEM images of biofilm by sputtering the surface region of root canal wall, in order to sight morphologically the biofilm formation architecture. Methods: Immediately after the tooth had been extracted, they were rinsed in distilled water and split with chisel into two specimens. The specimens were fixed in 2% glutaraldehyde fixative and kept in it for 2 h at 4°C, and then post-fixed in 2% osmium tetroxide for 2 h at 4°C. Dried specimens by critical point drying were sputter-coated with osmium. Next, a beam of gallium ions was focused down to a diameter of 0.1 µm at a current density of 1.5 A/cm₂ (10¹⁹ ions/cm₂s). Sample was examined under a S-4500 (HITACHI, Tokyo, Japan) scanning electron microscope at 75 kV. Results: FIB images revealed that occoi and short rod bacteria organized the multi-layered bacterial condensation. That was almost from three to five µm thicknesses. Some region showed a combination of cocci, short rod and rod shaped bacteria. Conclusion: This study suggest that the application of FIB in biofilms research will give us a new strategies to manage biofilm in endodontic field.