An improved method to identify osmium-stained organic matter within soil aggregate structure by electron microscopy and synchrotron X-ray micro-computed tomography
An improved method to identify osmium-stained organic matter within soil aggregate structure by electron microscopy and synchrotron X-ray micro-computed tomography
复制标题
DOI：
10.1016/j.still.2019.04.010
复制
发表时间：
2019-08-01
影响因子：
6.5
通讯作者：
Wagai, Rota
中科院分区：
文献类型：
--
作者：
Arai, Miwa;Uramoto, Go-Ichiro;Wagai, Rota
表层土壤中的有机质（OM）主要存在于多孔矿物占主导地位的集群称为团聚体。最近的研究表明，基于同步加速器X射线显微计算机断层扫描（mu CT）结合气相锇（Os）染色预处理，OM在大型大团聚体结构中的定位。在这里，我们开发了一种新的方法，以确定OM的位置在亚毫米级的聚集体在更高的空间分辨率和评估其适用性，使用一个良好的特点，表面火山土壤，一个allophanic Andisol，免耕下与叶堆肥添加。将水稳定的聚集体（直径500-800 μ m）包埋在琼脂糖中，化学固定，并用四氧化锇和硫代碳酰肼反复染色，然后用乙醇逐渐脱水并树脂固定。扫描电子显微镜和μ CT观察显示样品制备过程中没有物理变化的迹象。能量色散X射线光谱分析的显微镜切割表面的聚集确定植物碎屑高度富集的Os和更小的无定形OM（< 10 μ m），其中Fe，Al，和Si以及Os。通过Os L-3 X射线吸收限以上和以下的光子能量的同步辐射μ CT成像（10.87 keV）和随后的图像计算显示了Os染色体素的分布下降到微米尺度。与以前的研究相比，分辨率的提高部分归因于液态染色和硫代碳酰肼桥接。讨论了该方法的其他优点和注意事项。
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.