pH-O₂ ratiometry: Correlative imaging of pH and oxygen saturation in dental biofilms

Oxygen and pH critically influence the metabolism and virulence of microbial biofilms. In dental biofilms, low pH drives the caries process, but the relationship between oxygen saturation and pH remains largely unexplored.

pH-O₂ ratiometry, a microscopy-based method for the combined quantification of biofilm pH and oxygen saturation was developed. pH was measured using the ratiometric dye C-SNARF-4, and oxygen saturation was assessed with a planar optode (oxygen-sensitive indicator: platinum(II) meso(2,3,4,5,6-pentafluoro)phenyl porphyrin; reference fluorophore: Macrolex Yellow). As a proof of concept, pH and oxygen were simultaneously monitored inside Streptococcus mutans biofilms under static and flow conditions (0.8 mm/min and 8.0 mm/min), as well as in complex salivary biofilms.

Anaerobically grown S. mutans biofilms showed greater pH drops and lower oxygen levels than aerobically grown biofilms. The latter exhibited high initial oxygen concentrations that decreased when exposed to 0.04% sucrose but increased when exposed to 0.4% or 4% sucrose, which indicates an impact of glycolysis on oxygen consumption. No correlations between pH and oxygen saturation were observed at the field-of-view level, under static and flow conditions. In more complex salivary biofilms, low pH and low oxygen saturation correlated strongly at the field-of-view level, for biofilms grown in the presence and in the absence of sucrose.

pH-O₂ ratiometry provides a valuable approach for investigating the interplay between pH and oxygen in biofilm metabolism. Further studies are warranted to investigate the relationship between pH and oxygen saturation in dental biofilms.