CN105784736A - Scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDX) detection method for distribution characteristics of nitrogen, phosphorus and potassium in crop leaf - Google Patents
Scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDX) detection method for distribution characteristics of nitrogen, phosphorus and potassium in crop leaf Download PDFInfo
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Abstract
本发明公开了一种作物叶片内部氮磷钾分布特征的SEM‑EDX检测方法，采用扫描电镜技术（SEM）和X射线能谱技术（EDX）相结合的方式，对叶片断面进行了面扫描和厚度方向的线扫描，获取了叶片内部氮磷钾的能谱图，再对能谱图像进行分析，得到氮、磷、钾在叶肉组织中的空间分布特征。氮磷钾在整个叶片断面范围内都有分布，氮和磷的分布特征具有相似性，都沿细胞壁轮廓周围分布密度明显高于其它部位；钾在整个叶片断面范围内呈现较均匀分布。本发明解决了叶片内部氮磷钾元素空间分布特征无法确定的问题，可应用于作物养分的微观检测。
The invention discloses a SEM‑EDX detection method for the distribution characteristics of nitrogen, phosphorus and potassium inside the leaves of crops. It uses scanning electron microscopy (SEM) and X-ray energy spectrum technology (EDX) to scan and analyze the leaves. Line scanning in the thickness direction obtained the energy spectrum of nitrogen, phosphorus, and potassium inside the leaf, and then analyzed the energy spectrum image to obtain the spatial distribution characteristics of nitrogen, phosphorus, and potassium in the mesophyll tissue. Nitrogen, phosphorus and potassium were distributed in the whole section of the leaf, and the distribution characteristics of nitrogen and phosphorus were similar, and the distribution density around the cell wall contour was significantly higher than that in other parts; potassium was more evenly distributed in the whole section of the leaf. The invention solves the problem that the spatial distribution characteristics of the nitrogen, phosphorus and potassium elements inside the leaves cannot be determined, and can be applied to the microscopic detection of crop nutrients.
Description
技术领域technical field
本发明涉及作物微观尺度养分检测方法，具体涉及一种作物叶片内部氮磷钾分布特征的SEM-EDX检测方法。The invention relates to a crop microscale nutrient detection method, in particular to a SEM-EDX detection method for the distribution characteristics of nitrogen, phosphorus and potassium inside crop leaves.
背景技术Background technique
作物养分信息的准确、快速检测是实现养分精确管理，减少环境污染的基础，因而近年来一直是农业工程领域的研究热点。Accurate and rapid detection of crop nutrient information is the basis for realizing precise nutrient management and reducing environmental pollution, so it has been a research hotspot in the field of agricultural engineering in recent years.
现有的养分检测方法主要有：图像法、光谱法、冠层温度、三维激光扫描法等。图像识别方法通常是基于颜色、形态和纹理特征的，但这些特征只有在作物出现胁迫症状后才变得明显；光谱法主要是根据因氮素变化而引起的叶绿素含量差异原理而进行测量，虽能精确测量氮营养水平，但对磷、钾营养水平的检测精度低。这主要是因为缺磷植物叶片细胞其伸长受影响的程度超过叶绿素所受的影响，因此其单位叶面积中叶绿素含量反而较高；又因为缺磷的植株，体内碳水化合物代谢受阻，有糖分积累，易形成花青素。所以，缺磷的植株的叶片的光谱反射率受叶绿素和花青素这两个色素的影响较大；另外钾营养水平的检测机理还不成熟，因而叶片光谱分析较为复杂。作物的冠层温度系指作物冠层茎、叶表面温度的平均值，与水分、养分、光照、通风等诸多因素有关，因此冠层温度和养分之间的直接相关性的表征存在一定难度。三维激光扫描可以通过记录被测物体表面大量点的三维坐标信息，快速复制出被测目标的三维模型及点、线、面、体等各种图件数据，在作物三维模型的建立方面有着广泛的应用，可以作为表征作物长势信息的重要指标，但作为养分检测方法，缺乏直接的生理依据。The existing nutrient detection methods mainly include: image method, spectrum method, canopy temperature, three-dimensional laser scanning method, etc. Image recognition methods are usually based on color, shape, and texture features, but these features become apparent only after the crops show stress symptoms; spectroscopic methods are mainly based on the principle of differences in chlorophyll content caused by nitrogen changes, although It can accurately measure the level of nitrogen nutrition, but the detection accuracy of phosphorus and potassium nutrition levels is low. This is mainly because the elongation of leaf cells of phosphorus-deficient plants is more affected than that of chlorophyll, so the chlorophyll content per unit leaf area is higher; Accumulation, easy to form anthocyanins. Therefore, the spectral reflectance of leaves of phosphorus-deficient plants is greatly affected by two pigments, chlorophyll and anthocyanin; in addition, the detection mechanism of potassium nutrition level is not yet mature, so the spectral analysis of leaves is more complicated. The canopy temperature of crops refers to the average temperature of the stem and leaf surface of the crop canopy, which is related to many factors such as water, nutrients, light, ventilation, etc. Therefore, it is difficult to characterize the direct correlation between canopy temperature and nutrients. Three-dimensional laser scanning can quickly copy the three-dimensional model of the measured object and various map data such as points, lines, surfaces, and volumes by recording the three-dimensional coordinate information of a large number of points on the surface of the measured object. It can be used as an important indicator to characterize crop growth information, but as a nutrient detection method, it lacks direct physiological basis.
另一方面，这些宏观特征的差异，是作物因吸收的养分差异而导致的微观结构以及各种合成物质等微观差异的综合体现。因此，研究人员将研究重点转至微观尺度的研究，旨在从作物生理特征的本质入手，寻求养分的早期、快速、准确的检测方法。On the other hand, the differences in these macroscopic characteristics are a comprehensive reflection of the microscopic differences in the microstructure and various synthetic substances caused by the differences in the nutrients absorbed by crops. Therefore, the researchers shifted their research focus to micro-scale research, aiming to start with the essence of crop physiological characteristics and seek early, rapid and accurate detection methods for nutrients.
植物的营养分布在植物器官内存在空间分布差异，在微观尺度上如何表征这种空间差异，并确定各营养元素的分布特征，未见相关研究报道。The nutrient distribution of plants has spatial distribution differences in plant organs. How to characterize this spatial difference at the microscopic scale and determine the distribution characteristics of each nutrient element has not been reported.
X射线能谱能快速、同时对各种试样的微区内Be-U的所有元素进行定性、定量分析。对试样和探测器的几何位置要求低，对W.D的要求不是很严格，可以在低倍率下获得X射线的线扫描、面扫描结果，能谱所需探针电流小，对电子束照射后易损伤的试样（如，生物试样、快离子导体试样等）损伤小，能对固体材料表面元素进行定性分析。X射线技术广泛应用于微区化学成分的分析，最小的分析区10nm甚至更小，测试过程中X射线对样品产生的原子序数效应、吸收效应及荧光效应均由计算机自动校正，采用归一法对测试对象中的元素的含量进行计算。由于能谱中Si（Li）探头可以放在离发射源很近的地方（10cm左右），无需经过晶体衍射，信号强度几乎没有损失，所以灵敏度高。由于能谱仪没有运动部件，稳定性好，且没有聚焦要求，所以重复性好，适合比较粗糙表面的分析工作。X-ray energy spectroscopy can quickly and simultaneously conduct qualitative and quantitative analysis of all elements of Be-U in the micro-regions of various samples. The requirements for the geometric position of the sample and the detector are low, and the requirements for W.D. are not very strict. The X-ray line scanning and surface scanning results can be obtained at low magnification. The probe current required for the energy spectrum is small. Samples that are easily damaged (such as biological samples, fast ion conductor samples, etc.) have little damage, and can perform qualitative analysis on the surface elements of solid materials. X-ray technology is widely used in the analysis of micro-area chemical composition. The smallest analysis area is 10nm or even smaller. During the test, the atomic number effect, absorption effect and fluorescence effect of X-ray on the sample are automatically corrected by the computer, and the normalization method is adopted. Calculate the content of elements in the test object. Since the Si(Li) probe in the energy spectrum can be placed very close to the emission source (about 10cm), there is no need for crystal diffraction, and there is almost no loss in signal strength, so the sensitivity is high. Since the energy spectrometer has no moving parts, it has good stability and no focusing requirements, so it has good repeatability and is suitable for the analysis of relatively rough surfaces.
目前该方法主要用于各种材料成分的分析中。但未见借助该方法分析叶片组织内营养元素分布的研究报道。At present, this method is mainly used in the analysis of various material components. However, there is no research report on analyzing the distribution of nutrient elements in leaf tissue by this method.
本方法采用能谱技术和扫描电镜技术相结合对作物叶片内部氮、磷、钾的分布特征进行了检测，提供了氮、磷、钾在叶片组织内的空间分布特征，解决了目前无法实现氮、磷、钾在叶片内分布特征表征的问题。This method uses the combination of energy spectrum technology and scanning electron microscope technology to detect the distribution characteristics of nitrogen, phosphorus and potassium in the leaves of crops, and provides the spatial distribution characteristics of nitrogen, phosphorus and potassium in leaf tissues, which solves the problem that nitrogen cannot be realized at present. , phosphorus and potassium distribution characteristics in leaves.
发明内容Contents of the invention
本发明目的在于提供一种作物叶片内部氮磷钾分布特征的SEM-EDX检测方法，以获得叶肉组织范围内氮、磷、钾元素的分布特征，提供基于能谱技术的叶片内部氮磷钾分布特征的确定方法，实现氮、磷、钾在叶片内分布特征表征。The purpose of the present invention is to provide a SEM-EDX detection method for the distribution characteristics of nitrogen, phosphorus and potassium inside the leaves of crops, so as to obtain the distribution characteristics of nitrogen, phosphorus and potassium elements within the mesophyll tissue, and provide the distribution of nitrogen, phosphorus and potassium inside the leaves based on energy spectrum technology. The determination method of the characteristics realizes the distribution characteristics of nitrogen, phosphorus and potassium in the leaves.
针对目前微观尺度养分检测大都是基于统计分析，检测机理不明确的问题，本发明提出了一种基于能谱技术的叶片内部氮磷钾分布特征的确定方法，可以获得叶肉组织范围内氮、磷、钾元素的分布特征；提供基于能谱技术的叶片内部氮磷钾分布特征的确定方法为了解决以上技术问题，本发明采用扫描电镜技术（SEM）和X射线能谱技术（EDX）相结合的方式，对叶片断面进行了面扫描和厚度方向的线扫描，获取了叶片内部氮磷钾的能谱图，再对能谱图像进行分析，得到氮、磷、钾在叶肉组织中的空间分布特征。具体技术方案如下：Aiming at the problem that most of the current micro-scale nutrient detection is based on statistical analysis and the detection mechanism is not clear, the present invention proposes a method for determining the distribution characteristics of nitrogen, phosphorus, and potassium inside the leaf based on energy spectrum technology, which can obtain nitrogen, phosphorus, and nitrogen within the range of mesophyll tissue. , the distribution characteristics of potassium elements; providing a method for determining the distribution characteristics of nitrogen, phosphorus and potassium inside the blade based on energy spectrum technology. By means of surface scanning and line scanning in the thickness direction of the leaf section, the energy spectrum of nitrogen, phosphorus, and potassium inside the leaf was obtained, and then the energy spectrum image was analyzed to obtain the spatial distribution characteristics of nitrogen, phosphorus, and potassium in the mesophyll tissue . The specific technical scheme is as follows:
一种作物叶片内部氮磷钾分布特征的SEM-EDX检测方法，其特征在于包括以下步骤：A SEM-EDX detection method for the distribution characteristics of nitrogen, phosphorus and potassium inside a crop leaf is characterized in that it comprises the following steps:
步骤一：避开主脉切取叶片得叶片样品，叶片样品大小为1.0cm*1.0cm，将叶片样品平铺在铝盒进行速冻固定，然后取出样本立即进行真空冷冻干燥，得干燥后的样本；Step 1: Cut the leaves away from the main veins to obtain leaf samples. The size of the leaf samples is 1.0cm*1.0cm. Spread the leaf samples flat in an aluminum box for quick freezing and fixation. Then take out the samples and immediately perform vacuum freeze-drying to obtain dried samples;
步骤二：将所述干燥后的叶片样本用刀片切开后，断面朝上用固定在样品台上，对所述断面表面用离子溅射仪喷镀黄金，得经过喷金处理的叶片样本；Step 2: After cutting the dried blade sample with a blade, fix the cross-section upward on the sample stage, and spray gold on the surface of the cross-section with an ion sputtering instrument to obtain a gold-sprayed blade sample;
步骤三：将经过喷金处理的叶片样本，喷金处理断面朝上，用导电胶带固定在扫描电镜的样品台上，并抽真空，得抽真空处理叶片样本；Step 3: Fix the gold-sprayed leaf sample with the gold-sprayed section facing upwards on the sample stage of the scanning electron microscope with conductive tape, and vacuumize the blade sample;
步骤四：设置能谱仪工作参数，使X射线的入射电子束尽可能垂直于所述抽真空处理叶片样本断面；Step 4: Set the working parameters of the energy spectrometer so that the incident electron beam of X-rays is as perpendicular as possible to the section of the vacuum-treated blade sample;
步骤五：设置扫描区域，在叶片厚度范围内对叶片断面微区进行面扫描，分别获得叶片断面整个区域内的氮、磷、钾元素的分布能谱图像；Step 5: Set the scanning area, scan the micro-area of the blade cross-section within the range of blade thickness, and obtain the distribution energy spectrum images of nitrogen, phosphorus, and potassium elements in the entire area of the blade cross-section;
步骤六：在叶片厚度方向，进行线扫描，分别获得叶片断面厚度方向氮磷钾的能谱线；Step 6: Carry out line scanning in the thickness direction of the blade, and obtain the energy spectrum lines of nitrogen, phosphorus and potassium in the thickness direction of the blade section;
步骤七：分析叶片断面氮、磷、钾元素的空间分布，确定氮、磷、钾元素在叶片断面的分布特征，包括分布位置特征和分布含量特征，从而检测到作物叶片内部氮磷钾分布特征。Step 7: Analyze the spatial distribution of nitrogen, phosphorus, and potassium elements in the leaf section, and determine the distribution characteristics of nitrogen, phosphorus, and potassium elements in the leaf section, including the distribution position characteristics and distribution content characteristics, so as to detect the distribution characteristics of nitrogen, phosphorus, and potassium in the crop leaves .
所述步骤一中的固定为液氮，将新鲜的叶片样本平铺在铝盒里迅速投入液氮进行速冻固定，固定时间约为1.5min，然后立即进行真空冷冻干燥。The fixation in said step 1 is liquid nitrogen, and the fresh leaf samples are spread flat in an aluminum box and quickly put into liquid nitrogen for quick-freezing and fixing. The fixing time is about 1.5 minutes, and then vacuum freeze-drying is carried out immediately.
所述步骤二具体为：对所述样品表面喷镀黄金，喷镀时间为1min，电流为15mA。The second step specifically includes: spraying gold on the surface of the sample, the spraying time is 1 min, and the current is 15 mA.
所述步骤三具体为：用电阻率小于5ohms/mm2、基底材料为绝缘无纺布，导电物质为碳粉的碳导电胶；扫描电镜是由美国fei公司生产的quanta200型环境扫描电子显微镜的样品台上，在15kV加速电压下进行观察。The third step is specifically: use a carbon conductive adhesive with a resistivity of less than 5 ohms/mm 2 , an insulating non-woven fabric as a base material, and carbon powder as a conductive substance; the scanning electron microscope is a quanta200 environmental scanning electron microscope produced by an American fei company. On the sample stage, observe at an accelerating voltage of 15kV.
所述步骤四具体为：用英国牛津公司生产的Inca X-Act型电制冷能谱仪进行叶片断面的各元素分布分析，加速电压为20Kv，能谱仪的计数率为1500cps，采谱时间为600s。将能谱仪的探测器插入样品室，检测样品出射的特征X射线，通过电缆馈送到电镜右侧的脉冲处理器和计算机，获取能谱图像。Described step 4 is specifically: carry out each element distribution analysis of blade section with the Inca X-Act type electric cooling energy spectrometer produced by British Oxford Company, the accelerating voltage is 20Kv, the counting rate of energy spectrometer is 1500cps, and the spectrum acquisition time is 600s. Insert the detector of the energy spectrometer into the sample chamber to detect the characteristic X-rays emitted by the sample, and feed them to the pulse processor and computer on the right side of the electron microscope through the cable to obtain the energy spectrum image.
所述步骤五具体为：让电子束在叶片断面微区反复做光栅扫描分析。The fifth step is specifically: let the electron beam repeatedly perform raster scanning analysis on the micro-area of the blade cross section.
所述步骤六具体为：让电子束在叶片断面厚度做线扫描，分别采集氮、磷、钾元素的线分布图。The sixth step is specifically: let the electron beam perform line scanning on the thickness of the blade section, and collect line distribution diagrams of nitrogen, phosphorus, and potassium elements respectively.
所述步骤七具体为：根据步骤五、六中获得的元素分布图，分析氮、磷、钾在叶片内部的空间分布特征，在面分布图像中颜色越亮，表示元素分布含量越多，在线分布图像中峰值越高，表示元素分布含量越高。The step seven is specifically: according to the element distribution map obtained in steps five and six, analyze the spatial distribution characteristics of nitrogen, phosphorus, and potassium inside the blade. The brighter the color in the surface distribution image, the more elements are distributed. Online The higher the peak in the distribution image, the higher the element distribution content.
本发明具有有益效果。本发明借助扫描电镜技术和能谱技术相结合，可以获取叶片内部结构特征和氮磷钾元素的分布特征；本发明从微观尺度出发，探求了氮磷钾元素在叶片内部细胞尺度上的分布特征，能定性描述它们在叶片内部的空间分布特征，为作物养分信息的微观尺度测量提供依据；本发明提供了一种叶片内部氮磷钾元素分布特征的测量方法。The invention has beneficial effects. The present invention combines scanning electron microscope technology and energy spectrum technology to obtain the internal structural characteristics of leaves and the distribution characteristics of nitrogen, phosphorus and potassium elements; the present invention explores the distribution characteristics of nitrogen, phosphorus and potassium elements on the cell scale inside leaves from the microscopic scale , can qualitatively describe their spatial distribution characteristics inside the leaves, and provide a basis for the micro-scale measurement of crop nutrient information; the invention provides a method for measuring the distribution characteristics of nitrogen, phosphorus and potassium elements inside the leaves.
附图说明Description of drawings
下面结合附图和具体实施方法对本发明做出进一步详细的说明。The present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation methods.
图1是本发明测量原理示意图；Fig. 1 is a schematic diagram of the measurement principle of the present invention;
图2是叶片断面的扫描电镜图片；Fig. 2 is the scanning electron microscope picture of blade section;
图3是叶片断面氮元素面扫描结果图；Fig. 3 is the scanning result map of the nitrogen element surface of the blade section;
图4是叶片断面磷元素面扫描结果图；Fig. 4 is the surface scanning result diagram of the phosphorus element of the blade section;
图5是叶片断面钾元素面扫描结果图；Fig. 5 is the scanning result figure of potassium element surface of blade section;
图6是叶片断面线扫描位置图；Fig. 6 is the scanning position map of blade section line;
图7是叶片断面氮元素线扫描结果图；Fig. 7 is the line scan result diagram of nitrogen element in blade section;
图8是叶片断面磷元素线扫描结果图；Fig. 8 is a line scan result diagram of phosphorus element in blade section;
图9是叶片断面钾元素线扫描结果图。Fig. 9 is a line scan result diagram of potassium element in the leaf section.
具体实施方式detailed description
下面结合附图和具体实施例对本发明的技术方案做进一步详细描述。The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.
本发明具体实施方式中所采用的扫描电子显微镜系统是由美国Fei公司生产的quanta200型，利用扫描电子显微系统中的显微图像采集系统采集温室番茄叶片的断面微观结构。本发明具体实施方式中所采用的能谱测量系统是由英国OXFORD公司INCA X-Act型，利用能谱测量系统采集番茄叶片断面各元素的分布和含量信息。本发明于2015年8月至2015年12月在江苏大学现代农业装备与技术教育部重点实验室玻璃温室中进行实验，番茄品种选用粉霞。为保证前期的基础性研究能够对番茄养分的有效特征进行准确提取，本发明采用无土栽培方式进行样本培育，采用山崎培方营养液进行浇灌。为了保证样本的代表性，对每个样本的倒七叶进行取样，5次重复。The scanning electron microscope system adopted in the specific embodiment of the present invention is the quanta200 type produced by Fei Company of the United States, and the microscopic image acquisition system in the scanning electron microscope system is used to collect the cross-sectional microstructure of the tomato leaves in the greenhouse. The energy spectrum measuring system adopted in the specific embodiment of the present invention is the INCA X-Act type of British OXFORD company, utilizes the energy spectrum measuring system to collect the distribution and content information of each element of the tomato leaf section. The present invention was tested in the glass greenhouse of the Key Laboratory of Modern Agricultural Equipment and Technology of the Ministry of Education of Jiangsu University from August 2015 to December 2015, and the tomato variety was Fenxia. In order to ensure that the effective characteristics of tomato nutrients can be accurately extracted in the previous basic research, the present invention adopts a soilless culture method for sample cultivation, and uses Yamazaki Peifang nutrient solution for watering. In order to ensure the representativeness of the samples, the inverted seven leaves of each sample were sampled and repeated 5 times.
（1）叶片样本固定(1) Leaf sample fixation
将新鲜的叶片样本装入大小适当的铝盒里迅速投入液氮进行速冻固定，固定时间约为1.5min，然后立即放入由德国Christ公司生产的Alpha-2-4型进行真空冷冻干燥，干燥时间为36h。Put the fresh leaf sample into an aluminum box of appropriate size and quickly put it into liquid nitrogen for quick-freezing and fixing. The fixing time is about 1.5 minutes, and then immediately put it into the Alpha-2-4 type produced by Christ Company of Germany for vacuum freeze-drying. The time is 36h.
（2）仪器参数设计及样品安装(2) Instrument parameter design and sample installation
将经过冷冻干燥处理的叶片样本用刀片沿厚度方向切开，断面朝上固定在在导电胶上，放入由日本HITACHI E1010离子溅射仪中喷镀黄金，喷镀时间为1min，电流为15mA。然后将处理好的叶片样品粘贴在由美国fei公司生产的quanta200型环境扫描电子显微镜的样品台上，在15kV加速电压下进行观察，并用英国牛津公司生产的Inca X-Act型电制冷能谱仪进行叶片断面的各元素分布分析，加速电压为20Kv，能谱仪的计数率为1500cps，采谱时间为600s。图1是测量原理示意图。Cut the freeze-dried leaf sample along the thickness direction with a blade, fix it on the conductive glue with the cross-section facing up, and put it into the Japanese HITACHI E1010 ion sputtering instrument to spray gold, the spraying time is 1min, and the current is 15mA . Then paste the treated blade samples on the sample stage of the quanta200 environmental scanning electron microscope produced by the American fei company, observe under the acceleration voltage of 15kV, and use the Inca X-Act electric refrigeration energy spectrometer produced by the British Oxford company The element distribution analysis of the blade section was carried out, the accelerating voltage was 20Kv, the counting rate of the energy spectrometer was 1500cps, and the spectrum acquisition time was 600s. Figure 1 is a schematic diagram of the measurement principle.
（3）观察叶片断面微结构(3) Observe the microstructure of the blade section
在扫描电子显微镜下观察叶片断面，测量表皮厚度、栅栏组织厚度、海绵组织厚度和整个叶片厚度。选取典型视野进行拍照。每个样本电镜观察统计至少10个视野，取平均值。The leaf section was observed under a scanning electron microscope, and the thickness of the epidermis, palisade tissue, spongy tissue and the whole leaf were measured. Select a typical field of view to take pictures. At least 10 fields of view were counted for each sample under the electron microscope, and the average value was taken.
叶片断面显微观察发现：番茄叶片厚度为123.23±2.5um，其中海绵组织厚度为69.15±6.5um，栅栏组织厚度为49.87±1.3um，如图2所示。Microscopic observation of the leaf section revealed that the thickness of the tomato leaf was 123.23±2.5um, of which the thickness of the spongy tissue was 69.15±6.5um, and the thickness of the palisade tissue was 49.87±1.3um, as shown in Figure 2.
（4）观察叶片断面元素分布(4) Observe the distribution of elements in the blade section
将能谱仪的探测器插入样品室，检测样品出射的特征X射线，对叶片断面分别进行面扫描和厚度方向的线扫描，将扫描结果通过电缆馈送到电镜右侧的脉冲处理器和计算机，获取氮磷钾分布的能谱图像。Insert the detector of the energy spectrometer into the sample chamber to detect the characteristic X-rays emitted by the sample, perform surface scanning and line scanning in the thickness direction on the blade section, and feed the scanning results to the pulse processor and computer on the right side of the electron microscope through the cable. Obtain a spectral image of the NPK distribution.
（5）分析叶片断面氮磷钾的空间分布特征；(5) Analyze the spatial distribution characteristics of nitrogen, phosphorus and potassium in the leaf section;
图3~图5分别是借助SEM-EDX对叶片断面进行面扫描得到的氮、磷、钾的分布情况，图像亮度越大，表示该元素分布也越多，亮度越小该元素元素分布越少。图6表示叶片断面线扫描的位置，图7~图9分别是对叶片断面进行厚度方向线扫描得到的氮、磷、钾的分布情况，谱线中峰值越高表示该处元素含量越多，峰值越低表示该处元素含量越少。根据上述原则，可得到氮磷钾的空间分布特征：Figures 3 to 5 are the distributions of nitrogen, phosphorus, and potassium obtained by scanning the leaf section with the help of SEM-EDX. The larger the brightness of the image, the more the element is distributed, and the smaller the brightness, the less the element is distributed. . Figure 6 shows the position of the line scan of the leaf section. Figures 7 to 9 show the distribution of nitrogen, phosphorus, and potassium obtained by line scanning the blade section in the thickness direction. The higher the peak value in the spectral line, the more element content there. The lower the peak, the lower the element content there. According to the above principles, the spatial distribution characteristics of nitrogen, phosphorus and potassium can be obtained:
1）氮元素的分布信号在整个叶片断面范围内都能检测到，但分布存在一定的区域特征，不论是表皮细胞、栅栏组织细胞、还是海绵组织细胞，沿细胞壁轮廓周围分布密度明显高于其它部位；1) The distribution signal of nitrogen element can be detected in the whole section of the leaf, but the distribution has certain regional characteristics. Whether it is epidermal cells, palisade tissue cells, or spongy tissue cells, the distribution density along the contour of the cell wall is significantly higher than that of other cells. part;
2）磷元素的分布信号和氮元素的分布信号在区域特征上具有较大的一致性，同样呈现沿细胞轮廓分布较多的特征，而在细胞内部分布较少；2) The distribution signal of phosphorus element and the distribution signal of nitrogen element have greater consistency in regional characteristics, and also show the characteristics of more distribution along the cell outline, but less distribution inside the cell;
3）钾元素的分布信号在整个叶片断面范围内也是可见的，但不同于氮、磷元素的分布特征，在整个叶片断面范围内呈现较均匀分布，在细胞壁轮廓周围分布却低于其它区域。3) The distribution signal of potassium element is also visible in the whole leaf section, but different from the distribution characteristics of nitrogen and phosphorus elements, it is more uniform in the whole leaf section, and the distribution around the cell wall outline is lower than other areas.
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