Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil

The bio to chemical cycling of elements in soil plays a crucial role in environmental systems. With this protocol, the distribution of plant available element fractions can be imaged in 2D using DGT mass spectrometry imaging. This method is unique in its ability to visualize and quantify ultra trace levels of multiple inorganic solute species at the solute interface, substantially exceeding the spatial resolution of alternative methods.

In addition to the investigation of labor and solute fluxes in soils and sediments, this method can be applied to investigate how plant roots take up nutrient and contaminant elements. For DGT gel fabrication, first coat a thin film of polyurethane based mixed anion and cation binding gel suspension onto a glass plate, and place the plate into an oven to initiate gel formation by solvent evaporation. After repeating the application and evaporation three times, hydrate the resulting triple coated glass plate in a water bath to obtain a 0.1 millimeter thin, tear proof, mixed anion and cation binding gel.

To assemble the rhizotron, use two clamps to attach one small acrylic blade at the bottom of the rise of rhizotron with the pressure of the clamps directed onto the rhizotron frame, so that the plate does not bend inwards. Incline the rhizotron slightly toward the small plastic plate and fill the rhizotron with pre-moistened soil up to an approximate height of four centimeters. Agitate the rhizotron slightly to evenly distribute the soil and use a compaction tool to gently compress the soil by a few millimeters.

Repeat the filling and compression until the rhizotron is filled with soil, leaving a three centimeter gap at the top. Use tape to carefully fix a 13 by 22 centimeter piece of PTFE foil to the rhizotron frame one corner at a time, applying tension to ensure a flat foil surface. When the PTFE foil is flat and contiguous with the soil surface, attach a second piece of foil on the lower end of the rhizotron, overlapping the upper PTFE foil piece by one centimeter in the same manner.

When the second piece has been secured, apply a protective plastic foil cover. Place a front plate onto the soil filled and foil covered rhizotron, and place one rail around each side of the rhizotron. Then tighten the screws by hand to fix the rails in front plate to the rhizotron with the screws position toward the closed side of the rhizotron.

To water the soil, push pipette tips into the watering holes and let the water flow into the soil by gravity. To grow plants, plant up to two seedlings into the rhizotron and add five milliliters of water directly to the seedlings to support their growth. Cover the top opening of the rhizotron for the first two days after planting with a transparent moisture retaining film and wrap the rhizotron in aluminum foil to prevent microphytic growth.

Then place the planted rhizotron into a growth room with the environmental conditions set to the specific plant requirements and incline the rhizotron 25 to 35 degrees to ensure root development along the front plate via gravitropism. To apply the fabricated DGT gel, cut a 10 micron thick polycarbonate membrane with a 0.2 micron pore size to an at least greater than one centimeter width and length of each side of the gel and place the membrane onto the gel. Apply water to remove air bubbles from the stack and use vinyl electric tape to fix the membrane to the plate along all four edges of the gel.

After removing the front plate and protective foils, align the viewfinder of a digital single lens reflex camera equipped with a macro lens to the center of the region of interest in the gel and including a scale bar in the image acquire an orthogonal photo of the region of interest. Then align one edge of the plate equipped with the gel membrane stack with an edge of the open rhizotron. Gently bend the plate toward the soil and use the rails and screws to attach the plate to the rhizotron.

After the solitude sampling period, transfer the front plate from the rhizotron to a laminar flow hood with the gel membrane stack side facing up, and carefully remove the tape and the polycarbonate membrane covering the gel. Apply water to help the gel to float freely on a thin film of water on the plate with the soil contact side facing up and transfer the gel onto a polyethersulfone membrane with a 0.45 micron pore size and blotting paper support. After covering the gel stack with protective foil, place the stack in a vacuum gel dryer.

When the gel has fully dried, use double-sided adhesive tape to fix the dry gel together with other gel samples on a glass plate. To perform a laser ablation inductively coupled plasma mass spectrometry line scan analysis of the dry DGT gels, first fix the sample blanks and standards onto the laser ablation sample stage, and lock the laser stage into the ablation cell of the laser ablation system. In the laser ablation software move the region of interest on the gel surface and draw a single, approximately one milliliter long line across the surface of the gel standard.

Right-lick the line in the scan patterns window to verify that the laser ablation parameters have been set and adopted and use the duplicate scans tool to duplicate this line four times with an interline distance larger than the spot diameter. After repeating this line for each gel standard calibration blank and method blank, draw a single line along the top edge of the rectangular area of the gel sample to be analyzed and duplicate the line to create parallel lines for the entire sample area as demonstrated, using an interline distance of 300 to 400 micrometers. Verify that each start and end point of each line is properly focused on the gel surface and click analyze batch to initiate the sample sequence on the inductively coupled plasma mass spectrometer.

Click emission in the laser energy window to recharge the laser head. Click run to open the run experiment window and select selected patterns only. Set the washout delay to 20 to 30 seconds.

Select the enabled laser during scans box and set the laser warm-up time to 10 seconds. Then click run and okay to start the line scan analysis and monitor the raw signal intensity in counts per second for each isotope on the inductively coupled plasma mass spectrometer in real time. Each line should start and end with a gas blank.

After the analysis import the raw data file for each ablated line into a spreadsheet. The raw data table shows the inductively coupled plasma mass spectrometry readings for each isotope in counts per second, and the corresponding time points in seconds. List all the lines next to each other in different columns.

Calculate an average gas blank for each isotope from all of the gas blank values recorded before the line ablations and subtract the average gas blank from the corresponding raw intensities for each isotope to correct for the background signal. To apply internal normalization, divide the gas blank corrected signal intensity of each isotope by the gas blank corrected signal intensity of the internal standard carbon 13 for each data point to correct for variations in the amount of material ablated and instrumental drift. Crop the data before the start and after the end of each ablated line to remove the gas blank background signal and transpose the data table to obtain a grid matrix in which each row corresponds to an ablated line and each column corresponds to a normalized isotope intensity value.

Then apply the calibration function obtained from the analysis of the gel standards and save the calibrated data matrix as a text file. To generate an image, import the calibrated sample data matrix into the image analysis software as a text image and apply the aspect ratio correction factor and a look up table to visualize the chemical gradients in the solute image. Adjust the image color balance to control the lower and upper limits of the display range, add a calibration bar and save the solute image as a TIF file.

Use the copy to system command to copy the solid image and paste the image into desktop publishing software. Then scale match, align, and compose the solid image with a photo of the region of interest and the other solute images. Alignment of the solute images with a photographic image of the region of interest reveals that the sub-millimeter 2D solid flux distribution of different elements is highly variable according to the soil structure and root morphology.

For example, in this analysis of a young buckwheat root grown in carbonate free soil, fertilized with ammonium nitrate, the sub-millimeter solute distribution showed zones of decreased aluminum, phosphorus and iron fluxes alongside older root sections due to root uptake, and highly increased magnesium, aluminum, phosphorus, manganese and iron fluxes at the root apex due to localize nutrient mobilization processes. In this analysis, a distinct depletion of zinc, cadmium and lead can be observed at the immediate root position, illustrating that the roots of the metal tolerant Willow species Salix smithiana act as a localized sink for labile trace metals in contaminated soil. In this analysis, the distribution of labile trace metals alongside roots of Salix smithiana was co-localized with the distribution of pH, using a combined single layer planar optode-DGT cation binding gel.

This method combination revealed that increased solute fluxes of manganese, iron, cobalt, nickel, copper and lead were associated with a pH decrease by approximately one unit, suggesting pH induced metal solubilization. It is critical to ensure a close and stable contact between the DGT tool and the solid surface to avoid analytical artifacts. If in doubt, repeat the gel application procedure.

This method can be combined with other diffusion based solid imaging techniques, such as planar optodes to simultaneously assess a range of parameters involved in plant element uptake.