Methods: Electron transparent specimens, <100nm in thickness, were prepared by dual beam FIBSEM milling using in situ lift out from samples of bovine enamel. Sample treatment consisted of (1) untreated, (2) treated with a strontium/fluoride solution (33,000ppm and 300ppm respectively). The specimens were attached to a specialist TEM Cu grid by means of platinum deposition for high resolution imaging, EDX analysis and SAED.
Results: FIB-milling was able to generate high-quality specimens that could be used for TEM examination. High resolution TEM imaging in the scanning mode revealed preferential alignment of the needle-like crystals, furthermore characteristic banding of the enamel crystals was observed. Electron diffraction patterns were taken from the samples, revealing a ring pattern. Measurements of the ring radius led to the conclusion that the needle-like crystals were hydroxyapatite. EDX analysis revealed the presence of Ca, P, O and F. Some Cu was detected due to the TEM grid. Pt and Ga were also detected, originating from the FIB milling procedure. The sample treated with a strontium fluoride solution also contained a small signal for strontium. The strontium L and K lines were used for semi-quantification.
Conclusion: High quality TEM sections of treated and untreated bovine enamel have been successfully prepared using FIB milling. Characteristic rod-like hydroxyapatite crystals were clearly observed in the FIB sections without the need for any additional preparatory steps. The uptake of strontium into the enamel as a result of a strontium/fluoride solution treatment is easily detected and quantifiable.