Our understanding of plant-microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants\uffe2\uff80\uff99 large volume of influence. Here, we present the development of 3D live microscopy for resolving plant-microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor made dual-illumination light-sheet system acquired scattering signals from the plant whilst fluorescence signals were captured from transparent soil particles and labelled microorganisms, allowing the generation of quantitative data on samples approximately 3600 mm3in size with as good as 5 \uffce\uffbcm resolution at a rate of up to one scan every 30 minutes. The system tracked the movement ofBacillus subtilispopulations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favoured small pore spaces over the surface of soil particles, colonising the root in a pulsatile manner. Migrations appeared to be directed towards the root cap, the point \uffe2\uff80\uff9cfirst contact\uffe2\uff80\uff9d, before subsequent colonisation of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant-microbe interactions.
", "keywords": ["0301 basic medicine", "570", "Microscopy", "Silicon", "0303 health sciences", "Temperature", "root-microbe interactions", "Equipment Design", "Biological Sciences", "Environment", "15. Life on land", "Plant Roots", "630", "Fluorescence", "Soil", "03 medical and health sciences", "Seedlings", "Calibration", "Rhizosphere", "Image Processing", " Computer-Assisted", "environmental imaging", "rhizosphere", "Soil Microbiology", "Bacillus subtilis", "Lactuca"]}, "links": [{"href": "https://eprints.whiterose.ac.uk/178939/18/e2109176118.full.pdf"}, {"href": "https://pnas.org/doi/pdf/10.1073/pnas.2109176118"}, {"href": "https://doi.org/10.1073/pnas.2109176118"}, {"rel": "related", "href": "https://repository.soilwise-he.eu/cat/collections/metadata:main/items/Proceedings%20of%20the%20National%20Academy%20of%20Sciences", "name": "related record", "description": "related record", "type": "application/json"}, {"rel": "self", "type": "application/geo+json", "title": "10.1073/pnas.2109176118", "name": "item", "description": "10.1073/pnas.2109176118", "href": "https://repository.soilwise-he.eu/cat/collections/metadata:main/items/10.1073/pnas.2109176118"}, {"rel": "collection", "type": "application/json", "title": "Collection", "name": "collection", "description": "Collection", "href": "https://repository.soilwise-he.eu/cat/collections/metadata:main"}], "time": {"date": "2021-02-13T00:00:00Z"}}, {"id": "10.1101/2023.06.28.546105", "type": "Feature", "geometry": null, "properties": {"updated": "2026-05-29T16:18:36Z", "type": "Journal Article", "created": "2023-06-29", "title": "Construction and Characterisation of a Structured, Tuneable, and Transparent 3D Culture Platform for Soil Bacteria", "description": "2.AbstractWe have developed a tuneable workflow for the study of soil microbes in an imitative 3D soil environment that is compatible with routine and advanced optical imaging, is chemically customisable, and is reliably refractive index matched based on the metabolic profile of the study organism. We demonstrate our transparent soil pipeline with two representative soil organisms,Bacillus subtilisandStreptomyces coelicolor, and visualise their colonisation behaviours using fluorescence microscopy and mesoscopy. This spatially structured, 3D approach to microbial culture has the potential to further study the behaviour of other difficult-to-culture bacteria in conditions matching their native environment and could be expanded to study microbial interactions, such as interaction, competition, and warfare.
3.Graphical AbstractA step-by-step method for creating a tailored 3D culture medium for study of soil microbes.
The complete workflow can be split into three parts: Growth and observation, metabolic profiling to provide a stable refractive index matching solution, and production of the 3D soil environment. The 3D culture scaffold was created by cryomilling Nafion\uffe2\uff84\uffa2 resin pellets and size filtration. Chemical processing altered the surface chemistry of Nafion\uffe2\uff84\uffa2 particles and facilitated nutrient binding by titration of a defined liquid culture medium. Metabolic profiling determined non-metabolisable sugars and provided an inert refractive index matching substrate, which was added to the final nutrient titration. Inoculation and growth of the test strain allowed for downstream assessment of colonisation behaviours and community dynamicsin situby, for example, optical microscopy.The rhizosphere hosts complex and abundant microbiomes whose structure and composition are now well described by metagenomic studies. However, the dynamic mechanisms that enable micro-organisms to establish along a growing plant root are poorly characterized. Here, we studied how a motile bacterium utilizes the microhabitats created by soil pore space to establish in the proximity of plant roots. We have established a model system consisting of Bacillus subtilis and lettuce seedlings co-inoculated in transparent soil microcosms. We carried out live imaging experiments and developed image analysis pipelines to quantify the abundance of the bacterium as a function of time and position in the pore space. Results showed that the establishment of the bacterium in the rhizosphere follows a precise sequence of events where small islands of mobile bacteria were first seen forming near the root tip within the first 12\uffe2\uff80\uff9324\uffe2\uff80\uff89h of inoculation. Biofilm was then seen forming on the root epidermis at distances of about 700\uffe2\uff80\uff931000\uffe2\uff80\uff89\uffc2\uffb5m from the tip. Bacteria accumulated predominantly in confined pore spaces within 200\uffe2\uff80\uff89\uffc2\uffb5m from the root or the surface of a particle. Using probabilistic models, we could map the complete sequence of events and propose a conceptual model of bacterial establishment in the pore space. This study therefore advances our understanding of the respective role of growth and mobility in the efficient colonization of bacteria in the rhizosphere.Our understanding of plant-microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants\uffe2\uff80\uff99 large volume of influence. Here, we present the development of 3D live microscopy for resolving plant-microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor made dual-illumination light-sheet system acquired scattering signals from the plant whilst fluorescence signals were captured from transparent soil particles and labelled microorganisms, allowing the generation of quantitative data on samples approximately 3600 mm3in size with as good as 5 \uffce\uffbcm resolution at a rate of up to one scan every 30 minutes. The system tracked the movement ofBacillus subtilispopulations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favoured small pore spaces over the surface of soil particles, colonising the root in a pulsatile manner. Migrations appeared to be directed towards the root cap, the point \uffe2\uff80\uff9cfirst contact\uffe2\uff80\uff9d, before subsequent colonisation of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant-microbe interactions.Our understanding of plant-microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants\uffe2\uff80\uff99 large volume of influence. Here, we present the development of 3D live microscopy for resolving plant-microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor made dual-illumination light-sheet system acquired scattering signals from the plant whilst fluorescence signals were captured from transparent soil particles and labelled microorganisms, allowing the generation of quantitative data on samples approximately 3600 mm3in size with as good as 5 \uffce\uffbcm resolution at a rate of up to one scan every 30 minutes. The system tracked the movement ofBacillus subtilispopulations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favoured small pore spaces over the surface of soil particles, colonising the root in a pulsatile manner. Migrations appeared to be directed towards the root cap, the point \uffe2\uff80\uff9cfirst contact\uffe2\uff80\uff9d, before subsequent colonisation of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant-microbe interactions.The rhizosphere hosts complex and abundant microbiomes whose structure and composition are now well described by metagenomic studies. However, the dynamic mechanisms that enable micro-organisms to establish along a growing plant root are poorly characterized. Here, we studied how a motile bacterium utilizes the microhabitats created by soil pore space to establish in the proximity of plant roots. We have established a model system consisting of Bacillus subtilis and lettuce seedlings co-inoculated in transparent soil microcosms. We carried out live imaging experiments and developed image analysis pipelines to quantify the abundance of the bacterium as a function of time and position in the pore space. Results showed that the establishment of the bacterium in the rhizosphere follows a precise sequence of events where small islands of mobile bacteria were first seen forming near the root tip within the first 12\uffe2\uff80\uff9324\uffe2\uff80\uff89h of inoculation. Biofilm was then seen forming on the root epidermis at distances of about 700\uffe2\uff80\uff931000\uffe2\uff80\uff89\uffc2\uffb5m from the tip. Bacteria accumulated predominantly in confined pore spaces within 200\uffe2\uff80\uff89\uffc2\uffb5m from the root or the surface of a particle. Using probabilistic models, we could map the complete sequence of events and propose a conceptual model of bacterial establishment in the pore space. This study therefore advances our understanding of the respective role of growth and mobility in the efficient colonization of bacteria in the rhizosphere.Our understanding of plant-microbe interactions in soil is limited by the difficulty of observing processes at the microscopic scale throughout plants\uffe2\uff80\uff99 large volume of influence. Here, we present the development of 3D live microscopy for resolving plant-microbe interactions across the environment of an entire seedling growing in a transparent soil in tailor-made mesocosms, maintaining physical conditions for the culture of both plants and microorganisms. A tailor made dual-illumination light-sheet system acquired scattering signals from the plant whilst fluorescence signals were captured from transparent soil particles and labelled microorganisms, allowing the generation of quantitative data on samples approximately 3600 mm3in size with as good as 5 \uffce\uffbcm resolution at a rate of up to one scan every 30 minutes. The system tracked the movement ofBacillus subtilispopulations in the rhizosphere of lettuce plants in real time, revealing previously unseen patterns of activity. Motile bacteria favoured small pore spaces over the surface of soil particles, colonising the root in a pulsatile manner. Migrations appeared to be directed towards the root cap, the point \uffe2\uff80\uff9cfirst contact\uffe2\uff80\uff9d, before subsequent colonisation of mature epidermis cells. Our findings show that microscopes dedicated to live environmental studies present an invaluable tool to understand plant-microbe interactions.We have developed a tuneable workflow for the study of soil microbes in an imitative 3D soil environment that is compatible with routine and advanced optical imaging, is chemically customisable, and is reliably refractive index matched based on the metabolic profile of the study organism. We demonstrate our transparent soil pipeline with two representative soil organisms,Bacillus subtilisandStreptomyces coelicolor, and visualise their colonisation behaviours using fluorescence microscopy and mesoscopy. This spatially structured, 3D approach to microbial culture has the potential to further study the behaviour of other difficult-to-culture bacteria in conditions matching their native environment and could be expanded to study microbial interactions, such as interaction, competition, and warfare.
3.Graphical AbstractA step-by-step method for creating a tailored 3D culture medium for study of soil microbes.
The complete workflow can be split into three parts: Growth and observation, metabolic profiling to provide a stable refractive index matching solution, and production of the 3D soil environment. The 3D culture scaffold was created by cryomilling Nafion\uffe2\uff84\uffa2 resin pellets and size filtration. Chemical processing altered the surface chemistry of Nafion\uffe2\uff84\uffa2 particles and facilitated nutrient binding by titration of a defined liquid culture medium. Metabolic profiling determined non-metabolisable sugars and provided an inert refractive index matching substrate, which was added to the final nutrient titration. Inoculation and growth of the test strain allowed for downstream assessment of colonisation behaviours and community dynamicsin situby, for example, optical microscopy.