Transcriptomic analysis of bacterial microcolonies has been drastically limited by the use of bulk approaches that average thousands of different bacteria and ignore spatial heterogeneity. A new generation of imaging-based spatial transcriptomics methods is emerging as a powerful approach for studying gene expression at the single-cell level. Many sophisticated methods based on fluorescence in situ hybridization (FISH) have been developed in recent years, that are capable of capturing individual transcripts, but their implementation for studying bacteria is still limited. Furthermore, the inherently small size of bacteria makes the imaging challenging. Here, we aim to integrate FISH with expansion microscopy [1] to detect individual mRNAs with high spatial resolution.

We have successfully implemented expansion microscopy, enhancing the resolution of our imaging. This technique was seamlessly combined with HCR-FISH, providing a comprehensive approach to visualize RNA within individual bacterial cells. Future efforts involve optimizing gel chemistry to increase the expansion factor and applying this technique to investigate the dynamics of microcolonies formed on the intestinal epithelium by enteropathogenic E. coli.

Furthermore, we want to extend the protocol to explore diverse sample types, including gram-positive bacteria, biofilms, and infected cell cultures. Utilizing Par-Seq-FISH [2] will additionally allow us to increase the throughput of RNA detection in the future.

[1] Cheng Z, Stefani C, Skillman T, Klimas A, Lee A, DiBernardo EF, K. Brown KM, Milman T, Wang Y, Gallagher BR, Lagree K, Jena BP, Pulido JS, Filler SG, Mitchell AP, Hiller NL, Lacy-Hulbert A, Zhao Y; MicroMagnify: A Multiplexed Expansion Microscopy Method for Pathogens and Infected Tissues. Adv. Sci. 2023, 10 (30)

[2] Dar D, Dar N, Cai L, Newman DK; Spatial transcriptomics of planktonic and sessile bacterial populations at single-cell resolution. Science 2021, 373 (6556)