An improved method to identify osmium-stained organic matter within soil aggregate structure by electron microscopy and synchrotron X-ray micro-computed tomography

Organic matter (OM) in surface soil is largely present within porous mineral-dominant clusters called aggregates. Recent studies have shown the localization of OM within large macroaggregate structure based on synchrotron X-ray micro-computed tomography (mu CT) coupled with a vapor-phase, osmium (Os)-staining pretreatment. Here we developed a new approach to identify OM locations within submillimeter-sized aggregates at higher spatial resolution and assessed its applicability using a well-characterized surface volcanic soil, an allophanic Andisol, under no-tillage with leaf compost addition. Water-stable aggregates (500-800 mu m in diameter) were embedded in agarose, chemically fixed, and repeatedly stained by osmium tetroxide and thiocarbohydrazide, followed by gradual dehydration with ethanol and resin fixation. Scanning electron microscopy and mu CT observation showed no signs of physical alteration during sample preparation. Energy-dispersive X-ray spectroscopy analysis of the microtome-cut surface of the aggregate identified plant detritus highly enriched in Os and much smaller amorphous OM ( < 10 mu m) that were high in Fe, Al, and Si as well as Os. Synchrotron mu CT imaging by photon energies above and below Os L-3 X-ray absorption edge (10.87 keV) and subsequent image calculation showed the distribution of Os-stained voxels down to micrometer scale. The improved resolution compared to previous studies was partly attributable to liquid-state staining and thiocarbohydrazide bridging. Other advantages and some cautions of the proposed method were discussed.