Testing Contrast Agents to Improve Micro Computerized Tomography (μCT) for Spatial Location of Organic Matter and Biological Material in Soil

Abstract

Soil carbon is essential for soil and ecosystem functioning. Its turnover and storage in soil are multifaceted processes that involve microbial activity in complex physical matrices. Biological litter, which include plants, animals, and microorganisms, is decomposed in soil stimulating soil biota (archaea, bacteria, fungi, protists, and animals) activity and yielding soil organic matter (SOM). Such decomposition processes are influenced by local physico-chemical characteristics including the spatial distribution of aggregates and pores. More refined analytical tools are needed to better understand these processes, especially considering the spatial 3D structure of soil matrices. Using synchrotron radiation (X-ray) micro computerized tomography (SR-µCT), we tested different contrast agents (staining methods) based on silver (Ag), eosin (Br based), and liquid and gaseous iodine (I) in order to spatially image biological material and SOM in soil samples. We also performed K-edge SR-µCT for the Ag and I 2 treatments and conventional µCT for additional soil samples applying the I 2 treatment. Our results indicated that I 2 was the most efficient contrast method for SR-µCT imaging of soil samples. I 2 qualitatively improved the images, but mainly, by using the K-edge SR-µCT, this method provided a powerful tool to determine the spatial location of SOM. We acknowledge that the use of SR-µCT is an expensive technique to study soil samples, which comes with bottlenecks in terms of access to facilities and measurement time. Nevertheless, we show that the I 2 treatment improved soil images also using standard µCT. In conventional µCT the I 2 treatment improved the visualization of biological material and consequently improved the qualitative analysis of fine plants roots and micro-fauna (Collembola). This improvement may have a positive implication in soil biology, by improving a non-destructive method to detect fungi (SR-µCT), soil fauna (conventional µCT) and roots in undisturbed soil samples. An unexpected finding was that the I 2 treatment also stained the plastic sample containers (nylon and polyimide), indicating the potential for the I 2 staining procedure to be applied for the detection of plastic pollution in soil samples.

Chemical staining of particulate organic matter for improved contrast in soil X-ray µCT images

“…While manual pin-pointing of the PMA stained POM was not impeded, automated POM extraction from the CT volume may still prove to be challenging due to the lack of image contrast between POM and mineral soil www.nature.com/scientificreports/ particles. Very recent work by Lammel et al 38 applied a machine learning segmentation tool in synchrotron-based soil CT volumes but experienced limited success. Piccoli et al 39 suggested that an operator-based ability for the selection of thresholds may still result in the most accurate segmentation of POM in soil.…”

“…However, the K-edge of Mo is situated at 20 keV, an energy level at which most X-rays may be attenuated by the soil mineral fraction, thereby probably making a dual energy approach similar to that used for Os challenging for non-synchrotron scanners and probably also for synchrotrons. Very recently, Lammel et al 38 identified gaseous iodide (I 2 ) as a plausible candidate for selective staining of OM in soil for use with synchrotron scanners. However, they did not fully demonstrate www.nature.com/scientificreports/ its selectivity for OM versus silt and clay sized mineral particles, nor in X-ray µCT soil volumes obtained with non-synchrotron scanners.…”

A Review of X‐Ray Imaging at the BAMline(BESSY II)

“…To increase the contrast between the two organic materials, Lammel et al stained a whole sample with iodine (I 2 ), which mostly interacts with SOM instead of the fungi material. [ 39 ] Differential absorption edge tomography at the I 2 ‐K‐edge (33.2 keV) finally emphasized the SOM, see Figure c. This is rendered in cyan color in Figure 8d.…”

Opportunities and limits in imaging microorganisms and their activities in soil microhabitats

“…A number of heavy metal stains have been tested in order to accentuate the contrast (Peth et al, 2014;Van Loo et al, 2014;Maenhout et al, 2021). So far, only osmium (Peth et al, 2014;Zheng et al, 2020;Maenhout et al, 2021) and iodine (Lammel et al, 2019) stains have proved to specifically stain organic matter (though Schlüter et al (2022) suggest that Osmium binds to some minerals), to provide a detectable staining and to diffuse through the soil matrix. Osmium was successfully used to map soil organic matter (Rawlins et al, 2016).…”