Investigating 3D microbial community dynamics in the rhizosphere using complex-field and fluorescence microscopy

Microbial interactions in the rhizosphere contribute to soil health. Understanding these interactions thus has the potential to advance sustainable agriculture, ecosystem management, and environmental stewardship. Yet it is difficult to understand what we cannot see; amongst the limitations in rhizosphere imaging are challenges associated with rapidly and non-invasively imaging microbial cells over field depths relevant to plant roots. Here, we present a novel bimodal imaging technique called Complex-field and Fluorescence microscopy using the Aperture Scanning Technique (CFAST) that begins to address these limitations by integrating quantitative phase and 3D fluorescence imaging. We showcase CFAST’s practicality and versatility in two ways. First, by harnessing its depth of field of more than 100 microns, we significantly reduce the number of captures required for 3D imaging of plant roots and bacteria in the rhizoplane, thereby minimizing potential photobleaching and phototoxicity. Second, by leveraging CFAST’s phase sensitivity and fluorescence specificity, we track early bacterial aggregate development, bacterial competition, and gene expression under varying environmental conditions. Specifically, we resolve bacterial growth dynamics of mixed populations at the early stages of colonization without the need for genetically labeling environmental isolates. Moreover, we find that the expression of genes of interest to rhizosphere chemistry (e.g. representative genes involved in phosphorus-sensing and antibiotic production) varies spatiotemporally within microbial populations that are surface-attached and appears distinct from their expression in planktonic cultures. Together, CFAST’s attributes overcome commercial imaging platform limitations and enable new insights to be gained into microbial behavioral dynamics in experimental systems of relevance to the rhizosphere. ### Competing Interest Statement The authors have declared no competing interest.