Clear as mud redefined: Tunable transparent mineral scaffolds for visualizing microbial processes below ground
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1.
California Institute of Technology
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.
Copyright and License (English)
© 2025, © The Author(s) 2025. Published by Oxford University Press on behalf of National Academy of Sciences. This article is available under the Creative Commons CC-BY-NC license and permits non-commercial use, distribution and reproduction in any medium, provided the original work is properly cited.
Acknowledgement (English)
The authors thank Julia Schwartzman and Otto Cordero (MIT) for providing the bacterial strains used in this study, Shaelyn Silverman (Caltech) for assistance with bacterial cultivation, George Rossman (Caltech) for use of his Raman instrument, Julie Kornfield for inspiration and discussions about this work, Emmanuelle Botté (Manuscribe) for editorial assistance, and two anonymous reviewers whose suggestions improved this manuscript. The authors additionally acknowledge Caltech's Kerckhoff Marine Laboratory for facility support and access to the seagrass field site. Collection of Z. marina sediment cores for incubation experiments was carried out under permit ID SC-13962 issued from the State of California–Department of Fish and Wildlife. V.J.O. is a CIFAR Fellow in the Earth 4D Subsurface Science and Exploration program.
Funding (English)
This work was supported through a collaborative pilot research grant from the Caltech’s Donna and Benjamin M. Rosen Bioengineering Center (ROSENBE.PROJECTS-6.VJO-ENDOW.ROSENBE to K.T.F. and V.J.O.) as well as support from the US Department of Energy, Office of Science, Office of Biological and Environmental Research under Award Number [DE-SC0022991], from the Simons Foundation Principles of Microbial Ecosystems (PriME) consortium (V.J.O.), and partial support from the Resnick Sustainability Institute (RSI.DIRINIT-VJO-RESNICK.SRA; to V.J.O.).
Contributions (English)
Laura K. Quinn (Resources, Data curation, Formal analysis, Investigation, Visualization, Methodology, Writing—original draft), Kriti Sharma (Supervision, Validation, Investigation, Visualization, Methodology, Writing—original draft), Katherine T. Faber (Conceptualization, Supervision, Funding acquisition), and Victoria J. Orphan (Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Project administration, Writing—review & editing).
Data Availability (English)
The authors confirm that the data supporting the findings of this study are available within the article and its Supplementary material. The raw data files are openly available in Figshare at https://doi.org/10.6084/m9.figshare.27173274.v1.
Supplemental Material
Supplementary material is available at PNAS Nexus online.
pgaf118_Supplementary_Data - zip file
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Additional details
- California Institute of Technology
- ROSENBE.PROJECTS-6.VJO-ENDOW.ROSENBE
- Office of Biological and Environmental Research
- DE-SC0022991
- Simons Foundation
- Principles of Microbial Ecosystems (PriME) -
- Resnick Sustainability Institute
- RSI.DIRINIT-VJO-RESNICK.SRA
- Available
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2025-05-09Corrected and typeset
- Caltech groups
- Division of Engineering and Applied Science (EAS), Division of Geological and Planetary Sciences (GPS)
- Publication Status
- Published