The authors acknowledge a joint seed grant to Seyfferth and Tappero to support collaboration between the University of Delaware and Brookhaven National Laboratory. Parts of this research used the XFM (4-BM) Beamline of the National Synchrotron Light Source II, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704.

1. Preparation of slam-freezing equipment
<ol>
	<li>Place two copper blocks (~5 cm x 5 cm x 15 cm) horizontally inside of a clean cooler capable of holding liquid nitrogen and pour enough liquid nitrogen to submerge the blocks. Once the bubbling subsides, place two spacers on top of one copper block at each end. 
	NOTE: The spacer height determines the height of the sample to be frozen; this example uses a 2 cm spacer to create cubes approximately 3 cm x 3 cm x 2 cm. The volume of the liquid nitrogen will depend o...

This paper describes a protocol to obtain preserved bulk soil + rhizospheres of wetland plant roots using a slam-freezing technique that can be used for elemental imaging and/or chemical speciation mapping.
There are several benefits of this method over existing methods. First, this method allows the simultaneous investigation of roots and the surrounding rhizospheres. Methods currently exist to preserve and chemically image roots out of their soil environment by washing away the soil and pres...

Roots and their rhizospheres are dynamic, heterogeneous, and critically important for understanding how plants obtain mineral nutrients and contaminants1,2,3. Roots are the primary pathway by which nutrients (e.g., phosphorus) and contaminants (e.g., arsenic) move from soil to plants and thus understanding this process has implications for food quantity and quality, ecosystem functioning, and phytoremediation. However, roots are dynamic in space and time growing in response to nutrient acquisition needs and they often vary in function, diameter, and structure (e.g., lateral roots, adventitious roots, root hairs)2. Heterogeneity of root systems can be studied on spatial scales from cellular to ecosystem-level and on temporal scales from hourly to decadal. Thus, the dynamic and heterogeneous nature of roots and their surrounding soil, or rhizosphere, poses challenges for capturing rhizosphere chemistry over time. Despite this challenge, it is imperative to study roots in their soil environment to characterize this critical plant-soil relationship.
The rhizosphere chemistry of wetland plants is particularly challenging to investigate because of steep oxygen gradients that exist from bulk soil to the roots, which change in space and time. Because roots need oxygen to respire, wetland plants have adapted to the low oxygen conditions of wetland soils by creating aerenchyma4, 5. Aerenchyma are hollowed cortical tissues that extend from shoots to roots, allowing the diffusion of air through the plant into the roots. However, some of this air leaks into the rhizosphere in less suberized parts of the roots particularly near lateral root junctions, less mature root tips and elongation zones6,7,8,9. This radial oxygen loss creates an oxidized zone in the rhizosphere of wetland plants that affects rhizosphere (bio-geo)chemistry and is distinct from the reduced bulk soil10,11,12. To understand the fate and transport of nutrients and contaminants in wetland rhizospheres and roots, it is critical to preserve the chemically reduced bulk soil, the oxidized rhizosphere, and roots of wetland plants for analysis. However, because the bulk soil contains reduced soil constituents that are oxygen-sensitive, root and soil preservation methods must preserve root structures and minimize oxygen-sensitive reactions.
Methods exist to fix plant tissues and preserve the ultrastructure for imaging, but those methods cannot be applied to chemically preserve roots growing in wetland soil. For investigations where only the elemental distribution within plant cells is desired, plants are typically grown hydroponically and roots can be easily removed from solution, fixed under high-pressure freezing and freeze substitution and sectioned for a variety of imaging applications including high-resolution secondary ion mass spectrometry (nanoSIMS), electron microscopy, and synchrotron X-ray fluorescence (S-XRF) analysis13,14,15. To investigate Fe plaque on the outside of wetland roots, these hydroponic studies must artificially induce Fe plaque formation in solution16, which does not accurately represent the heterogeneity of the distribution and mineral composition of Fe plaque formation and associated elements in situ17,18,19,20. Methods exist to preserve wetland soil and associated microorganisms with freeze-coring21, but it is difficult to obtain roots with this technique. Current methods to visualize roots growing in soil and their rhizospheric chemistry consist of two primary measurement types: elemental fluxes and total elemental concentration (and speciation). The former is typically measured using diffusive gradients in thin films (DGT)22,23,24, in which soil is placed into rhizoboxes to support plant growth in a laboratory setting and labile elements in the soil diffuse through a gel into a binding layer. This binding layer can then be imaged to quantify the labile elements of interest. This technique can successfully illustrate relationships between roots and the rhizosphere24,25,26,27, but artefacts from root-bounding may exist by growing plants in rhizoboxes, and information on the root interior is not captured with DGT. The latter involves sampling of the roots and rhizosphere, preserving the sample, and directly analyzing elemental distribution on a sample section. For this environmental sampling of wetland plant roots and their surrounding rhizosphere, careful sample handling is required to avoid artefacts from sample preparation.
Here a protocol is described that effectively preserves root structures and rhizosphere chemistry of wetland plants by slam-freezing and freeze drying. Flash-freezing can drastically slow down transformations of oxygen sensitive solutes but may damage roots and may cause mobilization when samples dry out. However, slam-freezing where the sample is frozen between copper blocks pre-cooled with liquid nitrogen minimizes root damage and sample distortion28. The preserved samples are then embedded in an epoxy resin that preserves As speciation20, 29 and can be cut and polished for imaging of roots within their rhizosphere soil. The samples in this report were analyzed by S-XRF chemical speciation imaging after thin sectioning. However, other imaging techniques could also be used, including laser ablation-inductively coupled plasma mass spectrometry (LA-ICP-MS), particle induced x-ray emission (PIXE), secondary ion mass spectrometry (SIMS), and laser induced breakdown spectroscopy (LIBS) imaging.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Copper blocks</td>
			<td>McMaster Carr</td>
			<td>89275K42</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond blade</td>
			<td>Buehler</td>
			<td>15 LC, 102 mm x 0.3 mm</td>
			<td>operation speed: 225 rpm</td>
		</tr>
		<tr>
			<td>Epoxy forms</td>
			<td>Struers</td>
			<td>40300085</td>
			<td>FixiForm</td>
		</tr>
		<tr>
			<td>Epoxy</td>
			<td>Epotek</td>
			<td>301-2FL</td>
			<td></td>
		</tr>
		<tr>
			<td>Superglue</td>
			<td>Loctite</td>
			<td>404</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin sectioning machine</td>
			<td>Buehler</td>
			<td>PetroThin</td>
			<td></td>
		</tr>
		<tr>
			<td>Wet saw</td>
			<td>Buehler</td>
			<td>IsoMet 1000</td>
			<td></td>
		</tr>
	</tbody>
</table>

a method to preserve wetland roots and rhizospheres for elemental imaging

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The rhizosphere chemistry of wetland plants is particularly challenging to capture because of steep oxygen gradients from the roots to the bulk soil. Here a protocol is described that effectively preserves root structure and rhizosphere chemistry of wetland plants through slam-freezing and freeze drying. Slam-freezing, where the sample is frozen between copper blocks pre-cooled with liquid nitrogen, minimizes root damage and sample distortion that can occur with flash-freezing while still minimizing chemical speciation changes. While sample distortion is still possible, the ability to obtain multiple samples quickly and with minimal cost increases the potential to obtain satisfactory samples and optimizes imaging time. The data show that this method is successful in preserving reduced arsenic species in rice roots and rhizospheres associated with iron plaques. This method can be adopted for studies of plant-soil relationships in a wide variety of wetland environments that span concentration ranges from trace-element cycling to phytoremediation applications.

This method allows for preservation of roots and chemical species in the roots and rhizosphere of wetland plants and into the bulk soil. In this work, the method was used to evaluate As speciation and co-localization with Fe and Mn oxides and plant nutrients in the rhizosphere of rice (Oryza sativa L.). Rice was grown at the RICE Facility at the University of Delaware where 30 rice paddy mesocosms (2 m x 2 m, 49 plants each) are used to grow rice under various soil and water management conditions with the goal o...

Watch this Scientific Journal Video about A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging at JoVE.com

A Method to Preserve Wetland Roots and Rhizospheres for Elemental Imaging

We describe a protocol to sample, preserve, and section intact roots and the surrounding rhizosphere soil from wetland environments using rice (Oryza sativa L.) as a model species. Once preserved, the sample can be analyzed using elemental imaging techniques, such as synchrotron X-ray fluorescence (XRF) chemical speciation imaging.

Roots extensively interact with their soil environment but visualizing such interactions between roots and the surrounding rhizosphere is challenging. The ...