Revolutionary Bacterial Research Unlocks New Insights Through Advanced Imaging Techniques

By combining MERFISH imaging with expansion microscopy, researchers have unlocked a new way to study bacteria at the single-cell level. This groundbreaking approach allows them to observe how bacteria activate different genes in response to various environments, offering invaluable insights into microbial behavior, antibiotic resistance, and infection strategies.

Understanding Bacterial Activities

How do bacteria, whether beneficial or harmful, organize and coordinate their activities? Recent research has shed light on these processes by utilizing advanced genomic-scale microscopy in combination with an innovative technique to track gene activation in bacteria under different conditions.

Jeffrey Moffitt, PhD, and his team at Boston Children’s Hospital used MERFISH, a molecular imaging technique developed by Moffitt, to analyze messenger RNAs (mRNAs) in thousands of individual bacteria simultaneously. This method not only provided a comprehensive map of gene expression on a large scale but also revealed how spatial factors influence which genes bacteria activate—a first in the field.

Overcoming Imaging Challenges

The team initially faced a significant challenge: the dense packing of bacterial RNAs inside tiny cells made them difficult to distinguish and image. “It was a complete disaster, we couldn’t see anything,” says Moffitt.

To address this issue, they borrowed a technique from the laboratory of Ed Boyden, PhD, at MIT—expansion microscopy. The researchers embedded the bacterial samples in a special hydrogel, anchoring the RNAs within it and adjusting the chemical buffer. This process caused the sample to swell, expanding it 50 to 1000 times in volume, making individual RNAs resolvable.

Importance of Measuring Gene Expression

Traditionally, scientists averaged bacterial behavior across populations. However, this approach masks individual variations. The ability to determine which genes individual bacteria activate provides powerful new insights into their interactions, virulence, stress responses, antibiotic resistance, biofilm formation, and more.

“We now have the tools to answer fascinating questions about host-microbe and microbe-microbe interactions,” Moffitt says. “We can explore how bacteria might communicate and compete for resources and define the structure of microbial communities. And we can ask how pathogenic bacteria adjust their gene expression during infections.”

This imaging technique also allows researchers to study bacteria without culturing them, enabling direct observation of natural bacterial environments.

Single-Cell Insights into Survival Strategies

The team conducted several experiments to demonstrate the potential of bacterial-MERFISH. In one study, they observed that individual E. coli bacteria, when deprived of glucose, sequentially switch to alternative food sources. By taking genomic snapshots over time, they were able to piece together this specific survival strategy.

The researchers also gained insights into how bacteria organize their RNAs within their cells, a factor that may play a crucial role in gene expression regulation. Additionally, they discovered that intestinal bacteria activate different genes depending on their location in the colon.

A New Era in Bacterial Research

“The same bacteria could be doing very different things over a space of tens of microns,” Moffitt says. “They are exposed to different environments and respond accordingly. It was very difficult to address such variation before, but now we can answer questions that have long puzzled scientists.”

This breakthrough opens up new possibilities in the study of bacteria, enabling researchers to explore questions about how bacteria interact with each other and their hosts, how they adapt to various environments, and how they cause infections.

Reference: “Highly Multiplexed Spatial Transcriptomics in Bacteria” by Ari Sarfatis, Yuanyou Wang, Nana Twumasi-Ankrah, and Jeffrey R. Moffitt. Published in Science, January 24, 2025. DOI: 10.1126/science.adr0932.

The paper’s co-authors were Ari Sarfatis, Yuanyou Wang, PhD, and Nana Twumasi-Ankrah in the Moffitt Lab.

Conclusion

This revolutionary imaging technique represents a significant step forward in bacterial research. By allowing scientists to study bacteria at the single-cell level, it provides unprecedented insights into their behavior, gene expression, and survival strategies, opening up new avenues for understanding and addressing bacterial infections and antibiotic resistance.

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