Highly multiplexed spatial transcriptomics in bacteria.

Single-cell decisions made in complex environments underlie many bacterial phenomena. Image-based transcriptomics approaches offer an avenue to study such behaviors, yet these approaches have been hindered by the massive density of bacterial messenger RNA. To overcome this challenge, we combined 1000-fold volumetric expansion with multiplexed error-robust fluorescence in situ hybridization (MERFISH) to create bacterial-MERFISH. This method enables high-throughput, spatially resolved profiling of thousands of operons within individual bacteria. Using bacterial-MERFISH, we dissected the response of Escherichia coli to carbon starvation, systematically mapped subcellular RNA organization, and charted the adaptation of a gut commensal Bacteroides thetaiotaomicron to micrometer-scale niches in the mammalian colon. We envision that bacterial-MERFISH will be broadly applicable to the study of bacterial single-cell heterogeneity in diverse, spatially structured, and native environments.