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

Abstract

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 >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.

Directed evolution of a far-red fluorescent rhodopsin

“…Arch(DETC+P196S) showed the most pH-sensitive fluorescence of the mutants we fully characterized and had the lowest Schiff-base pK a (6.8) ( Table 1). E. coli is able to robustly buffer its cytosolic pH against changes in environmental pH up to pH ∼9 (34); in contrast, environmental pH can change drastically, and complex microenvironments such as biofilms can form distinct local heterogeneities in pH (35). A surface-displayed pH-sensitive GFP variant (pHluorin) was recently used to monitor pH changes surrounding Streptococcus mutans microcolonies during sucrose fermentation (36).…”

Discovery of a biofilm electrocline using real-time 3D metabolite analysis

“…Numerous technological advancements have allowed for the examination of chemical gradients within biofilms, primarily through the use of in situ responsive fluorophores or microprobes (20)(21)(22)(23). However, a fundamental question still exists regarding the nature of such gradients in bulk solution surrounding a biofilm.…”

Ratiometric Imaging of Extracellular pH in Bacterial Biofilms with C-SNARF-4

“…Although none of these techniques rely on a uniform distribution of the fluorescent dye in the biofilms for pH calculations, the dye concentration has to be high enough in all areas of the biofilm to allow for quantification. This proved to be a problem for pHsensitive nanoparticles, which mainly bound to bacterial surfaces and left channels and voids filled with extracellular matrix unstained (32). In dental biofilms, pH in these areas is of crucial importance for the outcome of caries.…”

“…While small dye molecules readily diffuse into the biofilm, penetration proved to be a considerable problem for pH-sensitive nanoparticles. Hidalgo et al reported that staining of the biofilm was not possible with nanoparticles of 70 nm and 30 nm in diameter, and 10-nm-sized particles mostly adhered to bacterial cells, which made it impossible to record pH in cell-free areas of the biofilm (32).…”

“…Two-photon excitation followed by time-gated fluorescence life-time imaging (FLIM) uses pH-dependent differences in the fluorescence lifetime decay of, for example, carboxyfluorescein to determine local pH in a biofilm sample (31). pH-sensitive nanoparticles comprising both pH-sensitive and pH-insensitive dye molecules have been developed to quantify pH in biofilms by comparing the fluorescent emission of both dyes (32). Yet another approach is the use of ratiometric dyes, such as C-SNARF-4, that display different fluorescent emission spectra depending on the state of protonation of the dye.…”