Spatial profiling of microbial communities by sequential FISH with error-robust encoding

Spatial analysis of microbiomes at single cell resolution with high multiplexity and accuracy has remained challenging. Here we present spatial profiling of a microbiome using sequential error-robust fluorescence in situ hybridization (SEER-FISH), a highly multiplexed and accurate imaging method that allows mapping of microbial communities at micron-scale. We show that multiplexity of RNA profiling in microbiomes can be increased significantly by sequential rounds of probe hybridization and dissociation. Combined with error-correction strategies, we demonstrate that SEER-FISH enables accurate taxonomic identification in complex microbial communities. Using microbial communities composed of diverse bacterial taxa isolated from plant rhizospheres, we apply SEER-FISH to quantify the abundance of each taxon and map microbial biogeography on roots. At micron-scale, we identify clustering of microbial cells from multiple species on the rhizoplane. Under treatment of plant metabolites, we find spatial re-organization of microbial colonization along the root and alterations in spatial association among microbial taxa. Taken together, SEER-FISH provides a useful method for profiling the spatial ecology of complex microbial communities in situ.

Automated summary

Spatial analysis of microbiomes at single cell resolution with high multiplexity and accuracy is challenging, but can be achieved using sequential error-robust fluorescence in situ hybridization (SEER-FISH). SEER-FISH allows mapping of microbial communities at micron-scale and increases multiplexity of RNA profiling through sequential rounds of probe hybridization and dissociation. With error-correction strategies, SEER-FISH enables accurate taxonomic identification in complex microbial communities and provides a useful method for profiling the spatial ecology of these communities in situ.