Droplet microfluidics for single-cell studies: a frontier in ecological understanding of microbiomes.

Recent advances in single-cell technologies have profoundly impacted our understanding of microbial communities-shedding light on cell-to-cell variability in gene expression, regulatory dynamics, and metabolic potential. These approaches have shown that microbial populations are more heterogeneous and functionally complex than previously thought. However, direct probing of single-cell physiology-arguably more ecologically relevant by focusing on functional traits such as growth, metabolic activity, and enzymatic activity-remains underexplored. Droplet microfluidics provides a practical and high-throughput approach to address this gap, allowing functional characterization of individual microbial cells within complex communities and offering new opportunities to study ecological processes at high resolution. In this review, we look at the state of droplet microfluidics for single-cell microbial ecology. We revisit the fundamentals of microbial droplet workflows, we overview the current capabilities of droplet microfluidics that exist for microbial ecology and we look at the phenomena these workflows have uncovered and understanding they have generated. Finally, we integrate these capabilities to envision future droplet workflows that could enhance our understanding of single-cell physiology and discuss the fundamental limitations that go together with the droplet format.