![]() Cryolite has previously been utilized to gain insights into the burrowing behaviors of marine invertebrates ( Flessa, 1972 Francoeur and Dorgan, 2014 Dorgan, 2018), the swimming behavior of Bacillus subtilis ( Zhu et al., 2014), and the spatial patterns of oxygen consumption by a bacterial pseudomonad species ( Oates et al., 2005), but its potential for microbial ecology studies remains largely unexplored. Cryolite’s RI (=1.339) ( Lewis, 2007) renders it effectively entirely transparent in aqueous solutions. We further show that cryolite (a naturally-occuring sodium aluminum fluoride crystal) is another promising biocompatible TS substrate with high potential for microbial studies. These hydrogel beads, however, are poorly suited for microbial visualization due to their large size (~500 µm) ( Ma et al., 2019), and their susceptibility to degradation by microbes as a carbon source ( Rice et al., 1992 Lin et al., 2018).ĭespite its limitations for plant root imaging studies, as we show here, Nafion remains a useful and versatile model for soil microbial ecology studies when microcosms utilizing only a few micrograms of TS are used these chambers only require imaging depths of tens or hundreds of microns (and thus are tolerant of slight RI mismatches) and are inexpensive to build. Indeed, Ma and colleagues cited these reasons as their motivation for developing an inexpensive hydrogel-bead-based TS system for root phenotyping ( Ma et al., 2019). This may be due to (a) the high cost of Nafion and (b) the RI of Nafion (RI = 1.35 Leis et al., 2005) not matching water closely enough (RI = 1.333) to allow deep (millimeter to centimeter) imaging. Surprisingly, given the novel visualization abilities it provides, the Nafion TS system has only been sparsely utilized for studying biological systems ( Downie et al., 2014 Downie et al., 2012 O'Callaghan et al., 2018). Downie et al., 2012 Downie et al., 2014 then demonstrated that plant roots with native-like architectures could be grown and visualized in Nafion-based TS ( Downie et al., 2012). In 2005, however, work by Leis et al., 2005 introduced the synthetic fluoropolymer Nafion (Chemours, Wilmington, DE) as a TS that could be RI-matched to water-based solutions that are compatible with culturing microorganisms in situ. In part, this is due to soil physicists favoring TS systems that use RI-matching liquids that do not support life, such as silicone oil. Despite their importance in that field, however, TS systems have only recently and rarely been used for applications in biology. TS systems have been used for over 25 years in geoengineering and hydrology, where they have helped solve challenging problems in soil physics ( Iskander et al., 2015). ‘Transparent soils’ (TS) are model systems where particles with a similar refractive index (RI) as their saturating liquids allow transmission of light and render a porous, ‘soil-like’ system optically transparent ( Iskander, 2010). However, the opacity and complexity of natural soils present a formidable challenge to the study of soil microbes in their native habitats. The metabolic activities of these microbes drive critical biogeochemical processes with biosphere-level effects ( Pold and DeAngelis, 2013 Wieder et al., 2013). Terrestrial soils are habitats to an unparalleled abundance and diversity of bacteria and fungi ( Delgado-Baquerizo et al., 2016 Fierer and Jackson, 2006 Horner-Devine et al., 2004). These data underscore the impact fungi have facilitating bacterial survival in fluctuating conditions and how these microcosms can yield insights into microscale microbial activities. We applied this system to ascertain that after a dry-down/rewetting cycle, bacteria on and near dead fungal hyphae were more metabolically active than those far from hyphae. We demonstrated that both substrates enable the growth, maintenance, and visualization of microbial cells in three dimensions over time, and are compatible with stable isotope probing using Raman. We assessed the polymer Nafion and the crystal cryolite as optically transparent soil substrates. ![]() To advance the study of soil processes, we constructed transparent soil microcosms that enable the visualization of microbes via fluorescence microscopy and the non-destructive measurement of microbial activity and carbon uptake in situ via Raman microspectroscopy. Microscale processes are critically important to soil ecology and biogeochemistry yet are difficult to study due to soil’s opacity and complexity.
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