Molecular rotors reveal the 3D viscous habitat of mucus-colonizing bacteria.

Interactions between bacteria and particulate organic matter, algae, coral reefs, fish, plant root systems, animals, and humans occur primarily through a dynamic interface of viscous mucus or mucilage. While mucus influences fundamental rates of bacterial infection, respiration, and carbon and nutrient cycling, our observations of this physical habitat of bacteria are limited by methods that damage the material and obfuscate spatial relationships. We present a technique using confocal microscopy of molecular rotors to reveal the 3D viscous structure of undisturbed mucus and associated bacteria. Quantification of the internal viscosity of mucus from different sources highlights variations in microscale morphologies that structure microbial distributions and ecological interactions. Individual examples of mucus aggregates from cultures of Chaetoceros affinis and Pseudo-nitzschia sp. diatoms exhibit consolidated versus patchy viscous morphologies, respectively, along with distinct patterns of microbial colonization. A viscous mucus layer surrounding an ephyra of the upside-down jellyfish Cassiopea xamachana may maintain a local microbiome while preventing direct contact with the animal. By quantifying the complex "mucoscape" shaping bacteria-organic matter interactions, this method provides a physical context for chemical fluxes and microbial activity in diverse ecosystems.