Profiling the heterogeneity of microbial populations and communities at the single-cell level.

Recent advancements in single-cell genomic and transcriptomic sequencing, in situ sequencing, and molecular imaging-based technologies have facilitated the examination of heterogeneity within microbial communities at the single-cell level. These cutting-edge methodologies permit the capture of phenotypic and genotypic heterogeneity, as well as the spatial organization within microbial communities. This enables in-depth investigation into microbial dark matter, the evaluation of microbial responses to perturbations, and a comprehensive exploration of spatial functions involved in community assembly and social interactions within microbial communities. These interactions include inter-microbial relationships, bacteria-phage interactions, and host-microbe interactions. Here, we highlight the key technological breakthroughs achieved, elucidating the perspectives from which these technologies enable us to interpret microbial heterogeneity at the single-cell level. Additionally, we critically examine the limitations associated with these technologies. Furthermore, we explore how these methods could be combined and also their applications in future studies. The integration of these approaches holds great promise for increasing our understanding of the organization and function of microbes in complex ecosystems.