When FLOW-FISH met FACS: Combining multiparametric, dynamic approaches for microbial single-cell research in the total environment

In environmental microbiology, the ability to assess, in a high-throughput way, single-cells within microbial communities is key to understand their heterogeneity. Fluorescence in situ hybridization (FISH) uses fluorescently labeled oligonucleotide probes to detect, identify, and quantify single cells of specific taxonomic groups. The combination of Flow Cytometry (FLOW) with FISH (FLOW-FISH) enables high-throughput quantification of complex whole cell populations, which when associated with fluorescence-activated cell sorting (FACS) enables sorting of target microorganisms. These sorted cells may be investigated in many ways, for instance opening new avenues for cytomics at a single-cell scale. In this review, an overview of FISH and FLOW methodologies is provided, addressing conventional methods, signal amplification approaches, common fluorophores for cell physiology parameters evaluation, and model variation techniques as well. The coupling of FLOW-FISH-FACS is explored in the context of different downstream applications of sorted cells. Current and emerging applications in environmental microbiology to outline the interactions and processes of complex microbial communities within soil, water, animal microbiota, polymicrobial biofilms, and food samples, are described.