Spatial mapping of microbial communities by an integrated automation platform of sequential FISH

Spatial mapping of microbial communities at single-cell resolution is opening up dimensions to understand microbiome organization and function. However, current fluorescence in situ hybridization (FISH) methods for microbiomes are limited by multiplexity and scalability. Here, we present the sequential error-robust FISH spatial mapping platform (SEER-Map) for fully automated imaging of complex microbial communities. We show that an integrated platform of fluidics control and fluorescence microscopy can perform 40 rounds of sequential FISH. We apply SEER-Map to profile complex microbial communities colonized on plant roots and identify distinct spatial patterns and species co-occurrence at the micron-scale. Our work establishes SEER-Map as a high-throughput and scalable platform for high-resolution spatial profiling of microbiomes.