Dentin was visualized using a new fluorescence technique and confocal laser scanning microscopy. Thirty extracted human teeth showing no clinical signs of caries were investigated. All teeth were horizontally sectioned to approximately 200 μm thickness and sections were subjected to different pretreatment conditions as follows: vacuum only, ultrasonication only, sodium hypochlorite only, sodium hypochlorite and vacuum, sodium hypochlorite and ultrasonication, and a combination of sodium hypochlorite, vacuum, and ultrasonication. Some samples were left untreated to serve as control. Following pretreatment, rhodamine 123 fluorescent
dye was used for staining at concentrations ranging from 10-3 to 10-7M for 1 to 24 h at pH 6.0, 6.5, or 7.4. Optical staining occurred at pH 7.4 and concentrations ≥ 10-5 M over 3 h or longer. Surface images obtained using confocal laser scanning microscopy were similar to those observed by scanning electron microscopy without the need for sample-altering conventional scanning electron microscope preparation techniques. Subsurface imaging to a depth of approximately 60 μm was achieved using confocal laser microscope techniques. This fluorescence technique offers a useful new alternative for visualization and quantification of dentin.