3D visualisation of dentine tubule occlusion by dual beam FIB SEM/EDS

Dentine hypersensitivity (DH) is sharp pain derived from exposed dentine in response to stimuli that can not be ascribed to any other dental diseases1. It is highly prevalent and has a significant impact on quality of life. Occlusion of dentine tubules by brushing teeth with a desensitising toothpaste is a common but effective method that offers quick alleviation of DH.

The effectiveness of dentine tubule occlusion is often studied at dentine surfaces or cross session using SEM, which provides a snapshot of local dentine tubule occlusion, but is not capable of capturing the characteristic occlusion depth in a larger area and the 3D distribution of occlusion. Non-contacting confocal laser scanning microscopy provides 3D visualisation, however, can not record the chemical distribution of occlusion which is important for long term occlusion. X-ray CT is an effective method for 3D visualisation of occlusion that also provides information on chemical distribution but at a limited resolution. Focused ion beam (FIB) milling with SEM imaging and Energy Dispersive Spectrometer (EDS) mapping2 is therefore proposed for 3D visualisation and chemical mapping of dentine tubule occlusion.

A de-sensitising toothpaste (containing SiO2 and bioactive glass) was applied on citric acid etched dentine disc by mechanical brushing with an electrical toothbrush. The sample was then placed at the coincident point of the electron beam and ion beam of a duel beam FIB (Helios 660, ThermoFisher) for ion beam milling and electron beam imaging. The sample was milled leaving a block for 3D EDS analysis as shown in Fig. 1a. A Pt layer was deposited on the top surface of the 3D block to protect the sample from ion beam damage. A fiducial marker was made for image correlation controlling the sequential milling position (Fig. 1). Ion beam operated at 30 kV was applied to sequentially mill away slices with a thickness of 220 nm. Electron image and EDS mapping were collected on each milled slice faces. Si was used as a chemical marker to differentiate toothpaste against dentine tubules. Image processing, segmentation, visualisation and calculation were performed using Avizo 9.0 (ThermoFisher). Marker based segmentation technique was applied to assist segmentation of electron images of toothpaste and dentine tubules which have similar grey level.

Electron image (Fig. 2a) of the milled slice face demonstrates the presence of dentine tubules and a layer of occlusion on dentine surface. The Si EDS mapping at the same area confirms the presence of Si on the occluded layer and within dentine tubules. The resconstructed 3D volume (Fig. 3) shows the chemical distribution of the desensitising toothpaste (yellow represents Si). Depth of the Si marker indicates the occlusion depth of the toothpaste used.

In conclusion, FIB SEM/EDS technique successfully provided 3D visualisation and chemical mapping of dentine tubule occlusion at a high resolution that is not currently available by other characterisation techniques.