Real-time pH Measurement within Enamel Caries Lesions Using Confocal Microscopy

Whilst the dynamics of protons in plaque above caries lesions has been the subject of numerous studies, little is known of the dynamics of pH changes within the lesion itself. Objectives: We used the confocal laser scanning microscope with the pH-sensitive fluorochrome SNARF-4F (Molecular Probes Inc, USA) to map pH changes within caries lesions in real-time. Methods: SNARF-4F has two maxima in its emission spectrum: 575nm and 650nm. At pH 6.8 the peak ratio (650:575) is approximately 2. At pH 4.6 the ratio is approximately 0.7. Thus pH changes can be followed by plotting changes in the peak ratio with time. Smooth surface white spot lesions were infiltrated with a solution of 5µg/ml SNARF-4F at pH 6.8. The lesion was then imaged using a Leica SP2 AOBS CLSM with water ‘dipping’ lens. The intensity of the two maxima was determined at a number of points throughout the depth of the lesion. The pH of the bathing solution was then dropped to pH 4.6 and the lesion reanalysed over a 60min period. Results: Throughout the bulk of the lesion the pH dropped steadily over a 15min period.. In the deepest part of the lesion penetrated by the dye this fall in pH did not reach a minimum until at least 30min had elapsed. In some lesions the ‘surface zone’ demonstrated little if any net decrease in pH, even after 60min. Conclusion: These results would indicate that even within the most superficial zones of a caries lesion the pH does not appear to immediately equilibrate with the overlying plaque and a period of up to 30 minutes may be required in the deepest regions before the pH falls to a minimum. This is likely to have significant consequences for the dynamics of lesion demineralisation and remineralisation. Supported by BBSRC (Grant:24/REI18424)