Locked Expansion Microscopy to in Situ Analyze Microbial Communities

Abstract

There is a global race in microbiome research to map spatial organization of microbial species in densely packed communities, but progress is limited by optical imaging resolution and our ability in characterizing the diversity of microbial species within a population in situ. Here we provide a new super-resolution imaging method, based on recently developed expansion microscopy (ExM), to address this knowledge gap. Expansion microscopy is an optical imaging technique by physically expanding tissues anchored to a hydrogel. The current method relies on a polyelectrolyte hydrogel that is expanded by electrostatic repulsion; as a result, the hydrogel shrinks in any ionic buffers. This technical limitation precludes the possibility of using most anti-photobleaching systems (which typically require buffering) and thereby restricting the application of ExM in thick tissues with weak fluorescence signals, as these demand long imaging times. Here, we developed a novel method, locked ExM, in which the first expanded polyelectrolyte hydrogel is embedded in a second interpenetrating hydrogel mesh that expands through entropic forces. The second interpenetrating mesh retains the size of expanded tissues even in buffers with extreme ionic strengths. This method has allowed us to use anti-photobleaching systems in ExM to achieve sub-diffraction-limit resolution with high photostability. We have applied this method to image host-associated microbial communities, and, to our surprise, found that the expansion ratios of microbial species depend on their specific cell wall structures. We demonstrated the utilities of locked ExM to visualize and analyze spatial distributions of different microbial species within gut microbiota in a variety of organisms (worm, mouse, and human).