Microbial Identification, High-Resolution Microscopy and Spectrometry of the Rhizosphere in Its Native Spatial Context

Abstract

During the past decades, several stand-alone and combinatorial methods have been developed to investigate the chemistry (i.e., mapping of elemental, isotopic, and molecular composition) and the role of microbes in soil and rhizosphere. However, none of these approaches are currently applicable to characterize soil-root-microbe interactions simultaneously in their spatial arrangement. Here we present a novel approach that allows for simultaneous microbial identification and chemical analysis of the rhizosphere at micro− to nano-meter spatial resolution. Our approach includes (i) a resin embedding and sectioning method suitable for simultaneous correlative characterization of Zea mays rhizosphere, (ii) an analytical work flow that allows up to six instruments/techniques to be used correlatively, and (iii) data and image correlation. Hydrophilic, immunohistochemistry compatible, low viscosity LR white resin was used to embed the rhizosphere sample. We employed waterjet cutting and avoided polishing the surface to prevent smearing of the sample surface at nanoscale. The quality of embedding was analyzed by Helium Ion Microscopy (HIM). Bacteria in the embedded soil were identified by Catalyzed Reporter Deposition-Fluorescence in situ Hybridization (CARD-FISH) to avoid interferences from high levels of autofluorescence emitted by soil particles and organic matter. Chemical mapping of the rhizosphere was done by Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray analysis (SEM-EDX), Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), nano-focused Secondary Ion mass Spectrometry (nanoSIMS), and confocal Raman spectroscopy (μ-Raman). High-resolution correlative characterization by six different techniques followed by image registration shows that this method can meet the demanding requirements of multiple characterization techniques to identify spatial organization of bacteria and chemically map the rhizosphere. Finally, we presented individual and correlative workflows for imaging and image registration to analyze data. We hope this method will be a platform to combine various 2D analytics for an improved understanding of the rhizosphere processes and their ecological significance.

Direct Imaging of Plant Metabolites in the Rhizosphere Using Laser Desorption Ionization Ultra-High Resolution Mass Spectrometry

“…Freeze-drying of the soil samples resulted in a major volume reduction and a brittle structure of the soil. Conventional resin-embedding requires chemical fixation and the removal of water before the embedding ( Mueller et al, 2013 ; Bandara et al, 2021 ) and is prone to extract or delocalize small molecules from the soil. Gelatin was previously used for the stabilization of soil samples for microscopic examination of soil structure ( Anderson, 1978 ), and a mixture of gelatin and CMC was recently used for sediment samples ( Alfken et al, 2019 ; Wörmer et al, 2019 ).…”

“…• The sampling and sample preparation strategy has to preserve the structural integrity of the root-soil system, even in the vacuum of the ion source (Mueller et al, 2013;Veličković et al, 2020b;Bandara et al, 2021). • The chemical composition of the potential target molecules must not be altered by agents used for fixation, embedding media, or solvents that could selectively remove compounds (Alfken et al, 2019).…”

“…The sampling and sample preparation strategy has to preserve the structural integrity of the root-soil system, even in the vacuum of the ion source ( Mueller et al, 2013 ; Veličković et al, 2020b ; Bandara et al, 2021 ).…”

Editorial: Microscale Modelling of Soil Processes: Recent Advances, Challenges, and the Path Ahead

“…The progressive shift in emphasis from the macroscale to the microscale over the last few years has been rendered possible by tremendous technological advances, such as the advent of dedicated table-top X-ray computed tomography scanners, and the development of various synchrotron-based techniques (such µXRF or NEXAFS) as well as NanoSIMS. Experimental research in this field has been very active and is currently undergoing a major expansion around the world, in particular in work that combines several different observation techniques (e.g., Schlüter et al, 2019;Lucas et al;Bandara et al, 2021;Gerke et al, 2021).…”

“…This has been illustrated schematically by Baveye et al (2018), in a diagram that we reproduce here in Figure 1. There has been some progress achieved since 2018 in several respects, for example in terms of spatial measurements of the distribution of bacteria in soils (e.g., Juyal et al, 2019;Juyal et al, 2020;Juyal et al, 2021) and in the combination of physical, chemical, and biological measurements to obtain a full picture of microenvironments at the scale of microorganisms (e.g., Schlüter et al, 2019;Lucas et al;Bandara et al, 2021;Gerke et al, 2021). Nevertheless, one can argue that a "half-empty-glass" perspective remains appropriate, and there is still some way to go before enough microscale measurements will be available to reasonably rigorously assess microscale models.…”