High-Resolution Chemical Mapping and Identifying Spatial Distribution of Microbes in the Zea mays Rhizosphere using Correlative Microscopy

Studying the spatial distribution of bacteria and characterizing the soil chemistry (i.e., elemental, isotopic and molecular composition) underpins the comprehensive understanding of rhizosphere associated processes. During the past decades, several stand-alone methods have been developed to investigate soil chemistry, nutrient cycling and plant nutrition. However, individual approaches as stand-alone are not capable of providing an overall rhizosphere processes involving soil, root and microbes in a spatial context, as there is no common sample preparation method available to satisfy individual needs of each technique. Here, we present i) a sample preparation method, which includes soil embedding, sectioning and ii) a correlative imaging and image registration workflow, which allows for characterization of minerals, roots and microbes by different high-resolution imaging and microanalytical techniques. This allows for conducting rhizosphere studies on different scales, focusing on root-soil-microbe interfaces with spatial resolution of nano-meter scale. Hydrophilic, immunohistochemistry compatible, low viscosity LR White resin was used to embed and stabilize the soil and make it ultra-high vacuum compatible. We employed water-jet cutting as a novel approach to slice the embedded samples, and, by doing so, avoided polishing of the surface which often leads to translocation of sample material (smearing). The quality of this embedding was analyzed by and Helium Ion Microscopy (HIM). Epifluorescence microscopy in combination with Catalyzed Reporter Deposition-Fluorescence in-situ Hybridization (CARD-FISH) was employed to accurately identify and determine the spatial distribution of bacteria in the embedded sample, thus avoiding ambiguities from high levels of auto-fluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was acquired by SEM/EDX, ToF-SIMS, nanoSIMS for elemental, molecular and isotopic characterization, respectively, and µ-Raman microscopy for specific identification of minerals.

In summary, we demonstrate that LR White embedding and water-jet cutting of soil in combination with CARD-FISH and a correlative microscopic workflow, allows for a comprehensive characterization of biotic and abiotic components in the rhizosphere. The developed sample preparation method can facilitate the various requirements of involved microscopy techniques and individual workflows for imaging and image registration to analyze data. We foresee that this approach will establish an excellent platform to study various soil processes and synergistic understanding of complex rhizosphere processes.