Investigating 3D microbial community dynamics of the rhizosphere using quantitative phase and fluorescence microscopy

Microbial interactions in the rhizosphere contribute to soil health, making understanding these interactions crucial for sustainable agriculture and ecosystem management. Yet it is difficult to understand what we cannot see; among the limitations in rhizosphere imaging are challenges associated with rapidly and noninvasively imaging microbial cells over field depths relevant to plant roots. Here, we present a bimodal imaging technique called complex-field and fluorescence microscopy using the aperture scanning technique (CFAST) that addresses these limitations. CFAST integrates quantitative phase imaging using synthetic aperture imaging based on Kramers–Kronig relations, along with three-dimensional (3D) fluorescence imaging using an engineered point spread function. We showcase CFAST’s practicality and versatility in two ways. First, by harnessing its depth of field of more than 100 μm, we significantly reduce the number of captures required for 3D imaging of plant roots and bacteria in the rhizoplane. This minimizes potential photobleaching and phototoxicity issues. Second, by leveraging CFAST’s phase sensitivity and fluorescence specificity, we track microbial growth, competition, and gene expression at early stages of colony biofilm development. Specifically, we resolve bacterial growth dynamics of mixed populations without the need for genetically labeling environmental isolates. Moreover, we find that gene expression related to 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 insights to be gained into microbial behavioral dynamics in experimental systems of relevance to the rhizosphere.