Raman spectra and confocal raw .czi files associated with Figures 3,4,5,6 (confocal) and figures 1,7 (raman).

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 biocompatible, optically transparent mineral scaffolds to visualize and investigate microbial colonization and activities occurring in these environments, in laboratory settings and <i>in situ</i>. 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 fluorescence imaging down to 100 µm depth, as well as Raman spectroscopy, allowing the identification of colonizing microbes, combined with the quantification of microbial activity at the single-cell level, using D₂O- or ¹³C-labeling. 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, using fluorescence 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.