Imaging flow cytometry enables label-free cell sorting of morphological variants from unculturable bacterial populations

Bacterial populations often display remarkable morphological heterogeneity. Fluorescence activated cell sorting (FACS) is an important tool for understanding the biological significance of this morphological diversity, as it permits the separation and study of distinct morphological variants (morphotypes) from each other and their environmental milieu. In FACS, cells are first labeled with fluorescent markers such as antibodies or transgenic constructs, and then sorted based on their possession of such labels. However, since the development of fluorescent labels requires a priori knowledge of bacterial biology, it is often impossible to apply FACS to understudied and/or unculturable bacteria. This challenge has limited our capacity to investigate the role of bacterial size and shape in all but a small, largely culturable subset of bacterial taxa. Here, we present an innovative strategy that permits label-free cell sorting of bacterial morphotypes, using an unculturable, pleiomorphic pathogen ( Pasteuria ramosa ) as a model bacterium. We show that imaging flow cytometry (IFC) can be used to systematically identify light-scattering and autofluorescence “signatures” of bacterial morphotypes, on which basis cell sorting can be conducted. Critically, our IFC-enabled cell sorting strategy yields samples of sufficient purity (>90%) for common downstream analyses e.g., “-omics” analyses. Our work represents an innovative application of IFC and provides an economical, widely applicable solution to a central problem in the study of bacterial diversity. ### Competing Interest Statement Daniel Vocelle is on the scientific advisory board for the ThermoFisher Attune CytPix and for the NanoCellect VERLO.