A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

This method provides researchers with a new way to prepare aquatic plant roots, the surrounding rhizosphere and bulk soil for elemental imaging techniques. The main advantage of this method is that it's relatively quick and affordable while also accurately preserving the distribution and speciation of elements of interest. This technique can be used to answer questions regarding many elements and plant systems from nutrients in agricultural systems to fate and transport of contaminants in fettermiation systems.

Be sure to work quickly while also be mindful of the desired area or areas of the root system for sampling. And take multiple samples per root system. To prepare the slam freezing equipment for an experiment, place two, five by five by 15 centimeter copper blocks horizontally into a clean cooler capable of holding liquid nitrogen.

And wearing the appropriate PPE, pour enough liquid nitrogen into the cooler to submerge the blocks. Once the bubbling subsides, place one spacer on each end of one copper block. Then, using tongs and cryogenic gloves, stand the other copper blog on its end to make retrieval easier when the sample is in place.

To collect the sample, either use a potted plant or use a shovel to begin extracting the desired plant and rhizosphere from the wet soil, taking care of that the dug hole is much larger than the desired root volume. Use a steel blade to cut away any excess soil, taking care not to disturb the soil within the desired area. When the desired areas reach, cut an approximately three by three by two centimeter root cube, and immediately placed the cube between the two spacers on the horizontal copper block.

For a slam freezing of the sample, used the cryogenic gloves to place the vertical copper block onto the spacers for about five minutes. When the bubbling subsides transfer the slam frozen rhizosphere cube sample into a piece of pre-labeled aluminum foil square. To freeze dry the soil cubes, when the freeze dryer has reached the proper vacuum pressure and temperature, place one frozen rhizosphere cube sample for clean and acid washed 50 milliliter tube or directly into the freeze dryer and use a clean disposable wipe to prevent dust from entering the vacuum pump.

Place the samples into the freeze dryer vessel for at least a few days until dry. When the tissues have been freeze dried, use a steel blade to dried soil cubes to an appropriate size for the analysis before placing the cubes into labeled forms inside a vacuum desiccator. After preparing epoxy according to manufacturer's instructions, use a dropper to slowly add epoxy to the form on one side of the soil, until the epoxy entirely covers the sample.

The soil will darken in color as the epoxy wets the soil. Once the forms are filled with epoxy, close the desiccator and turn on the vacuum. Check the level of a epoxy every 30 to 90 minutes for the first one to four hours, adding additional epoxy if necessary.

Once the epoxy has hardened, remove the sample. After embedding, use a diamond blade precision wet saw to cut the sample. Cut the sample in a different direction if no roots are obtained in the previous cut.

After trimming, manually sand the cut side of each sample with progressively finer sandpaper for 30 seconds per grit size. In this figure, several root diameters can be observed within the soil matrix as transverse sections. The roots may demonstrate varying levels of quality.

For example, in this image, a well-preserved root, a root distorted by the freeze drying process, and a root that was pulled out during the thin sectioning process can be observed. Analysis of this root transverse section with a lateral root in longitudinal section by synchrotron x-ray fluorescence imaging as demonstrated allows the detection of iron, manganese, and arsenic. The presence of iron within the soil and surrounding the root in the iron plaque is also visible in the light micrograph images.

Manganese is uniquely present within the cortex of the lateral root, but also co-localizes with iron in some areas within the iron plaque as a green-blue hue. Arsenic is mostly found within the vasculature of the lateral root merging into the vasculature of the primary root. Chemical speciation imaging reveals a variability in the localization of the arsenic species.

As observed in this tri-color plot, arsenite and arsenite glutathione are closely associated in the vasculature, while arsenate primarily localizes within the exterior of the root, associated with the iron plaque. Prior to sample collection, determine the desired part of the root system for elemental imaging. This will dictate the location orientation of the root key removed.

Following this protocol, samples can be imaged at micron scale or coarser using a variety of techniques, depending on the specific research questions, elements of the interest, or instrumentation available.