Characterization of lipid in mature enamel using confocal laser scanning microscopy

Objective. To characterize the lipid components of organic matrix in mature human enamel using Confocal Laser Scanning Microscopy (CLSM) coupled with a hydrophobic fluorescent probe. Methods. Twenty-four longitudinal sections of human enamel were fixed with 3.7% paraformaldehyde (PFA), partially decalcified with 0.5 M of EDTA, and labelled with a fluorescent probe (Nile red) before CLSM characterization. Based on the fluorescence spectra of Nile red in ethanol (1 μg/ml), each enamel section was evaluated with 543 nm light source and with 590 nm long pass filter. Spectrophotometric analysis was carried out to characterize the autofluorescence and Nile red lipid fluorescence in mature enamel. Special optical parameters of the microscope were chosen to rule out the intrinsic fluorescence of the samples, and that induced by PFA. Results. The intensity of autofluorescence and PFA−induced fluorescence were negligible above 565 nm; whereas the fluorescence of Nile red peaked at around 600 nm. Lipid material was identified in the cross-striations, the lines of Retzius, the Hunter–Schreger bands, inter-prismatic spaces, and intraprismatic spaces in the mature human enamel. Conclusions. This technique successfully revealed the distribution of lipid components of organic matrix in mature human enamel and may be promising in assessing the changes of enamel organic elements in the developmental, pathological or experimental conditions.