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

1. Fabrication of DGT gels

NOTE: Several DGT gel types are available for 2D imaging of labile solute species at high (sub-mm) spatial resolution³⁵. Here, the fabrication of three well-characterized high resolution (HR)-DGT binding gels used in solute imaging applications is briefly summarized. Laboratory procedures for trace element analysis, as well as detailed fabrication procedures of all presented HR-DGT gels are described in the Supporting Information (SI) sections S1 and S2.

1. Polyurethane-based mixed anion and cation binding gel (HR-MBG; SI S2.1)³¹
   1. Prepare a polyurethane gel suspension with homogeneously dispersed zirconium (IV) hydroxide and iminodiacetate (IDA) phases.
   2. Coat the gel suspension in a thin film onto a glass plate and initiate gel formation by solvent evaporation to obtain a 0.1 mm-thin, tear-proof mixed anion and cation binding gel (HR-MBG).
2. Polyacrylamide-zirconia anion binding gel (HR-ABG; SI S2.2)³⁶
   1. Fabricate a 0.4 mm-thin agarose cross-linked polyacrylamide gel (APA) following established gel casting procedures³⁷ (see SI S2.4 for a detailed protocol of the APA fabrication).
   2. Precipitate zirconium (IV) hydroxide phases into the precast APA gel to obtain a 0.4 mm-thin anion binding gel (HR-ABG).
3. Polyacrylamide-iminodiacetate cation binding gel (HR-CBG; SI S2.3)³⁸
   1. Prepare a polyacrylamide gel suspension with homogeneously dispersed IDA phases.
   2. Cast the gel between two glass plates and initiate the polymerization reaction to obtain a 0.4 mm-thin cation binding gel (HR-CBG) where the IDA phases are settled to one side of the gel.

2. Plant cultivation

NOTE: The experimental system uses rhizotrons⁴ (Figure 1) to grow plants in unsaturated soil for solute imaging. First, rhizotron soil filling and watering are described, then details on the experimental plant growth are given. Details on the rhizotron design and soil substrate preparation before filling into the rhizotron are presented in the SI section S3.

Figure 1: Rhizotron design (not to scale). (A) Exploded view of a rhizotron growth container. (B) Assembled rhizotron during plant growth. Please click here to view a larger version of this figure.

1. Rhizotron soil filling
   1. Before filling the rhizotron with pre-moistened soil (gravimetric water content, , is known; see SI S3.2), close the watering holes at the back of the rhizotron using adhesive tape and remove the front plate and its fixation rails and screws.
   2. Weigh the empty rhizotron (excluding the front plate, rails, and screws), eight 5 cm x 11 cm acrylic plates and 16 clamps (Table of Materials) and record the sum of weights.
   3. Attach one small acrylic plate at the bottom of the rhizotron using two clamps on each side, with the pressure of the clamps directed onto the rhizotron frame, so the plate does not bend inwards and the volume is constant.
   4. Incline the rhizotron slightly towards the small plastic plate and fill with pre-moistened soil up to a height of ~4 cm (Figure 2A). Distribute the soil inside the rhizotron by agitating the rhizotron slightly and gently compress the soil by a few mm (depending on the specific soil characteristics) with a compaction tool (Figure 2B).
   5. Repeat 2.1.3 – 2.1.4 until the rhizotron is filled with soil (Figure 2C). Leave a gap of ~3 cm at the top for the subsequent planting of seedlings into the rhizotron.
   6. Weigh the soil-filled rhizotron (including 8 small plates and 16 clamps) and record the weight. From this, subtract the empty rhizotron weight obtained in 2.1.2 and record the weight difference, i.e., the mass of moist soil, (g), in the rhizotron.
   7. Calculate the mass of dry soil in the rhizotron, (g), according to Eq. 1, and then calculate the dry soil bulk density, (g cm^-3), in the rhizotron according to Eq. 2.
      
      
      Here, (cm³) is the total inner volume of the rhizotron.
      NOTE: Typical values in the rhizotron are between 1.0-1.4 g cm^-3. Do not exceed 1.5 g cm^-3 as root growth might be impeded above this value.
   8. Place the soil-filled rhizotron on a support box and remove all clamps and small plates from the rhizotron (Figure 2D). Carefully clean the rhizotron frame (i.e., edges) using tissue paper, as remaining soil particles on the frame can cause leaks.
      NOTE: The exposed soil surface must be homogeneous and leveled with the rhizotron frame without any cracks or gaps. If not, empty the rhizotron and repeat 2.1.2 – 2.1.8.
   9. Cut two pieces of polytetrafluoroethylene (PTFE) (Table of Materials) foil to 22 cm x 13 cm each. In addition, cut a piece of plastic foil to 46 cm x 15 cm. Record the sum of PTFE and plastic foil weights. Place the first piece of PTFE foil on the top half of the exposed soil surface in the rhizotron, extending ~1 cm over the soil level at the top of the rhizotron.
   10. Carefully fix the PTFE foil to the rhizotron frame using adhesive tape (Figure 2E). Start by fixing one corner at the top of the rhizotron first, followed by its opposite corner and finally the two corners further down the rhizotron. Apply tension when fixing the corners 2-4 to ensure a flat foil surface. If folds emerge, open and re-fix the tape at individual corners (not all at once) until all folds are removed and the PTFE foil is flat and contiguous with the soil surface.
   11. Place the second piece of PTFE foil on the lower end of the rhizotron, overlapping the upper PTFE foil piece by ~1 cm. Repeat 2.1.10 for fixing the second PTFE foil to the rhizotron.
   12. Place the plastic foil (46 cm x 15 cm) onto the PTFE foils. Fix the plastic foil using the fixation procedure as detailed in 2.1.10.
   13. Place a front plate onto the soil-filled and foil-covered rhizotron. Place one rail around each side of the rhizotron and tighten the screws by hand to fix the rails and, thereby, the front plate to the rhizotron. The screws are positioned towards the closed side of the rhizotron, i.e., the side with the watering holes (Figure 1A).

Figure 2: Rhizotron assembly and filling to grow plants in soil for solute imaging in the rhizosphere. (A) Soil filling into the rhizotron. (B) Compaction of the filled soil using a compaction tool. (C) Soil-filled rhizotron with small acrylic plates and clamps. (D) Soil-filled rhizotron with exposed soil surface. (E) Soil-filled rhizotron partly covered with a protective PTFE foil. Please click here to view a larger version of this figure.

Figure 3: Rhizotron handling and DGT gel application. (A) Soil watering using 10 mL pipette tips in the watering holes in the back of the rhizotron. (B) Planting of seedlings (indicated as green spots) into the soil-filled and closed rhizotron. (C) Rhizotron planted with Salix smithiana cuttings and removed front plate and plastic foil cover. (D) Carefully peeling off the PTFE foil cover before DGT gel application. (E) High-resolution photo of the soil-root interface ROI. (F) Application of the front plate equipped with the DGT gel onto the rhizotron. (G) Photo of the ROI with the DGT gel applied during solute sampling. Please click here to view a larger version of this figure.

2. Watering the soil
   1. Determine the water holding capacity (WHC) of the experimental soil in the rhizotron. To this end, manufacture two rhizotrons with an open bottom and fill them with soil as specified in section 2.1. Fully saturate these open, soil-filled rhizotrons by immersion in a water container for 16 h and drain the rhizotrons for 8 h.
   2. Take a composite soil sample of ~50 g from random locations in the open-bottom rhizotrons and dry the sample at 105 °C to determine according to Eq. S1. The determined corresponds to the WHC of the soil and is, therefore, expressed as (g g^-1).
   3. Define the target in the experimental rhizotron. During the growth phase, set a of 60 % of the WHC (i.e., the WHC factor, ) to supply plants with an adequate amount of water while avoiding anoxic conditions in the rhizotron.
   4. Calculate the total mass of water to be added, (g), to irrigate the soil in the rhizotron at the target according to Eq. 3. Account for the mass of water present in the soil in the rhizotron, (g).
      
   5. Divide by the number of rhizotron watering holes (here 14) to obtain the mass of water to be added in each watering hole. Add water by pushing 10 mL pipette tips into the watering holes and letting the water flow into the soil by gravity (Figure 3A).
3. Plant growth
   1. Pre-germinate seeds of the experimental plant (e.g., on wet filter paper) according to the specific seed germination requirements until the radicle emerges (up to 1 cm long).
   2. Plant up to two seedlings into the rhizotron as indicated in Figure 3B. At planting, add ~5 mL 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 (Table of Materials). Wrap the rhizotron in aluminum foil to prevent microphytic growth.
      NOTE: Apart from seedlings, plant cuttings can also be cultivated in rhizotrons.
   3. Transfer the planted rhizotron into a growth room, with environmental conditions (i.e., temperature, humidity, light intensity) set to the specific plant requirements. Incline the rhizotron at 25°-35° to ensure root development alongside the front plate via gravitropism.
   4. During plant growth, gravimetrically maintain the target water content in the rhizotron by periodical watering every 2-4 days using the rhizotron watering holes as detailed in 2.2.5. Keep the soil surface at the top opening moist by regular additions of ~5 mL water.
      NOTE: If plants are grown for extended periods and the plant biomass is expected to substantially decrease the amount of water added to the rhizotron by the proposed method, account for the weight of the plant biomass by growing plants in separate rhizotron replicates and harvesting and weighing the plant tissues at defined intervals.
   5. Once roots reach a suitable location along the front plate, preferentially in the center of the rhizotron, apply DGT gels for sampling the rhizospheric solute distribution.

3. Sampling the solute distribution

1. Gel application
   1. Increase in the rhizotron from 60 % to 80 % 24 h before gel application as detailed in 2.2.4.-2.2.5. This ensures good soil-gel contact and allows solute diffusion into the gel while avoiding anoxic soil conditions during solute sampling.
   2. Take a new, acid-cleaned front plate, align it on the rhizotron used for sampling, and mark the regions of interest (ROIs) on the plate. Transfer the plate into a laminar flow bench or any other dust- and metal-free environment, unmarked side facing up.
   3. Cut the DGT binding gel on an acrylic support to the required rectangular size corresponding to the ROI, usually around 3 cm × 5 cm, using PTFE-coated razor blades. Cut the gel by pressing rather than sliding the razor blade to ensure a clear cut. Place the rectangular gel piece on the unmarked side of the plate at the marked location of the ROI.
      NOTE: If HR-MBG is used, a 100 µm-thin spacer foil may be added underneath the gel to ensure that the gel is in good contact with the soil-root system.
   4. Cut a 10 µm-thin polycarbonate membrane (0.2 µm pore size; Table of Materials) to a size which extends the gel size by ≥1 cm at each side and place the membrane onto the gel. Apply some water to remove air bubbles from the stack.
   5. Fix the membrane along all four edges using vinyl electrical tape (Table of Materials). In the process, carefully remove air bubbles trapped in between the gel and the membrane using plastic tweezers. The tape must only get in contact with the membrane and not with the gel.
      NOTE: If the tape comes in contact with the gel, air bubbles are trapped between the gel and the membrane, or the final membrane surface shows folds, the gel/membrane stack needs to be reassembled as the diffusive flux of solutes into the gel can be impaired³⁵ (repeat 3.1.3 – 3.1.5).
   6. Place the rhizotron on a stand, remove the rails and carefully lift off the front plate (Figure 3C). Remove the plastic foil, cut off the PTFE foil at the edges of the rhizotron and slowly peel off the PTFE foil to avoid disturbance of the soil-root system (Figure 3D).
   7. Take an orthogonal photo of the ROI using a digital single-lens reflex (DSLR) camera (Table of Materials) to facilitate the interpretation and presentation of the solute distribution based on the soil structure and root morphology (Figure 3E). Use a camera stand and, if available, a macro lens. Align the camera so that the center of the photo corresponds to the center of the ROI, and the camera’s focus plane is parallel to the soil surface. Include a scale bar (e.g., a ruler) in the photo.
   8. Attach the plate equipped with the gel/membrane stack to the open rhizotron (Figure 3F). Therefore, align one edge of the plate with an edge of the rhizotron and gently ‘bend’ the plate towards the soil. This mode of application helps to avoid air bubbles between the gel/membrane stack and the soil-root system. Fix the front plate using the rails and screws.
      NOTE: This step is critical and needs to be carried out carefully. The plate cannot be moved after establishing contact between the gel/membrane stack and the soil-root system without displacing soil and roots.
   9. Record the exact starting time of gel deployment and take a photo of the deployed gel at the ROI as given in 3.1.7 (Figure 3G). Wrap the rhizotron in aluminum foil and transfer into the growth room until the end of the solute sampling period (often 24 h).
2. Gel retrieval
   1. Place the rhizotron on a support box, carefully lift off the front plate and rinse the gel/membrane stack on the plate with water to wash off adhering particles (Figure 4A). Record the exact end time of gel deployment. Sample soil from the ROI to determine the actual w_soil in the rhizotron according to Eq. S1.
   2. Transfer the front plate into the laminar flow bench, gel/membrane stack facing up. Retrieve the gel from the front plate by carefully removing first the tape along all four edges and then the polycarbonate membrane covering the gel (Figure 4B). 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.
      NOTE: It is critical to keep track of the gel orientation. The gel side exposed to the soil and roots must always face up (toward the user).
3. Gel drying
   1. Cut a rectangular piece of gel blotting paper (Table of Materials) and place a slightly smaller piece of a polyethersulfone membrane (0.45 µm pore size; Table of Materials) on top.
   2. Transfer the gel from the plate onto the gel blotting paper/membrane stack using plastic tweezers, soil-contact side facing up. The gel must be relaxed (i.e., not stretched) and completely flat, without any air bubbles between gel and membrane. Apply some water onto the gel blotting paper/membrane stack to facilitate the gel transfer and positioning.
   3. Cover the gel blotting paper/membrane/gel stack completely with a piece of plastic foil and label the gel sample and its orientation on the plastic foil (Figure 4C). Place the gel blotting paper/membrane/gel/plastic foil stack in a vacuum gel dryer (Table of Materials) and dry until the stack is fully desiccated (typically 48-72 h). For HR-MBG, set the temperature to 50-55 °C, for HR-ABG and HR-CBG best dry at room temperature.
   4. Remove the gel blotting paper from the dried stack and transfer the gel, which is now inseparably merged with the polyethersulfone membrane, into a zip bag. The plastic foil cover stays on the gel until shortly before the LA-ICP-MS analysis.
   5. Include a gel piece from the original gel sheet as a method blank, which does not get exposed to soil. Process the method blank gel identical to the sample gel following 3.3.2 – 3.3.4.

Figure 4: DGT gel retrieval and preparation for drying upon solute sampling. (A) Plate with the DGT gel and rhizotron directly after solute sampling. (B) Retrieval of the DGT gel from the plate in a laminar flow bench. (C) Stack of gel blotting paper/polyethersulfone membrane/DGT gel/plastic foil cover for gel drying. Note that the gel is slightly colored after its deployment on the rhizosphere soil. Please click here to view a larger version of this figure.

4. Chemical analysis of the DGT binding gel

NOTE: In this protocol, analysis of the solute distribution on the DGT binding gel is accomplished by LA-ICP-MS using a nanosecond 193 nm ArF excimer LA system equipped with a two-volume ablation cell coupled to a quadrupole ICP-MS (Figure 5). All instruments are listed in the Table of Materials. Alternatively, nanosecond 213 nm or 266 nm solid-state LA systems can be applied^{36,39,40,41,42,43}. If enhanced sensitivity or mass resolution is required, sector field ICP-MS is an alternative to quadrupole ICP-MS^15,44. Details on the preparation of DGT gel standards for external calibration and coupling of the LA system to the quadrupole ICP-MS are presented in the SI sections S4 and S5.

Figure 5: LA-ICP-MS setup for DGT gel analysis. (A) Nanosecond 193 nm ArF excimer LA system and quadrupole ICP-MS. (B) Dried gels mounted onto glass plates and fixed on the LA sample stage ready for introduction into the ablation cell. (C) Nebulizer gas (Ar) from ICP-MS and aerosol carrier gas (He or Ar) from ablation cell connected to the ICP via a two-way Y-splitter and torch adapter fitting. Please click here to view a larger version of this figure.

1. Sample preparation for LA-ICP-MS
   1. Transfer the dried gel samples, standards, and method blanks (which are merged with the polyethersulfone membrane support) onto individual pieces of double-sided adhesive tape (Table of Materials), gel side facing up. Crop excess tape parts to save space in the ablation cell.
   2. Mount the dried gels onto glass plates. Use individual glass plates for each gel sample, standard series, or method blank to allow for flexible arrangement on the LA sample stage (typical size of 10 cm × 10 cm). Use a glasscutter to adjust the glass plate size as needed. Fix the glass plates with the gels on the LA sample stage (Figure 5B) using plasticine (Table of Materials). Level the gel surface by adjusting the stage floor and lock the sample stage into the ablation cell.
2. LA-ICP-MS line-scan analysis
   1. Couple the LA system to the ICP-MS as specified in the SI section S5. In the LA software (Table of Materials), set the camera light settings (i.e., ‘Ring’, ‘Coax’ and ‘Transmitted’) to illuminate the gel surface in the ablation cell and focus onto the gel surface by adjusting the z-axis distance. Move to random locations across the cell to ensure that all gel surfaces are in focus.
   2. Set the LA parameters in the ‘Laser Setup’ window of the LA software. Typical settings used in LA-ICP-MS line-scan analysis of DGT gels are: mode continuous; energy output 20-30%; repetition rate 10-20 Hz; spot diameter 100-200 µm; scan speed 150-250 µm s^-1.
      NOTE: The parameters need to be optimized for the type of gel used and may vary. Parameters can also be enhanced for increasing signal to noise ratio, spatial resolution, or reducing acquisition times depending on the experimental and instrumental setup. Use a relatively low energy output (≤40 %) and repetition rate (≤25 Hz) to avoid penetrating through the gels into the backing materials³⁹. Make sure that the laser scan speed is ≤ the spot diameter divided by the total ICP-MS scan cycle duration (see 4.2.6) to avoid compression of the datapoints and thus a loss of resolution in scan direction⁴⁵. For example, when setting a spot diameter of 200 µm and a total ICP-MS scan cycle duration of 0.25 s, the scan speed should be ≤800 µm s^-1.
   3. Select the line tool and draw a single, ~1 mm-long line pattern across a gel standard. Right click the line pattern in the ‘Scan Patterns’ window and verify that the LA parameters set in 4.2.3. have been adopted. Use the ‘Duplicate Scans’ tool to duplicate this line four times, with a interline distance (distance between the line center) larger than the spot diameter (Figure 6). This approach offers a total of five parallel lines per gel standard (n = 5). Repeat this step for each gel standard, calibration blank and method blank.
   4. Move to the gel sample and draw a single line along the top edge of the rectangular area to be analyzed. Duplicate the line to create parallel lines for the entire sample area as specified in 4.2.3. Use an interline distance of 300-400 µm. Make sure the focus (z-axis distance) is set correctly for each start and end point of each line.
      NOTE: The spatial resolution of the analysis is higher along the scan direction compared to the interline distance. Therefore, start and end points of the lines may best follow the direction of the analyte gradients. For rhizosphere gradients, this is usually perpendicular to the root axis (Figure 6).
   5. In the ICP-MS software (Table of Materials), set up a time-resolved ‘Data Only’ method in the ‘Method’ screen, select one or more suitable isotope(s) for every analyte and include ¹³C as internal normalization standard^{31,36,40,41,42}. Verify that detection of the isotopes is not impaired by interferences⁴⁶.
   6. Set the total scan cycle duration of the ICP-MS method (‘Est. Reading Time’) to ≤0.5 s with appropriate dwell times per isotope (typically between 10-50 ms). Set the ICP-MS ‘Readings’ to 1 and change the ‘Readings/Replicate’ value to set the total measurement time per sample (‘Est. Sampling Time’), i.e., per individual ablation line. This depends on the specific LA parameters and line distances set in 4.2.2 – 4.2.4.
   7. For data reporting use an intensity versus time data collection mode and set the ‘File Write Option’ to ‘New Per Sample’ to create an individual data file (here .xl) for each ablation line.
   8. Set up a ‘Batch’ sample sequence in the ICP-MS ‘Sample’ screen, with each sample entry corresponding to an individual ablation line set in 4.2.3 – 4.2.4.
   9. Click ‘Analyze Batch’ to initiate the sample sequence on the ICP-MS, which will wait with data acquisition until it is triggered by the first laser pulse (see SI S5 for details on the trigger configuration).
   10. In the LA software, select all lines to be analyzed and verify that the ICP-MS method (‘Est. Sampling Time’, see 4.2.6.) matches the duration of the individual line ablations, which is typically different for gel samples, standards and method blanks. Repeat 4.2.6. to adjust the ICP-MS method if necessary.
   11. Click ‘Emission’ in the ‘Laser Energy’ window to recharge the laser head, and then click ‘Run’ to open the ‘Run Experiment’ window. Here, select ‘Selected Patterns Only’, set the ‘Washout Delay’ to 20-30 s, tick the ‘Enable Laser During Scans’ box, and set the ‘Laser Warmup Time’ to 10 s.
   12. Click ‘Run’ in the ‘Run experiment’ window to start the line-scan analysis and monitor the raw signal intensity in counts per second (cps) for each isotope on the ICP-MS in real time. Each line should start (‘Laser Warmup Time’, 10 s) and end (‘Washout Delay’, 20-30 s) with a gas blank.
   13. Monitor the laser fluence (J cm^-2) during analysis to assess the laser stability. If the fluence varies largely, abort the analysis, and verify that the laser source and/or its mirror system are fully functional.
   14. After the analysis, stop the ICP-MS plasma and set the carrier gas flow rate on the LA system to 0 mL min^-1. Remove the gels from the ablation cell and store in zip bags for further use.

Figure 6: Schematic of the DGT LA-ICP-MS experimental design (not to scale). The illustration depicts the DGT-based in situ solute sampling in the rhizosphere and the LA-ICP-MS mapping of the solute distribution on the gel surface, including a close-up showing exemplary line-scan dimensions and parameters. Note that the DGT gel is horizontally flipped when transferred from the rhizosphere soil onto the glass plate, as indicated by the position of the rectangle at the bottom corner of the DGT gel. Please click here to view a larger version of this figure.

3. Data processing and calibration
   1. Import the raw data file (.xl) for each ablated line in spreadsheet software (Table of Materials). The raw data table shows the ICP-MS readings (datapoints) for each isotope in cps and the corresponding time points in s. List all lines next to each other in different columns.
   2. Evaluate the line-scan data for signal stability of the internal standard (¹³C) and adequate washout times.
   3. Calculate an average gas blank for each isotope from all gas blank values recorded before the line ablations (i.e., during the laser warm-up time) and subtract the average gas blank from the corresponding raw intensities for each isotope to correct for the background signal.
   4. Apply internal normalization by dividing the signal intensity of each isotope (cps) by the signal intensity of the internal standard (cps) for each datapoint to correct for variations in the amount of material ablated and instrumental drift.
   5. Crop data before the start and after the end of each ablated line to remove the background signal. Transpose the data table to obtain a grid matrix where each row corresponds to an ablated line and each column to a normalized isotope intensity value. Separate the matrices for each isotope into individual worksheets.
   6. Calculate the average normalized signal intensity ratio per isotope for the calibration standards and calibration blank and compute the calibration function (y = ax + b) using a linear regression model with the gel standard analyte loadings (µg cm^-2; see SI S4) as x-values. Evaluate the calibration function of each isotope for linearity⁴⁷.
   7. Apply the calibration function to the sample data matrix. Convert the normalized signal intensity ratios, , to gel analyte loadings, (µg cm^-2), and subsequently to time-averaged solute fluxes, (pg cm^-2 s^-1), for each isotope and datapoint according to Eq. 4 and Eq. 5:
      
      
      Here, a is the slope of the calibration line, b is the intercept of the calibration line, and t (s) is the gel deployment time during solute sampling.
   8. Save the calibrated sample data matrix for each analyte as txt-file.
4. Image generation
   NOTE: Make sure to avoid any pixel interpolation operation during all steps of image generation, as this may lead to artificially smoothed solute flux gradients in the resulting images.
   1. Import the calibrated sample data matrix (.txt) as text image in the image analysis software (Table of Materials).
   2. Calculate the distance per pixel in x-direction (i.e., lateral resolution) by multiplying the laser scan speed with the total ICP-MS scan cycle duration (e.g., assuming a scan speed of 200 µm s^-1 and a total scan cycle duration of 0.25 s, the lateral resolution equates to 50 µm). The distance per pixel in y-direction equates to the interline distance (Figure 6).
   3. Calculate the aspect ratio correction factor of the image. Therefore, divide the distance per pixel in y-direction (e.g., 300 µm) by the distance per pixel in x-direction (e.g., 50 µm). Apply the obtained y/x correction factor (in this example 6) under ‘Scale’. Apply the distance per pixel (in µm or mm) in x-direction as scaling under ‘Set scale’.
   4. Apply a ‘Look Up Table’ i.e., pseudo-color scale for better visualization of chemical gradients in the solute image and adjust the image ‘color balance’ to control the lower/upper limits of the display range. Add a ‘Calibration bar’ and save the solute image as tiff-file.
   5. Copy the solute image using the ‘Copy to System’ command in the image analysis software and paste into desktop publishing software (Table of Materials). Scale-match, align and compose the solute image with the photo of the ROI obtained in 3.1.7.
      NOTE: Before copying, resize the solute image by e.g., factor 10 via applying a ‘X Scale’ of 10 and a ‘Y Scale’ of 10 under ‘Set scale’ to ensure sufficient pixels for high-resolution publishing.

Table 1: Approximate times for general steps of the DGT LA-ICP-MS technique.