这项研究由奥地利科学基金（FWF）:P30085-N28（托马斯·普罗哈斯卡）和奥地利科学基金（FWF）以及下奥地利联邦州共同资助:P27571-BBL（雅科布·桑特纳）。

作者没有什么可透露的。

此处介绍的溶质成像方案是一种多功能方法，可可视化和量化土壤植物环境中的二维养分和污染物通量。它具有独特的能力，能够产生亚毫米比例的多元素图像的腹腔溶质物种在土壤根界面，超过可实现的空间分辨率的替代方法测量溶质梯度在rhizosphere基本上4。DGT 的定点现场采样方法，结合 LA-ICP-MS 等高度敏感的化学分析方法，有助于详细研究土壤或类似基质中生长的单个植物根部周围的溶质通量动力学。由于基于水槽的取样过程，获得的图像反映了可视化溶解物的易感性，因此估计其植物可用性为10。虽然溶质通量的方法固有的测量具有相当大的优势，如可解释为植物可用的营养成分，但通量测量远不如孔水浓度测量更直接理解。散装土壤应用中的标准 DGT 采样几何形状（特别是该设置中使用的 0.8 毫米厚扩散凝胶）允许通过批量 DGT 测量cDGT和解释这些参数对溶质物种的再补给动力学进行比较实际孔隙水浓度、csoln和时间平均孔水浓度估计值。但是，这种比较不能基于具有非常薄扩散层的成像 DGT 应用程序进行，因为派生的cDGT值是不切实际的小34。因此，DGT成像结果并不总是简单和快速的解释，往往不能直接与更传统的孔隙水浓度测量相提并论。
在应用该方法时，需要仔细考虑一些关键步骤，主要涉及灌装和浇注 rhizotron 生长容器。在将土壤填入 rhizotron 时，避免过多压实土壤非常重要，因为植物根不能穿透强压实的土壤，根的生长将受到抑制。我们观察到根部避免强烈压实的土壤，沿着rhistron生长容器的内缘生长，那里的土壤通常不太紧凑。在这种情况下，位于rhigzotrons中心的单个根，其中DGT凝胶可以方便地应用，可能根本不开发，有效地抑制了成功的凝胶应用。在我们的实验室中，经验表明，1.0-1.4 gcm-3 的干燥土壤散装密度允许不受阻碍的根部发育。此外，过度的土壤压实也是有关氧化物敏感元素和生物地球化学相关物种溶解性的潜在人工来源。由于毛孔总体积减少，毛孔直径分布向高度压实土壤中的低直径转移，因此可用的气填充量较小，从而可能导致局部的还原条件。因此，MnIII/IV- 和 FeIII- 氧化物可能会减少，导致 Mn2+ 和 Fe2+ 通量增加。Fe-oxides的溶解是磷酸盐和微量营养素的重要吸附点，可以解放冰山和/或共同沉淀的物种，从而导致生物地球化学相关物种的人工增高通量。如果生长容器浇水过多，也可能会出现类似的问题。通过生长容器顶部的小土壤表面积蒸发量较低，土壤在种植后可能保持水饱和长达几个星期，这也可能导致重氧化人工制品。
另一个重要考虑因素是制造 HR-DGT 结合凝胶的化学功能。通过遵循协议，获得具有均匀分布的绑定相的薄凝胶。如果凝胶具有不均匀的材料分布区域（例如凝胶或结合相聚合的孔），则需要去除这些区域，或者，如果范围太广，则需要重复凝胶制造协议。如果准备正确，凝胶必须能够结合目标溶胶物种，立即和定量地扩散到凝胶27，这是由分析特异性凝胶结合能力决定的。虽然在未受污染的土壤中，超过凝胶容量的问题较小，但在金属污染土壤和盐碱土壤环境中应予以考虑。凝胶结合相的饱和不仅会损害定量溶胶采样，而且还会导致凝胶中结合相之间的溶解物横向扩散，导致小规模溶胶通量特征的无限期本地化。因此，如果目标土壤环境中预期有大量阴唇养分/污染物物种，则应进行初步测试。对于估计预期的DGT负荷，散装土壤DGT活塞采样，其次是凝胶elution和湿化学分析可以应用15，49。如有必要，可调整 DGT 部署时间以减少凝胶接触时间，从而避免凝胶饱和度超过容量阈值。相反，初步测试也有助于确定所需的凝胶接触时间和/或LA-ICP-MS灵敏度，如果非常低的溶质负荷预期，这可能是重要的映射微量元素溶解物在自然土壤背景水平15。此外，在制定DGT LA-ICP-MS校准标准时，应通过对凝胶的可控加载进行实验应用前验证正确的DGT凝胶功能。凝胶标准提供矩阵匹配的参考凝胶分析加载，可用于评估 LA-ICP-MS 确定的样品凝胶装载是否在预期范围内。如果无法获得与气体和方法空白背景噪声不同的信号，操作员必须确保实施跟踪元素分析的实验室程序，并正确执行所有协议步骤。有时，DGT 凝胶在用土壤暴露的、加载的侧面朝向玻璃板而不是激光束进行溶胶采样后意外翻转，导致最终溶胶通量图像中信号强度低且错误地翻转特征。
在洛杉矶-ICP-MS分析期间，生成了大量数据，这需要相当长的时间进行评估。在我们的实验室中，我们使用针对目标数据输出格式定制的内部数据评估脚本，使用标准电子表格软件。半自动排序和校准后，使用开源、开放访问图像分析工具（ImageJ，斐济50）进行图像绘图。这种方法允许完全控制数据排序、评估和演示，这一点至关重要，因为收集的数据对应于矩形，而不是二次像素，这需要正确地显示在生成的 solute 地图中。此外，在数据处理过程中，应小心避免任何像素插点。像素插点导致化学图像的梯度平滑，导致软化，通常是圆形元素分布特征，因此是原始数据的不良变化。像素插管是许多图像处理软件产品重新缩放和重新格式化操作的标准程序，但通常可以取消选择。
总之，上述方法是了解天然土壤-里佐圈-植物系统中的养分和污染物动力学的重大进展。除了 DGT 专用应用外，该方法还可以与其他基于扩散的成像技术（如平面光学 3、33、42、43、48、51 和 zym 光谱20、21、22、23、24）相结合，并可进一步开发以包括附加元素和土壤参数。

作物植物的营养获取是决定作物产量的关键因素。对作物有效吸收养分的过程进行了深入的研究，特别是通过土壤根系（rhizosphere）的植物根控制养分供应和养分内化的机制，因其在作物养分获取中的作用而得到认可。植物养分吸收的重要过程包括:向根部输送营养:溶解在土壤孔隙水中的物种与与固体土壤表面相连的物种之间的动态吸附平衡:微生物对营养物质的竞争;土壤有机物中所含养分的微生物矿化:和营养内化到根部症状。无机微量金属（oid）污染物的吸收在很大程度上由相同的机制控制。
根据养分和污染物的供应、植物需求和土壤中的扩散性，可以观察到岩石圈中的差异养分模式。对于内化率较高的强吸附元素（例如， P，Fe，Mn，Zn，As，Cd，Pb），与散装土壤相比，阴唇（即可逆吸附）元素部分的耗竭，耗竭区宽度通常≤1毫米，而对于更多的移动养分，如NO3-，耗竭区可以延伸至几厘米1。此外，当可用性超过工厂吸收率2、3时，还观察到 Al 和 Cd 等元素的积累。
鉴于 rhizosphere 过程在养分和污染物循环中的重要性，已开发出几种以高空间分辨率测量植物可用元素分数的技术。然而，测量小规模的腹腔溶胶分布已被证明是具有挑战性的，有几个原因。一个主要困难是在与活植物根相邻的固定位置对非常小（低 μL 范围）的土壤和/或孔水进行采样，以解决 rhizosphere 中陡峭的养分梯度。解决这个问题的方法之一是使用微吸杯提取孔隙水样品6。通过这种方法，A. Güttlein、A. Heim 和 E. Matzner7测量了奎尔库斯罗布尔L. 根附近的土壤孔水养分浓度，其空间分辨率为 +1 厘米。分析 μL 土壤或土壤溶液量的一个困难是，这些小样本量与除主要养分物种外的所有低浓度相结合，需要高度敏感的化学分析技术。
另一种系统，能够解决营养梯度在分辨率下降到〜0.5毫米，是种植根垫在土壤块的表面，与薄亲水膜层分离土壤从根8，9。在这种配置中，溶胶可以穿过膜，根可以从土壤中产生养分和污染物，而根渗出物可以扩散到土壤中。建立密集的根层后，可以对土壤块进行取样和切片，以获得土壤样本，以便随后提取元素部分。通过这种方式，可以分析在相对较大的区域（约100厘米2）平均的一维营养素和污染物梯度。
另一个挑战是获得实验室、植物可用元素部分的样本，因为大多数化学土壤提取技术的运作方式与植物获取养分和污染物的机制大相径庭。在许多土壤提取协议中，土壤与萃取溶液混合，目的是在溶解元素和冰激元素部分之间建立（伪）平衡。然而，植物不断内化养分，因此，往往逐渐耗尽了里佐圈的土壤。虽然均衡提取方案已被广泛采用作为土壤测试，因为它们很容易实施，提取的营养成分往往不代表植物可用的营养成分井10，11，12，13。不断耗尽被采样土壤养分的水槽方法已被建议为有利的方法，并可能更类似于潜在的养分吸收机制，模仿根吸收过程10，11，14，15。
除了上述方法外，还开发了能够测量分辨率为≤100μm的连续参数图的真人成像应用，用于特定元素和土壤（生物）化学参数5。自传可以用来成像元素分布在日光圈，只要有合适的放射性同位素16。平面光电图可可视化重要的土壤化学参数，如pH值和pO217、18、19，酶活性或蛋白质总分布可以使用荧光指示器成像技术（如土壤成像20、21、22、23和/或根部印迹方法24）进行映射。虽然酶成像和自传仅限于一次测量单个参数，但使用平面光电图进行 pH 和 pO2成像可以同时完成。更传统的根垫技术仅提供一维信息，而微吸盘提供点测量或低分辨率二维信息，但两种方法都允许多元素分析。最近，P.D. Ilhardt等人提出了一种利用激光诱导分解光谱（LIBS）绘制2D总多元素分布图的新方法，分辨率为+100μm的土壤根芯样品，通过仔细的样品准备保留了自然元素分布。
唯一能够以高空间分辨率对多种营养物质和污染物溶解物进行二维取样的技术是薄膜（DGT）技术中的扩散梯度，这是一种基于水槽的取样方法，可在嵌入在水凝胶层26、27层的结合材料上原位固定可移动的实验室微量金属（loid）物种。DGT作为测量沉积物和水域中腹腔溶解物的化学规格技术被引入，并很快被用于土壤28。它使亚毫米比例的多元素溶胶成像，这是最初在河流沉积物29中演示，并已进一步开发，以应用于植物里佐圈30，31，32，33。
对于 DGT 取样，在土壤块表面层生长的单个植物根上涂上一块大小约为 3 厘米 x 5 厘米的凝胶片，用亲水膜将凝胶与土壤分离。在接触期间，阴唇营养物质和/或污染物扩散到凝胶上，并立即被凝胶中加入的结合材料所束缚。通过这种方式，在采样过程中建立了浓度梯度，从而建立了对凝胶的连续净通量。取样后，可以使用分析化学技术去除和分析水凝胶，从而进行空间解析分析。为此目的，一项高度专业化且常用的技术是激光消融感应耦合等离子体质谱仪 （LA-ICP-MS）。在一些早期研究中，微粒子诱导的X射线发射（PIXE）也被使用29。DGT 采样与 LA-ICP-MS 分析相结合，可实现空间分辨率为 100μm 的多元素化学成像。如果采用高度敏感的 ICP-MS 技术（例如部门领域 ICP-MS），则可以达到极低的检测限制。在一项有关玉米15号对Zn和Cd吸收的影响的研究中，我们能够绘制出未受污染土壤中玉米里佐圈的阴唇CD，每个凝胶面积的检测限量为38 pgcm-2。DGT、平面光学和酶学依赖于目标元素从土壤扩散到凝胶层，这些凝胶层可用于这些方法的联合应用，以便同时或连续地成像与植物养分和污染物吸收相关的大量参数。关于DGT成像分析化学方面的详细信息，关于DGT和其他成像方法相结合的潜力，以及它的应用，在参考第34，35中进行了全面审查。
本文介绍了如何利用DGT技术对不饱和土壤环境中的陆生植物根部进行溶解成像实验，包括植物栽培、凝胶制造、凝胶应用、凝胶分析和图像生成。所有步骤都详细阐述，包括关于关键步骤和实验备选方案的说明。

<table>
	<tbody>
		<tr>
			<td>(NH4)2S2O8 (ammonium persulfate; APS)</td>
			<td>VWR</td>
			<td>21300.260</td>
			<td>&#8805;98.0%, analytical reagent</td>
		</tr>
		<tr>
			<td>2-(N-morpholino)-ethanesulfonic acid (MES)</td>
			<td>Sigma-Aldrich</td>
			<td>M8250-100G</td>
			<td>&#8805;99.5%</td>
		</tr>
		<tr>
			<td>Acrylamide solution</td>
			<td>Sigma-Aldrich</td>
			<td>A4058-100ML</td>
			<td>40%, for electrophoresis</td>
		</tr>
		<tr>
			<td>Analyte salts</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Use water soluble analyte salts of analytical grade or higher</td>
		</tr>
		<tr>
			<td>Buechner funnel</td>
			<td>VWR</td>
			<td>511-0065</td>
			<td>13 cm plate diameter</td>
		</tr>
		<tr>
			<td>Chemical equilibrium modelling software</td>
			<td>KTH Sweden</td>
			<td>n/a</td>
			<td>Visual MINTEQ</td>
		</tr>
		<tr>
			<td>Clamp</td>
			<td>Local warehouse</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Desktop publishing software</td>
			<td>Adobe Inc.</td>
			<td>n/a</td>
			<td>InDesign CS6</td>
		</tr>
		<tr>
			<td>DGT cross-linker</td>
			<td>DGT Research Ltd</td>
			<td>n/a</td>
			<td>2%, agarose derivative</td>
		</tr>
		<tr>
			<td>DGT piston sampler</td>
			<td>DGT Research Ltd</td>
			<td>n/a</td>
			<td>2 cm diameter exposure window</td>
		</tr>
		<tr>
			<td>Digital single-lens reflex (DSLR) camera</td>
			<td>Canon Inc.</td>
			<td>n/a</td>
			<td>Canon EOS 1000D</td>
		</tr>
		<tr>
			<td>Dispersion device</td>
			<td>IKA</td>
			<td>3737000</td>
			<td>Ultra-Turrax T10 Basic</td>
		</tr>
		<tr>
			<td>Double-sided adhesive tape</td>
			<td>Tesa</td>
			<td>56171</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>34923</td>
			<td>Puriss. p.a., absolute, &#8805;99.8%</td>
		</tr>
		<tr>
			<td>Gel blotting paper</td>
			<td>Whatman</td>
			<td>10426981</td>
			<td>Blotting Papers, Grade GB005, 20 &#215; 20 cm, 1.5 mm thickness</td>
		</tr>
		<tr>
			<td>Gel drier</td>
			<td>UniEquip</td>
			<td>n/a</td>
			<td>UNIGELDRYER 3545</td>
		</tr>
		<tr>
			<td>High-pressure microwave system</td>
			<td>Anton Paar</td>
			<td>n/a</td>
			<td>Multiwave 3000</td>
		</tr>
		<tr>
			<td>HNO3</td>
			<td>VWR</td>
			<td>1.00456.2500P</td>
			<td>65%, ISO for analysis</td>
		</tr>
		<tr>
			<td>Horizontal shaker</td>
			<td>GFL</td>
			<td>305</td>
			<td></td>
		</tr>
		<tr>
			<td>HydroMed D4</td>
			<td>AdvanSource Biomaterials Corp.</td>
			<td>n/a</td>
			<td>Ether-based hydrophilic urethane</td>
		</tr>
		<tr>
			<td>ICP-MS software</td>
			<td>Perkin Elmer</td>
			<td>n/a</td>
			<td>Syngistix</td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>National Institutes of Health (NIH)</td>
			<td>n/a</td>
			<td>ImageJ Fiji, freely available at https://fiji.sc/</td>
		</tr>
		<tr>
			<td>Knife-coating device</td>
			<td>BYK</td>
			<td>5561</td>
			<td>Single Bar 6&#8243;, 0.5 mils</td>
		</tr>
		<tr>
			<td>LA software</td>
			<td>Elemental Scientific Lasers</td>
			<td>n/a</td>
			<td>ActiveView</td>
		</tr>
		<tr>
			<td>LA system</td>
			<td>Elemental Scientific Lasers</td>
			<td>n/a</td>
			<td>NWR193</td>
		</tr>
		<tr>
			<td>Laminar flow bench</td>
			<td>Telstar Laboratory Equipment B.V.</td>
			<td>n/a</td>
			<td>Class II biological safety cabinet</td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA</td>
			<td>0003582400</td>
			<td>C-MAG MS 7</td>
		</tr>
		<tr>
			<td>Moisture-retaining film</td>
			<td>Bemis Company, Inc.</td>
			<td>PM999</td>
			<td>Parafilm M, 4&#34; x 250&#39;</td>
		</tr>
		<tr>
			<td>N,N,N&#8217;,N&#8217;-tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich</td>
			<td>T9281-50ML</td>
			<td>BioReagent, suitable for electrophoresis, ~99%</td>
		</tr>
		<tr>
			<td>NaNO3</td>
			<td>Sigma-Aldrich</td>
			<td>229938-10G</td>
			<td>99.995% trace metals basis</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>1064980500</td>
			<td>Pellets for analysis</td>
		</tr>
		<tr>
			<td>Overhead shaker</td>
			<td>GFL</td>
			<td>3040</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfluoroalkoxy alkane (PFA) vials</td>
			<td>Savillex</td>
			<td>200-015-20</td>
			<td>15 mL Standard Vial, Rounded Interior</td>
		</tr>
		<tr>
			<td>pH meter</td>
			<td>Thermo Scientific</td>
			<td>13-644-928</td>
			<td>Orion 3-Star Benchtop pH Meter</td>
		</tr>
		<tr>
			<td>pH probe</td>
			<td>Thermo Scientific</td>
			<td>8157BNUMD</td>
			<td>Orion ROSS Ultra pH/ATC Triode</td>
		</tr>
		<tr>
			<td>Plastic cutter</td>
			<td>DGT Research Ltd</td>
			<td>n/a</td>
			<td>Use empty cross-linker vials from DGT research Ltd</td>
		</tr>
		<tr>
			<td>Plastic tweezers</td>
			<td>Semadeni</td>
			<td>602</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasticine</td>
			<td>Local stationary shop</td>
			<td>n/a</td>
			<td>non-drying plastic modelling mass based on paraffin wax and bulking agents</td>
		</tr>
		<tr>
			<td>Polycarbonate membrane discs</td>
			<td>Whatman</td>
			<td>110606</td>
			<td>Nuclepore Hydrophilic Membrane, 25 mm diameter, 0.2 &#181;m pore size, 10 &#181;m thickness</td>
		</tr>
		<tr>
			<td>Polycarbonate membrane sheet</td>
			<td>Whatman</td>
			<td>113506</td>
			<td>Nuclepore Hydrophilic Membrane, 8 &#215; 10 in, 0.2 &#181;m pore size, 10 &#181;m thickness</td>
		</tr>
		<tr>
			<td>Polyethersulfone membrane discs</td>
			<td>Pall Corporation</td>
			<td>60172</td>
			<td>Supor 450 Membrane Disc Filters, 25 mm diameter, 0.45 &#181;m pore size, 0.14 mm thickness</td>
		</tr>
		<tr>
			<td>Polyethersulfone membrane sheet</td>
			<td>Pall Corporation</td>
			<td>60179</td>
			<td>Supor 450 Membrane Disc Filters, 293 mm diameter, 0.45 &#181;m pore size, 0.14 mm thickness</td>
		</tr>
		<tr>
			<td>PTFE foil</td>
			<td>Haberkorn</td>
			<td>n/a</td>
			<td>50 &#181;m thickness</td>
		</tr>
		<tr>
			<td>PTFE spacer</td>
			<td>Haberkorn</td>
			<td>n/a</td>
			<td>Variable thicknesses available</td>
		</tr>
		<tr>
			<td>PTFE-coated razor blades</td>
			<td>Personna GEM</td>
			<td>62-0178</td>
			<td>Stainless steel single edge blades (coated)</td>
		</tr>
		<tr>
			<td>PTFE-coated Tygon tubing</td>
			<td>S-prep GmbH</td>
			<td>SP8180</td>
			<td>0.32 cm inner diameter</td>
		</tr>
		<tr>
			<td>Quadrupole ICP-MS</td>
			<td>Perkin Elmer</td>
			<td>N8150044</td>
			<td>NexION 2000B</td>
		</tr>
		<tr>
			<td>Quantitative filter paper, 454</td>
			<td>VWR</td>
			<td>516-0854</td>
			<td>Particle retention 12-15 &#181;m</td>
		</tr>
		<tr>
			<td>Spreadsheet software</td>
			<td>Microsoft Corporation</td>
			<td>n/a</td>
			<td>Microsoft Excel 2016 (v16.0)</td>
		</tr>
		<tr>
			<td>Stainless-steel cutter</td>
			<td>Local locksmithery</td>
			<td>n/a</td>
			<td>2.5 cm diameter</td>
		</tr>
		<tr>
			<td>Suspended particulate reagent-iminodiacetate (SPR-IDA)</td>
			<td>Teledyne CETAC Technologies</td>
			<td>n/a</td>
			<td>10 &#181;m diameter polystyrene beads, 10 % (w/v) bead suspension</td>
		</tr>
		<tr>
			<td>Transistor-transistor logic (TTL) cable</td>
			<td>n/a</td>
			<td>n/a</td>
			<td>Consult ICP-MS technician to identify a suitable TTL cable for a specific instrument</td>
		</tr>
		<tr>
			<td>Two-volume cell</td>
			<td>Elemental Scientific Lasers</td>
			<td>n/a</td>
			<td>Two-volume cell 1</td>
		</tr>
		<tr>
			<td>Vinyl electrical tape</td>
			<td>3M</td>
			<td>n/a</td>
			<td>Scotch Super 33+</td>
		</tr>
		<tr>
			<td>Water purification system</td>
			<td>Termo Electron LED GmbH</td>
			<td>n/a</td>
			<td>TKA-GenPure</td>
		</tr>
		<tr>
			<td>ZrOCl2 &#215; 8H2O</td>
			<td>Alfa Aesar</td>
			<td>86108.30</td>
			<td>99.9 %, metals basis</td>
		</tr>
	</tbody>
</table>

two-dimensional visualization and quantification of labile, inorganic plant nutrients and contaminants in soil

我们描述了一种二维（2D）可视化和量化阴唇分布的方法（即 逆向吸收）无机养分（如P、Fe、Mn）和污染物（如Cd、Pb）以低于毫米（约100微米）的空间分辨率在植物根部（"rhizosphere"）相邻的土壤中溶解物种。该方法结合了薄膜（DGT）技术中扩散梯度的沉基溶胶采样与激光消融感应耦合等离子质谱（LA-ICP-MS）的空间解化学分析。DGT 技术基于具有均匀分布分析器选择性结合相的薄水凝胶。可用绑定阶段的多样性允许在简单的凝胶制造程序后制备不同的 DGT 凝胶类型。对于 Rhizosphere 中的 DGT 凝胶部署，植物生长在平坦、透明的生长容器 （rhizotrons） 中，从而能够以最小的侵入性进入土壤生长的根系。在生长前阶段后，DGT 凝胶应用于某些感兴趣的区域，用于在 rhizospher 中进行原位溶胶采样。之后，使用 LA-ICP-MS 线扫描成像检索并准备随后对绑定溶胶进行化学分析。使用 13C 应用内部规范化，使用矩阵匹配凝胶标准进行外部校准，进一步允许 2D 溶质通量的量化。该方法在土壤植物环境中生成多元素溶质通量的定量、亚毫米比例二维图像的能力方面独树一帜，大大超过了测量日光圈中溶质梯度的其他方法的可实现空间分辨率。我们介绍了在陆地植物的岩石圈中成像多成离子和肛门溶质物种的方法的应用和评价，并强调了将这种方法与补充溶质成像技术相结合的可能性。

为了证明DGT成像方法的能力和数据细节，我们汇编了与法戈皮鲁姆树脂和萨利克斯·史密西亚纳（图7）相邻的土壤中多种阴唇营养素和污染物溶质物种的亚毫米、二维通量分布。协议的一般程序步骤的大致时间在表1中。
图 7中的索卢特图像是在三种不同的研究中使用 HR-MBG 或 HR-CBG 绑定凝胶生成的。化学图像显示沿 x 轴的空间分辨率为 82-120 μm，沿 y 轴的 300-400 μm 的 2D 溶质通量分布，具体取决于所使用的 LA-ICP-MS 参数。由于在图像校准和调整过程中没有应用插点，因此单个像素表示已测量的数据点。将溶胶图像与 ROI 的摄影图像对齐后发现，根据土壤结构和根部形态，不同元素的亚毫米、二维溶质通量分布高度可变。这可以归因于土壤-红球-植物系统中元素的微分生化行为，以及它们与土壤矩阵和植物根的相互作用。
在 图7A 阴唇无机Mg，Al，P，Mn和Fe溶胶通量被可视化周围的一个年轻的 F.埃斯库伦特 根生长在无碳酸盐土壤施肥与NH4NO3.亚毫米溶质分布显示，由于根吸收，Al、P 和 Fe 通量与旧根部分一起减少，由于 F. esculentum 根的局部 P 动员过程，根部顶点的 Mg、Al、P、Mn 和 Fe 通量高度增加。请注意，根尖位于土壤表面后面，因此在照片图像中几乎看不到。 图 7B 显示了在受 Zn、Cd 和 Pb 适度污染的土壤中生长的耐金属 S. Smithiana 根部周围的阴唇微量金属的分布，包括 Mn、Fe、Zn、Cd 和 Pb。溶胶图像可视化了明显的消耗，特别是 Zn、Cd 和 Pb 的直接根部，表明 S. Smithiana 根部充当受污染土壤中腹腔微量金属的局部沉降器。此外，还可以观察到局部 Zn、Cd 和 Pb 通量增加，表明这些微量金属在直接土壤根接口的积累。
除了多元素溶胶成像外，该方法还可以与基于增释的辅助成像技术（如平面光学34）相结合。这一点在图7C中得到了证明，其中在S.史密斯尼亚纳的rhizospher中，阴唇微量金属的分布与pH值的分布是通过结合的单层平面光电-DGT cation结合凝胶33共同本地化的。土壤基板用 （NH4）2SO4施肥，导致与散装土壤相比，根轴沿线的 pH 降低 1 单位。pH 的下降与 Mn、Fe、Co、Ni、Cu 和 Pb 的溶质通量增加共同本地化，表明 pH 引起的金属溶解。
此外，这些示例结果显示了可能获得的一些潜在的成像人工制品。例如，结构土壤的不均匀性，例如，在 图 7A的 ROI 图像的下三分之一中观察到水平线，可导致土壤凝胶接触不连续性，导致此位置的有限扩散到粘合凝胶中。相反，在rhiozotron中过度的土壤压实会导致孔隙性差，导致土壤重氧化状态向厌氧的人工转移。 图 7B中说明了这一点，其中溶质图像中高度升高的 Mn 和 Fe 通量的大面积区域与 ROI 图像中的密集土壤层在视觉上相匹配。这表明，由于土壤压实，土壤重氧化的可能性降低，导致高度重氧化敏感元素的还原溶解和溶解。因此，建议在灌装后直接对土壤表面进行仔细的 rhizotron 填充和目视检查。
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/61661/61661fig07.jpg"> 图7:不同土壤根界面的阴唇养分和污染物溶质物种的亚毫米二维分布。（A） 在年轻的F. 埃斯库伦图姆根周围分布的食人性 P 和 cationic Mg、Al、Mn 和 Fe 溶质。在=75%WHC的土壤水饱和度下，使用HR-MBG对阴离子和阴离子溶胶进行了24小时的联合局部取样。Al、P 和 Mn 图像显示为校准fDGT值（pg cm-2 s-1），而 Mg 和 Fe 图像显示13C 规范化强度。比例尺条代表 1 厘米。这个数字是根据参考48改编的。（B） 在中度受Zn、Cd和Pb污染的土壤中生长的S.smithiana根周围分布Mn、Fe、Zn、Cd和Pb。所有图像都显示为校准的 fDGT值 （pg cm-2 s-1）。刻度条代表 0.5 厘米。这个数字是根据参考3改编的。（C） 通过修改HR-MBG协议，实现了在土壤中生长的S.Smithiana根部周围的pH和多个cationiana溶解物的分布，并采用CD.pH和溶胶动力学的联合定位，允许同时进行溶胶采样和平面光电成像33。 Mn、Cu、Zn 和 Cd 图像以校准fDGT值（pg cm-2 s-1）显示，而 Fe、Co、Ni 和 Pb 图像显示13个 C 规范化强度。比例尺条代表 1 厘米。这个数字是根据参考33改编的。所列数字转载自引用的第3条、第33条、第48条，这些条文根据CC BY获得许可。<a href="https://www.jove.com/files/ftp_upload/61661/61661fig07large.jpg" target="_blank">请点击这里查看此数字的较大版本。</a>
补充文件 1.<a href="https://www.jove.com/files/ftp_upload/61661/JoVE61661_Supporting%20Information_Updated%202021_.pdf" target="_blank">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Two-Dimensional Visualization and Quantification of Labile, Inorganic Plant Nutrients and Contaminants in Soil at JoVE.com

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

此协议提供了多个阴唇无机养分和污染物溶胶物种的亚毫米 2D 可视化工作流程，使用薄膜 （DGT） 中的扩散梯度与质谱成像相结合。详细描述了在陆地植物的岩石圈中对溶解物进行定量测绘的索卢特采样和高分辨率化学分析。

土壤中实验室、无机植物养分和污染物的二维可视化和量化

We describe a method for two-dimensional (2D) visualization and quantification of the distribution of labile (i.e., reversibly adsorbed) inorganic ...

two-dimensional-visualization-quantification-labile-inorganic-plant

Research

JoVE Journal

Environment

5.9K 次观看.  Montanuniversität Leoben. 此协议提供了多个阴唇无机养分和污染物溶胶物种的亚毫米 2D 可视化工作流程，使用薄膜 （DGT） 中的扩散梯度与质谱成像相结合。详细描述了在陆地植物的岩石圈中对溶解物进行定量测绘的索卢特采样和高分辨率化学分析。

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

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes and soil organic matter formation to small-scale stable-isotope biomarker probing. Coupling multiple stable isotopes such as 13C with 15N, 18O or&#160;2H has the potential to reveal even more information about complex stoichiometric relationships during biogeochemical transformations. Isotope labeled plant material has been used in various studies of litter decomposition and soil organic matter formation1-4. From these and other studies, however, it has become apparent that structural components of plant material behave differently than metabolic components (i.e. leachable low molecular weight compounds) in terms of microbial utilization and long-term carbon storage5-7. The ability to study structural and metabolic components separately provides a powerful new tool for advancing the forefront of ecosystem biogeochemical studies. Here we describe a method for producing 13C and 15N labeled plant material that is either uniformly labeled throughout the plant or differentially labeled in structural and metabolic plant components.
Here, we present the construction and operation of a continuous 13C and 15N labeling chamber that can be modified to meet various research needs. Uniformly labeled plant material is produced by continuous labeling from seedling to harvest, while differential labeling is achieved by removing the growing plants from the chamber weeks prior to harvest. Representative results from growing Andropogon gerardii Kaw demonstrate the system&#39;s ability to efficiently label plant material at the targeted levels. Through this method we have produced plant material with a 4.4 atom%13C and 6.7 atom%15N uniform plant label, or material that is differentially labeled by up to 1.29 atom%13C and 0.56 atom%15N in its metabolic and structural components (hot water extractable and hot water residual components, respectively). Challenges lie in maintaining proper temperature, humidity, CO2 concentration,&#160;and light levels in an airtight 13C-CO2 atmosphere for successful plant production. This chamber description represents a useful research tool to effectively produce uniformly or differentially multi-isotope labeled plant material for use in experiments on ecosystem biogeochemical cycling.

Design and Operation of a Continuous 13C and 15N Labeling Chamber for Uniform or Differential, Metabolic and Structural, Plant Isotope Labeling

This method explains how to build and operate a continuous 13C and 15N isotope labeling chamber for uniform or differential plant tissue labeling. Representative results from metabolic and structural labeling of Andropogon gerardii are discussed.

设计一个连续和操作<sup&gt; 13</sup&gt; C和<sup&gt; 15</sup&gt;Ñ贴标商会统一或差分，代谢和结构，植物同位素标记

Tracing rare stable isotopes from plant material through the ecosystem provides the most sensitive information about ecosystem processes; from CO2 fluxes ...

科研

环境科学

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

用于操纵植物诱导土壤异质性的实验协议

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

Quantification of Heavy Metals and Other Inorganic Contaminants on the Productivity of Microalgae

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages include high potential yield, use of non-arable land and integration with waste streams. The nutrient requirements of a large-scale microalgae production system will require the coupling of cultivation systems with industrial waste resources, such as carbon dioxide from flue gas and nutrients from wastewater. Inorganic contaminants present in these wastes can potentially lead to bioaccumulation in microalgal biomass negatively impact productivity and limiting end use. This study focuses on the experimental evaluation of the impact and the fate of 14 inorganic contaminants (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Sb, Se, Sn, V and Zn) on Nannochloropsis salina growth. Microalgae were cultivated in photobioreactors illuminated at 984 &#181;mol m-2 sec-1 and maintained at pH 7 in a growth media polluted with inorganic contaminants at levels expected based on the composition found in commercial coal flue gas systems. Contaminants present in the biomass and the medium at the end of a 7 day growth period were analytically quantified through cold vapor atomic absorption spectrometry for Hg and through inductively coupled plasma mass spectrometry for As, Cd, Co, Cr, Cu, Mn, Ni, Pb, Sb, Se, Sn, V and Zn. Results show N. salina is a sensitive strain to the multi-metal environment with a statistical decrease in biomass yieldwith the introduction of these contaminants. The techniques presented here are adequate for quantifying algal growth and determining the fate of inorganic contaminants.

Integration of microalgal cultivation with industrial flue gas will ultimately introduce heavy metals and other inorganic compounds into the growth media. This study presents a procedure used to determine the end fate and impact of heavy metals and inorganic contaminants on the growth of Nannochloropsis salina grown in photobioreactors.

重金属和微藻的产其他无机污染物的量化

Increasing demand for renewable fuels has researchers investigating the feasibility of alternative feedstocks, such as microalgae. Inherent advantages ...

Assessment of Labile Organic Carbon in Soil Using Sequential Fumigation Incubation Procedures

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is highly sensitive to disturbance. It is also the primary substrate for soil microorganisms, which is fundamental to nutrient cycling. Due to these attributes, labile organic carbon (LOC) has been identified as an indicator parameter for soil health. Quantifying the turnover rate of LOC also aids in understanding changes in soil nutrient cycling processes. A sequential fumigation incubation method has been developed to estimate soil LOC and potential C turnover rate. The method requires fumigating soil samples and quantifying CO2-C respired during a 10 day incubation period over a series of fumigation-incubation cycles. Labile organic C and potential C turnover rate are then extrapolated from accumulated CO2 with a negative exponential model. Procedures for conducting this method are described.

Labile organic carbon (LOC) and the potential carbon turnover rate are sensitive indicators of changes in soil nutrient cycling processes. Details are provided for a method based on fumigating and incubating soil in a series of cycles and using the CO2 accumulated during the incubation periods to estimate these parameters.

活性有机碳评估土壤中连续熏蒸孵化程序

Management practices and environmental changes can alter soil nutrient and carbon cycling. Soil labile organic carbon, a readily decomposable C pool, is ...

A Gusseted Thermogradient Table to Control Soil Temperatures for Evaluating Plant Growth and Monitoring Soil Processes

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a series of incubators. A temperature gradient is passively established across the surface of the table between the heated and cooled ends and is lost quickly at distances above the surface. Since temperature is only controlled on the table surface, experiments are restricted to shallow containers, such as Petri dishes, placed on the table. Welding continuous aluminum vertical strips or gussets perpendicular to the surface of a table enables temperature control in depth via convective heat flow. Soil in the channels between gussets was maintained across a gradient of temperatures allowing a greater diversity of experimentation. The gusseted design was evaluated by germinating oat, lettuce, tomato, and melon seeds. Soil temperatures were monitored using individual, battery-powered dataloggers positioned across the table. LED lights installed in the lids or along the sides of the gradient table create a controlled temperature chamber where seedlings can be grown over a range of temperatures. The gusseted design enabled accurate determination of optimum temperatures for fastest germination rate and the highest percentage germination for each species. Germination information from gradient table experiments can help predict seed germination and seedling growth under the adverse soil conditions often encountered during field crop production. Temperature effects on seed germination, seedling growth, and soil ecology can be tested under controlled conditions in a laboratory using a gusseted thermogradient table.

Traditional thermogradient tables create a range of temperatures across the surface. Welding gussets perpendicular to the surface of a thermogradient table will control temperature in depth increasing possible research applications.

一个插角Thermogradient表来控制土壤温度为评估植物生长和监测土壤过程

Thermogradient tables were first developed in the 1950s primarily to test seed germination over a range of temperatures simultaneously without using a ...

Isolation and Analysis of Microbial Communities in Soil, Rhizosphere, and Roots in Perennial Grass Experiments

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The purpose of this manuscript is to provide detail on how to rapidly sample soil, rhizosphere, and endosphere of replicated field trials and analyze changes that may occur in the microbial communities due to sample type, treatment, and plant genotype. The experiment used to demonstrate these methods consists of replicated field plots containing two, pure, warm-season grasses (Panicum virgatum and Andropogon gerardii) and a low-diversity grass mixture (A. gerardii, Sorghastrum nutans, and Bouteloua curtipendula). Briefly, plants are excavated, a variety of roots are cut and placed in phosphate buffer, and then shaken to collect the rhizosphere. Roots are brought to the laboratory on ice and surface sterilized with bleach and ethanol (EtOH). The rhizosphere is filtered and concentrated by centrifugation. Excavated soil from around the root ball is placed into plastic bags and brought to the lab where a small amount of soil is taken for DNA extractions. DNA is extracted from roots, soil, and rhizosphere and then amplified with primers for the V4 region of the 16S rRNA gene. Amplicons are sequenced, then analyzed with open access bioinformatics tools. These methods allow researchers to test how the microbial community diversity and composition varies due to sample type, treatment, and plant genotype. Using these methods along with statistical models, the representative results demonstrate there are significant differences in microbial communities of roots, rhizosphere, and soil. Methods presented here provide a complete set of steps for how to collect field samples, isolate, extract, quantify, amplify, and sequence DNA, and analyze microbial community diversity and composition in replicated field trials.

Excavation of plant roots from the field as well as processing of samples into endosphere, rhizosphere, and soil are described in detail, including DNA extraction and data analysis methods. This paper is designed to enable other laboratories to use these techniques for the study of soil, endosphere, and rhizosphere microbiomes.

多年生草试验中土壤、根际和根系微生物群落的分离与分析

Plant and soil microbiome studies are becoming increasingly important for understanding the roles microorganisms play in agricultural productivity. The ...

An Optimized Rhizobox Protocol to Visualize Root Growth and Responsiveness to Localized Nutrients

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive harvest or expensive equipment. We present a customizable and affordable rhizobox method that allows the non-destructive visualization of root growth over time and is particularly well-suited to studying root plasticity in response to localized resource patches. The method was validated by assessing maize genotypic variation in plasticity responses to patches containing 15N-labeled legume residue. Methods are described to obtain representative developmental measurements over time, measure root length density in resource-containing and control patches, calculate root growth rates, and determine 15N recovery by plant roots and shoots. Advantages, caveats, and potential future applications of the method are also discussed. Although care must be taken to ensure that experimental conditions do not bias root growth data, the rhizobox protocol presented here yields reliable results if carried out with sufficient attention to detail.

Visualizing and measuring root growth in situ is extremely challenging. We present a customizable rhizobox method to track root development and proliferation over time in response to nutrient enrichment. This method is used to analyze maize genotypic differences in root plasticity in response to an organic nitrogen source.

一种优化的 Rhizobox 协议, 用于可视化根系生长和对局部营养素的响应

Roots are notoriously difficult to study. Soil is both a visual and mechanical barrier, making it difficult to track roots in situ without destructive ...

High-throughput Siderophore Screening from Environmental Samples: Plant Tissues, Bulk Soils, and Rhizosphere Soils

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling in soils, to pathogen competition, plant growth promotion, and cross-kingdom signaling. Furthermore, siderophores are also of commercial interest in bioleaching and bioweathering of metal-bearing minerals and ores. A rapid, cost effective, and robust means of quantitatively assessing siderophore production in complex samples is key to identifying important aspects of the ecological ramifications of siderophore activity, including, novel siderophore producing microbes. The method presented here was developed to assess siderophore activity of in-tact microbiome communities, in environmental samples, such as soil or plant tissues. The samples were homogenized and diluted in a modified M9 medium (without Fe), and enrichment cultures were incubated for 3 days. Siderophore production was assessed in samples at 24, 48, and 72 hours (h) using a novel 96-well microplate CAS (Chrome azurol sulphonate)-Fe agar assay, an adaptation of the traditionally tedious and time-consuming colorimetric method of assessing siderophore activity, performed on individual cultivated microbial isolates. We applied our method to 4 different genotypes/Lines of wheat (Triticum aestivum L.), including Lewjain, Madsen, and PI561725, and PI561727 commonly grown in the inland Pacific Northwest. Siderophore production was clearly impacted by the genotype of wheat, and in the specific types of plant tissues observed. We successfully used our method to rapidly screen for the influence of plant genotype on siderophore production, a key function in terrestrial and aquatic ecosystems. We produced many technical replicates, yielding very reliable statistical differences in soils and within plant tissues. Importantly, the results show the proposed method can be used to rapidly examine siderophore production in complex samples with a high degree of reliability, in a manner that allows communities to be preserved for later work to identify taxa and functional genes.

We present a protocol for rapid screening of environmental samples for siderophore potential contributing to micronutrient bioavailability and turnover in terrestrial systems.

从环境样品中筛选高通量的安德森菌: 植物组织、散装土壤和根际土壤

Siderophores (low-molecular weight metal chelating compounds) are important in various ecological phenomenon ranging from iron (Fe) biogeochemical cycling ...

Measuring Phosphorus Release in Laboratory Microcosms for Water Quality Assessment

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine laboratory measures of P bioavailability are based on chemical extractions performed on dried samples under oxidizing conditions. While useful, these tests are limited with respect to characterizing P release under prolonged water saturation. Labile orthophosphate bound to oxidized iron and other metals can rapidly desorb to solution in reducing environments, increasing P mobilization risk to surface runoff and groundwater. To better quantify P desorption potential and mobility during extended saturation, a laboratory microcosm method was developed based on repeated sampling of porewater and overlying floodwater over time. The method is useful for quantifying P release potential from soils and sediments varying in physicochemical properties and can improve site-specific P mitigation efforts by better characterizing P release risk in hydrologically active areas. Advantages of the method include its ability to simulate in situ dynamics, simplicity, low cost, and flexibility.

Accurate quantification of phosphorus (P) desorption potential in saturated soils and sediments is important for P modeling and transport mitigation efforts. To better account for in situ soil-water redox dynamics and P mobilization under prolonged saturation, a simple approach was developed based on repeated sampling of laboratory microcosms.

测量实验室微量微生物中的磷释放,用于水质评估

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine ...

Manufacturing Simple and Inexpensive Soil Surface Temperature and Gravimetric Water Content Sensors

Quantifying temperature and moisture at the soil surface is essential for understanding how soil surface biota respond to changes in the environment. However, at the soil surface these variables are highly dynamic and standard sensors do not explicitly measure temperature or moisture in the upper few millimeters of the soil profile. This paper describes methods for manufacturing simple, inexpensive sensors that simultaneously measure the temperature and moisture of the upper 5 mm of the soil surface. In addition to sensor construction, steps for quality control, as well as for calibration for various substrates, are explained. The sensors incorporate a Type E thermocouple to measure temperature and assess soil moisture by measuring the resistance between two gold-plated metal probes at the end of the sensor at a depth of 5 mm. The methods presented here can be altered to customize probes for different depths or substrates. These sensors have been effective in a variety of environments and have endured months of heavy rains in tropical forests as well as intense solar radiation in deserts of the southwestern U.S. Results demonstrate the effectiveness of these sensors for evaluating warming, drying, and freezing of the soil surface in a global change experiment.

Accurately measuring temperature and water content of the upper 5 mm of the soil surface can improve our understanding of environmental controls on biological, chemical, and physical processes. Here we describe a protocol for manufacturing, calibrating, and conducting measurements with soil surface temperature and moisture sensors.

制造简单、廉价的土壤表面温度和重力含水量传感器

Quantifying temperature and moisture at the soil surface is essential for understanding how soil surface biota respond to changes in the environment. ...