New Technology for Visualizing Gene Activity in Single Cells

How do bacteria—whether harmless ones in our bodies or disease-causing strains—coordinate their activities? A recent study, published in Science, combines genomic-scale microscopy with a key technical breakthrough to reveal which genes bacteria activate in different environments, advancing bacterial research.

Jeffrey Moffitt, PhD, and colleagues at Boston Children’s Hospital’s Program in Cellular and Molecular Medicine (PCMM) used MERFISH, a molecular imaging technique Moffitt co-developed, to analyze thousands of bacterial mRNAs simultaneously. MERFISH also provided spatial data, showing how bacteria respond to their surroundings—a previously unattainable insight.

Overcoming Challenges in Bacterial RNA Imaging

Studying bacterial transcriptomes is challenging due to the small, densely packed nature of bacterial cells.

"It was a complete disaster—we couldn’t see anything," Moffitt admitted.

To solve this, the researchers adapted expansion microscopy from MIT’s Ed Boyden, PhD. By embedding bacterial samples in a hydrogel, anchoring RNAs, and altering the gel’s buffer, they expanded the sample 50- to 1,000-fold.

"All bacterial RNAs became individually resolvable," Moffitt explained.

Why Study Bacterial Gene Expression?

Traditionally, bacterial behavior has been averaged across populations. Identifying gene expression in individual bacteria offers deeper insights into interactions, virulence, stress responses, antibiotic resistance, and biofilm formation.

"We now have tools to explore host-microbe and microbe-microbe interactions, bacterial competition for spatial niches, and microbial community structures. We can also examine how pathogenic bacteria adjust gene expression during infection," Moffitt noted.

Bacterial-MERFISH is especially valuable for studying bacteria that are difficult to culture.

"Now, we don’t have to culture them—we can just image them in their native environment," Moffitt said.

Single-Cell Insights into Bacterial Behavior

The team demonstrated bacterial-MERFISH’s capabilities through several experiments. In one, they observed E. coli deprived of glucose, sequentially modifying gene expression to utilize alternative energy sources. By capturing genomic snapshots over time, they reconstructed this survival strategy.

They also found bacteria organize RNAs within cells to regulate gene expression and discovered intestinal bacteria activate distinct genes depending on their location in the colon.

"The same bacteria can behave very differently over small distances, responding to their environments in ways we couldn’t previously address. Now, we can answer long-standing questions," Moffitt said.

This breakthrough enables single-cell bacterial studies, providing deeper insights into microbial behavior in health and disease.