Clear as mud redefined: Tunable transparent mineral scaffolds for visualizing microbial processes below ground

Abstract

Microbes inhabiting complex porous microenvironments in sediments and aquifers catalyze reactions that are critical to global biogeochemical cycles and ecosystem health. However, the opacity and complexity of porous sediment and rock matrices have considerably hindered the study of microbial processes occurring within these habitats. Here we generated microbially-compatible, optically transparent mineral scaffolds to visualize and investigate microbial colonization and activities occurring in these environments, in laboratory settings and in situ. Using inexpensive synthetic cryolite mineral, we produced optically transparent scaffolds mimicking the complex three-dimensional structure of sediments and rocks, by adapting a suspension-based freeze-casting technique commonly used in materials science. Fine-tuning of parameters such as freezing rate and choice of solvent provided full control of pore size and architecture. The combined effects of scaffold porosity and structure on the movement of microbe-sized particles, tested using velocity-tracking of fluorescent beads, showed diverse yet reproducible behaviors. The scaffolds we produced are compatible with epifluorescence microscopy, allowing the fluorescence-based identification of colonizing microbes by DNA-based staining and fluorescence in situ hybridization to depths of 100 µm. Additionally, Raman spectroscopy analysis indicates minimal background signal in regions used for measuring deuterium and ¹³C enrichment in microorganisms, highlighting the potential to directly couple D₂O or ¹³C stable isotope probing and Raman-FISH for quantifying microbial activity at the single-cell level.

To demonstrate the relevance of cryolite scaffolds for environmental field studies, we visualized their colonization by diverse microorganisms within rhizosphere sediments of a coastal seagrass plant, using epifluorescence microscopy. The new tool presented here enables highly resolved, spatially explicit, and multi-modal investigations into the distribution, activities, and interactions of underground microbes typically obscured within opaque geological materials until now.

Accepted manuscripts

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Author notes

Laura Quinn and Kriti Sharma co-lead authors