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Attached bacterial communities can generate three-dimensional (3D) physicochemical gradients that create microenvironments where local conditions are substantially different from those in the surrounding solution. Given their ubiquity in nature and their impacts on issues ranging from water quality to human health, better tools for understanding biofilms and the gradients they create are needed. Here we demonstrate the use of functional tomographic imaging via confocal fluorescence microscopy of ratiometric core-shell silica nanoparticle sensors (C dot sensors) to study the morphology and temporal evolution of pH microenvironments in axenic Escherichia coli PHL628 and mixed-culture wastewater biofilms. Testing of 70-, 30-, and 10-nm-diameter sensor particles reveals a critical size for homogeneous biofilm staining, with only the 10-nm-diameter particles capable of successfully generating high-resolution maps of biofilm pH and distinct local heterogeneities. Our measurements revealed pH values that ranged from 5 to &gt;7, confirming the heterogeneity of the pH profiles within these biofilms. pH was also analyzed following glucose addition to both suspended and attached cultures. In both cases, the pH became more acidic, likely due to glucose metabolism causing the release of tricarboxylic acid cycle acids and CO(2). These studies demonstrate that the combination of 3D functional fluorescence imaging with well-designed nanoparticle sensors provides a powerful tool for in situ characterization of chemical microenvironments in complex biofilms.

Functional Tomographic Fluorescence Imaging of pH Microenvironments in Microbial Biofilms by Use of Silica Nanoparticle Sensors