In situ three-dimensional mapping of oxygen gradients in Staphylococcus epidermidis biofilms using a solution-based, ratiometric imaging platform

Staphylococcus epidermidis biofilm oxygen gradients are spatially mapped during biofilm development and after vancomycin treatment using a solution-based ratiometric imaging platform. By integrating the oxygen-sensitive, tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate with oxygen-insensitive, water-soluble CdSe/ZnS quantum dots, we achieve in situ microscale resolution of dissolved oxygen (DO) concentrations within biofilms. The oxygen-sensing platform is calibrated within alginate hydrogels to mimic probe confinement within biofilms and subsequently validated in biofilms using chemical oxygen depletion (sodium sulfite) and thermal inactivation (60{degrees}C). We demonstrate that the probes do not significantly alter planktonic bacterial growth or biofilm development. Using confocal laser scanning microscopy and quantitative image analysis, 3D microscale oxygen maps of biofilms are visualized and evaluated. During S. epidermidis biofilm development from 12 to 24 hours, average biofilm DO concentrations decrease from 2.62{+/-}0.22 mg/L to 2.02{+/-}0.47 mg/L, corresponding with increased S. epidermidis biofilm biomass and bacterial metabolic activity. While treatment of S. epidermidis biofilms with vancomycin at the minimum inhibitory concentration (MIC) (2 g/mL) results in negligible DO decreases and biofilm biomass and metabolic activity comparable to untreated biofilms, higher vancomycin concentrations (20, 200 g/mL) lead to increases in biofilm DO, higher dead-cell biovolumes, and decreased metabolic activity. This work establishes a microscale, solution-based ratiometric platform for quantifying the interplay between biofilm oxygen gradients, structure, and metabolic activity, providing a framework for understanding biofilm resilience during antimicrobial treatment.