Label-free visualisation of histological features in human teeth using autofluorescence imaging

We show that autofluorescence imaging can be used as a label-free modality to visualise histological features of human teeth. The autofluorescence emitted by enamel, dentin, predentin, and interglobular dentin (IGD) allows observing structural details that would otherwise require complex staining or even complementary electron microscopy or X-ray imaging. The simultaneous acquisition of autofluorescence and conventional staining fluorescence images by confocal microscope also provides important advantages to visualise occluded dentin porosity or the presence of porosity within IGD. However, autofluorescence imaging is practically limited by photobleaching. We, therefore, quantified the photobleaching extent in 3D and showed that, in confocal microscopy, the whole illuminated volume is bleached during acquisition. To mitigate this phenomenon, we analysed the photobleaching behaviour in enamel, dentin, predentin, and IGD as a function of laser power. We found that the measured decay rate cannot be modelled by a single population decay of fluorophores, highlighting the fact that autofluorescence probably arises from multiple sources. This analysis also allowed defining a laser excitation power where the photobleaching effect saturates.