Microbial single-cell applications under anoxic conditions

The field of microbiology traditionally focuses on studying microorganisms at the population level. Nevertheless, the application of single-cell level methods, including microfluidics and imaging techniques, has revealed heterogeneity within populations, making these methods essential to understand cellular activities and interactions at a higher resolution. Moreover, single-cell sorting has opened new avenues for isolating cells of interest from microbial populations or complex microbial communities. These isolated cells can be further interrogated in downstream single-cell "omics" analyses, providing physiological and functional information. However, applying these methods to study anaerobic microorganisms under in situ conditions remains challenging due to their sensitivity to oxygen. Here, we review the existing methodologies for the analysis of viable anaerobic microorganisms at the single-cell level, including live-imaging, cell sorting, and microfluidics (lab-on-chip) applications, and we address the challenges involved in their anoxic operation. Additionally, we discuss the development of non-destructive imaging techniques tailored for anaerobes, such as oxygen-independent fluorescent probes and alternative approaches.