Tips and tricks for gut microbiota investigation using scanning electron microscopy (SEM): going from sample preparation to imaging and landscape analysis.

The Gut Microbiota (GM) remains a complex microbial ecosystem with many unknown facets despite significant technologic advancement. This study introduces a novel rapid technique using tabletop scanning electron microscopy (SEM) for investigating GM composition, focusing on Clostridioides difficile infection (CDI) as a representative model for dysbiosis-related diseases. Six stool sample preparation protocols were tested on 40 stool samples to develop an optimized SEM protocol. Protocol stability was evaluated after four-month storage. The optimized protocol produced high-resolution micrographs while maintaining sample integrity over time. SEM investigation of GM was done by analyzing ten stool samples (5-control and 5-C. difficile groups), imaged at low and high magnifications. Object detection analysis generated a SEM-based GM components database helping describe and compare microbial diversity variation between the groups. CDI group revealed a reduction in microbial diversity, compared to the controls. Epithelial and red blood cells were more prevalent in CDI group. Statistical analyses of objects proved clear clustering of samples into CDI and control groups. This study pioneers the proof-of-concept for using tabletop SEM to investigate GM components in a dysbiosis-related disease model. This concept emerges as a complementary technique capable of providing deeper insight to describe GM components previously elusive with other methods.