WO2017156825A1 - Sem-edx method for detecting distribution characteristics of nitrogen, phosphorus and potassium in crop leaf - Google Patents

Sem-edx method for detecting distribution characteristics of nitrogen, phosphorus and potassium in crop leaf Download PDF

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WO2017156825A1
WO2017156825A1 PCT/CN2016/080089 CN2016080089W WO2017156825A1 WO 2017156825 A1 WO2017156825 A1 WO 2017156825A1 CN 2016080089 W CN2016080089 W CN 2016080089W WO 2017156825 A1 WO2017156825 A1 WO 2017156825A1
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nitrogen
phosphorus
potassium
blade
section
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PCT/CN2016/080089
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French (fr)
Chinese (zh)
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李青林
毛罕平
左志宇
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江苏大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
    • G01N23/223—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material by irradiating the sample with X-rays or gamma-rays and by measuring X-ray fluorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/02—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material
    • G01N23/04—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material and forming images of the material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01B—MEASURING LENGTH, THICKNESS OR SIMILAR LINEAR DIMENSIONS; MEASURING ANGLES; MEASURING AREAS; MEASURING IRREGULARITIES OF SURFACES OR CONTOURS
    • G01B15/00—Measuring arrangements characterised by the use of electromagnetic waves or particle radiation, e.g. by the use of microwaves, X-rays, gamma rays or electrons
    • G01B15/02—Measuring arrangements characterised by the use of electromagnetic waves or particle radiation, e.g. by the use of microwaves, X-rays, gamma rays or electrons for measuring thickness
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
    • G01N23/225—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
    • G01N23/22—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material
    • G01N23/225—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion
    • G01N23/2251—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by measuring secondary emission from the material using electron or ion using incident electron beams, e.g. scanning electron microscopy [SEM]
    • G01N23/2252—Measuring emitted X-rays, e.g. electron probe microanalysis [EPMA]

Definitions

  • the invention relates to a micro-scale nutrient detection method for crops, in particular to a SEM-EDX detection method for the distribution characteristics of nitrogen, phosphorus and potassium in crop leaves.
  • Existing nutrient detection methods include image method, spectroscopy, canopy temperature, and three-dimensional laser scanning.
  • Image recognition methods are usually based on color, morphology and texture characteristics, but these features become apparent only after the crop has developed stress symptoms; the spectroscopy method is mainly based on the principle of chlorophyll content difference caused by nitrogen changes, although It can accurately measure nitrogen nutrient levels, but has low detection accuracy for phosphorus and potassium nutrient levels. This is mainly because the elongation of leaf cells of phosphorus-deficient plants is affected more than the influence of chlorophyll, so the chlorophyll content per unit leaf area is higher; and because of phosphorus-deficient plants, carbohydrate metabolism is blocked and sugar is present.
  • the canopy temperature of crops refers to the average surface temperature of crop canopy stems and leaves, 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.
  • the 3D laser scanning can quickly copy the 3D model of the measured object and the various map data of points, lines, faces and bodies by recording the 3D coordinate information of a large number of points on the surface of the measured object, and has a wide range of crop 3D models.
  • the application can be used as an important indicator to characterize crop growth, but as a nutrient detection method, there is no direct physiological basis.
  • the difference in these macroscopic features is a comprehensive reflection of the microscopic structure of the crop due to the difference in nutrients absorbed and the microscopic differences of various synthetic substances. Therefore, the researchers turned their research focus to micro-scale research, aiming at the early, rapid and accurate detection methods of nutrients from the nature of crop physiological characteristics.
  • the nutrient distribution of plants has spatial distribution differences in plant organs. How to characterize such spatial differences on the microscopic scale and determine the distribution characteristics of various nutrient elements. No relevant research reports have been reported.
  • the X-ray energy spectrum can qualitatively and quantitatively analyze all the elements of Be-U in the micro-region of various samples quickly and simultaneously.
  • the geometrical position requirements of the sample and the detector are low, and the requirements for WD are not very strict.
  • the line scan and surface scan results of X-rays can be obtained at a low magnification, and the probe current required for the energy spectrum is small, after the electron beam is irradiated.
  • Damageable specimens eg, biological specimens, fast ion conductor specimens, etc.
  • X-ray The technology is widely used in the analysis of chemical composition in micro-areas. The smallest analysis area is 10nm or even smaller.
  • the atomic number effect, absorption effect and fluorescence effect of X-ray on the sample during the test are automatically corrected by computer, and the normalized test is used.
  • the content of the elements in the object is calculated. Since the Si(Li) probe in the energy spectrum can be placed very close to the emission source (about 10 cm), there is no need to undergo crystal diffraction, and the signal intensity is hardly lost, so the sensitivity is high. Since the spectrometer has no moving parts, good stability, and no focusing requirements, it has good repeatability and is suitable for analysis of rough surfaces.
  • the method uses the combination of energy spectrum and scanning electron microscopy to detect the distribution characteristics of nitrogen, phosphorus and potassium in crop leaves, and provides the spatial distribution characteristics of nitrogen, phosphorus and potassium in the leaf tissue, which solves the problem that nitrogen can not be realized at present.
  • the object of the present invention is to provide a SEM-EDX detection method for the distribution characteristics of nitrogen, phosphorus and potassium in crop leaves, to obtain the distribution characteristics of nitrogen, phosphorus and potassium in the mesophyll tissue, and to provide the distribution of nitrogen, phosphorus and potassium in the leaves based on energy spectrum technology.
  • the present invention proposes a method for determining the distribution characteristics of nitrogen, phosphorus and potassium in the leaves based on the energy spectrum technology, and can obtain nitrogen and phosphorus in the mesophyll tissue range.
  • the distribution characteristics of potassium elements the method for determining the distribution characteristics of nitrogen, phosphorus and potassium in the leaves based on the energy spectrum technology.
  • the present invention combines scanning electron microscopy (SEM) and X-ray energy spectroscopy (EDX). In this way, the surface cross-section of the blade section and the line scan in the thickness direction were obtained.
  • SEM scanning electron microscopy
  • EDX X-ray energy spectroscopy
  • a SEM-EDX detection method for the distribution characteristics of nitrogen, phosphorus and potassium in crop leaves which comprises the following steps:
  • Step 1 Avoid the leaves of the main vein and take the blade samples.
  • the sample size of the leaves is 1.0cm*1.0cm.
  • the leaf samples are laid in an aluminum box for quick-freezing fixation. Then the sample is taken out and vacuum-dried immediately to obtain a dried sample.
  • Step 2 After the dried blade sample is cut by a blade, the section is fixed upward on the sample stage, and the surface of the section is sprayed with gold by an ion sputtering apparatus to obtain a leaf sample subjected to gold spray treatment;
  • Step 3 The sample of the sprayed gold leaf is sprayed with gold facing up, and is fixed on the sample stage of the scanning electron microscope with a conductive tape, and vacuum is applied to obtain a vacuum sample of the blade;
  • Step 4 setting the working parameters of the spectrometer such that the incident electron beam of the X-ray is as perpendicular as possible to the section of the vacuum-treated blade sample;
  • Step 5 Set the scanning area, scan the blade section micro-area within the blade thickness range, and obtain the distribution energy spectrum images of nitrogen, phosphorus and potassium in the whole section of the blade section;
  • Step 6 Perform line scanning in the thickness direction of the blade to obtain the energy spectrum lines of NPK in the thickness direction of the blade section;
  • Step 7 Analyze the spatial distribution of nitrogen, phosphorus and potassium in the blade section, and determine the distribution characteristics of nitrogen, phosphorus and potassium in the leaf section, including the distribution location characteristics and distribution content characteristics, so as to detect the distribution of nitrogen, phosphorus and potassium in the crop leaves. .
  • the liquid nitrogen is fixed, and the fresh leaf sample is placed in an aluminum box and quickly put into liquid nitrogen for quick freezing and fixing, the fixing time is about 1.5 min, and then vacuum freeze-drying is immediately performed.
  • the second step is specifically: spraying gold on the surface of the sample, the spraying time is 1 min, and the current is 15 mA.
  • the step 3 is specifically: using a carbon conductive adhesive having a resistivity of less than 5 ohms/mm 2 , a base material is an insulating nonwoven fabric, and a conductive material is carbon powder; and a scanning electron microscope is a quanta 200 type environmental scanning electron microscope produced by the American fei company. On the sample stage, observation was carried out at an accelerating voltage of 15 kV.
  • the step 4 is specifically: using the Inca X-Act type electric refrigeration spectrometer produced by the Oxford Company of the United Kingdom to analyze the distribution of each element of the blade section, the acceleration voltage is 20Kv, the count rate of the spectrometer is 1500 cps, and the sampling time is 600s.
  • the spectrometer detector is inserted into the sample chamber, and the characteristic X-rays emitted by the sample are detected, and the energy spectrum image is obtained by feeding the cable to the pulse processor and the computer on the right side of the electron microscope.
  • the step 5 is specifically: the electron beam is repeatedly subjected to raster scanning analysis in the micro-area of the blade section.
  • the step 6 is specifically: the electron beam is scanned in the thickness of the blade section, and the line profiles of the nitrogen, phosphorus and potassium elements are respectively collected.
  • the step 7 is specifically: analyzing the spatial distribution characteristics of nitrogen, phosphorus and potassium in the blade according to the element distribution map obtained in steps 5 and 6.
  • the present invention has a beneficial effect.
  • the invention combines the scanning electron microscopy technique and the energy spectrum technology to obtain the internal structural features of the blade and the distribution characteristics of the nitrogen, phosphorus and potassium elements; the present invention explores the distribution characteristics of the nitrogen, phosphorus and potassium elements in the inner cell scale of the blade from the microscopic scale. It can qualitatively describe their spatial distribution characteristics inside the blade, and provide a basis for micro-scale measurement of crop nutrient information; the present invention provides a method for measuring the distribution characteristics of nitrogen, phosphorus and potassium elements in the blade.
  • Figure 1 is a schematic view of the measurement principle of the present invention
  • Figure 2 is a scanning electron microscope image of a blade section
  • Figure 3 is a diagram showing the result of scanning the nitrogen element surface of the blade section
  • Figure 4 is a diagram showing the result of scanning the phosphorus surface of the blade section
  • Figure 5 is a graph showing the results of the potassium element surface scanning of the blade section
  • Figure 6 is a scanning position diagram of a blade section line
  • Figure 7 is a diagram showing the result of scanning the nitrogen element line of the blade section
  • Figure 8 is a diagram showing the result of scanning the phosphorus element line of the blade section
  • Fig. 9 is a view showing the result of scanning the potassium element line of the blade section.
  • the scanning electron microscope system used in the specific embodiment of the present invention is a quanta 200 type manufactured by Fei Company of the United States, and the cross-sectional microstructure of greenhouse tomato leaves is collected by a microscopic image acquisition system in a scanning electron microscope system.
  • the energy spectrum measuring system used in the specific embodiment of the present invention is an INCAX-Act type of OXFORD, UK, which uses the energy spectrum measuring system to collect the distribution and content information of each element of the tomato leaf section.
  • the invention was carried out 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 selected from the powder.
  • the present invention adopts the soilless cultivation method for sample cultivation, and uses Yamazaki Peifang nutrient solution for watering.
  • the inverted seven leaves of each sample were sampled and repeated five times.
  • Fresh leaf samples were placed in an appropriately sized aluminum box and quickly placed in liquid nitrogen for quick freezing.
  • the fixing time was about 1.5 min, and then immediately placed in an Alpha ⁇ 2 ⁇ 4 type produced by Christ, Germany for vacuum freeze drying and drying. The time is 36h.
  • the lyophilized blade sample was cut with a blade in the thickness direction, and the cross section was fixed upward on the conductive paste, and the gold was sprayed by a Japanese HITACHI E1010 ion sputtering apparatus.
  • the spraying time was 1 min, and the current was 15 mA.
  • the processed blade samples were attached to a sample stage of a quanta 200 type environmental scanning electron microscope manufactured by American fei, and observed under an accelerating voltage of 15 kV, and were made of Inca X-Act type produced by Oxford, England.
  • the cold energy spectrometer analyzes the distribution of each element of the blade section.
  • the accelerating voltage is 20Kv
  • the counting rate of the spectrometer is 1500 cps
  • the sampling time is 600 s.
  • Figure 1 is a schematic diagram of the measurement principle.
  • the blade section was observed under a scanning electron microscope, and the thickness of the epidermis, the thickness of the palisade tissue, the thickness of the sponge tissue, and the thickness of the entire blade were measured. Take a typical view to take a photo. At least 10 fields of view were counted by electron microscopy of each sample and averaged.
  • the detector of the spectrometer is inserted into the sample chamber, the characteristic X-rays emitted by the sample are detected, the surface scan of the blade section and the line scan of the thickness direction are respectively performed, and the scan result is fed through the cable to the pulse processor and the computer on the right side of the electron microscope. Obtain an energy spectrum image of the distribution of NPK.
  • Figure 3 to Figure 5 show the distribution of nitrogen, phosphorus and potassium in the face of the blade by SEM-EDX.
  • Figure 6 shows the position of the blade cross-sectional line scan.
  • Figures 7 to 9 show the distribution of nitrogen, phosphorus and potassium in the thickness direction of the blade section. The higher the peak value in the line, the more the element content is. The lower the peak, the less the element content is. According to the above principles, the spatial distribution characteristics of NPK can be obtained:
  • the distribution signal of nitrogen element can be detected in the whole blade section, but the distribution has certain regional characteristics. Whether it is epidermal cells, palisade tissue cells or sponge tissue cells, the distribution density around the contour of the cell wall is significantly higher than other parts. ;
  • the distribution signal of phosphorus and the distribution signal of nitrogen have greater consistency in regional characteristics, and also exhibit more features along the contour of the cell, but less in the interior of the cell;
  • the distribution signal of potassium element is also visible in the whole blade section, but the distribution characteristics of nitrogen and phosphorus are more evenly distributed throughout the blade section, but lower than the other areas.

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Abstract

An SEM-EDX method for detecting distribution characteristics of nitrogen, phosphorus and potassium in a crop leaf. Surface scanning and line scanning in the direction of thickness are carried out in the cross section of the leaf by employing a combination of a scanning electron microscope (SEM) with energy dispersive X-ray spectroscopy (EDX) to obtain an energy spectrum diagram of nitrogen, phosphorus and potassium in the leaf, and then the energy spectrum diagram is analyzed to obtain the space distribution characteristics of nitrogen, phosphorus and potassium in a mesophyll tissue. Nitrogen, phosphorus and potassium are distributed throughout the cross section of the leaf. Nitrogen and phosphorus have similar distribution characteristics, they are both distributed with significantly higher densities along the periphery of the cell wall contour than other sites; and potassium is distributed relatively uniformly throughout the cross section of the leaf. The above-mentioned detection method solves the problem that the space distribution characteristics of nitrogen, phosphorus and potassium in the leaf cannot be determined, and can be applied to the microscopic detection of crop nutrients.

Description

一种作物叶片内部氮磷钾分布特征的SEM-EDX检测方法SEM-EDX detection method for distribution characteristics of nitrogen, phosphorus and potassium in crop leaves 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 micro-scale nutrient detection method for crops, in particular to a SEM-EDX detection method for the distribution characteristics of nitrogen, phosphorus and potassium in 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。The accurate and rapid detection of crop nutrient information is the basis for the accurate management of nutrients and the reduction of environmental pollution. Therefore, 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。Existing nutrient detection methods include image method, spectroscopy, canopy temperature, and three-dimensional laser scanning. Image recognition methods are usually based on color, morphology and texture characteristics, but these features become apparent only after the crop has developed stress symptoms; the spectroscopy method is mainly based on the principle of chlorophyll content difference caused by nitrogen changes, although It can accurately measure nitrogen nutrient levels, but has low detection accuracy for phosphorus and potassium nutrient levels. This is mainly because the elongation of leaf cells of phosphorus-deficient plants is affected more than the influence of chlorophyll, so the chlorophyll content per unit leaf area is higher; and because of phosphorus-deficient plants, carbohydrate metabolism is blocked and sugar is present. Accumulation, easy to form anthocyanins. Therefore, the spectral reflectance of leaves of phosphorus-deficient plants is greatly affected by the two pigments of chlorophyll and anthocyanin; in addition, the detection mechanism of potassium nutrition level is still immature, so the spectral analysis of leaves is more complicated. The canopy temperature of crops refers to the average surface temperature of crop canopy stems and leaves, 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. The 3D laser scanning can quickly copy the 3D model of the measured object and the various map data of points, lines, faces and bodies by recording the 3D coordinate information of a large number of points on the surface of the measured object, and has a wide range of crop 3D models. The application can be used as an important indicator to characterize crop growth, but as a nutrient detection method, there is no 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 difference in these macroscopic features is a comprehensive reflection of the microscopic structure of the crop due to the difference in nutrients absorbed and the microscopic differences of various synthetic substances. Therefore, the researchers turned their research focus to micro-scale research, aiming at the early, rapid and accurate detection methods of nutrients from the nature of crop physiological characteristics.
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 such spatial differences on the microscopic scale and determine the distribution characteristics of various nutrient elements. No relevant research reports have 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。The X-ray energy spectrum can qualitatively and quantitatively analyze all the elements of Be-U in the micro-region of various samples quickly and simultaneously. The geometrical position requirements of the sample and the detector are low, and the requirements for WD are not very strict. The line scan and surface scan results of X-rays can be obtained at a low magnification, and the probe current required for the energy spectrum is small, after the electron beam is irradiated. Damageable specimens (eg, biological specimens, fast ion conductor specimens, etc.) have small damage and can qualitatively analyze surface elements of solid materials. X-ray The technology is widely used in the analysis of chemical composition in micro-areas. The smallest analysis area is 10nm or even smaller. The atomic number effect, absorption effect and fluorescence effect of X-ray on the sample during the test are automatically corrected by computer, and the normalized test is used. The content of the elements in the object is calculated. Since the Si(Li) probe in the energy spectrum can be placed very close to the emission source (about 10 cm), there is no need to undergo crystal diffraction, and the signal intensity is hardly lost, so the sensitivity is high. Since the spectrometer has no moving parts, good stability, and no focusing requirements, it has good repeatability and is suitable for analysis of 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。This method is currently used primarily in the analysis of various material compositions. However, no research report on the distribution of nutrient elements in leaf tissue by this method has been found.
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。The method uses the combination of energy spectrum and scanning electron microscopy to detect the distribution characteristics of nitrogen, phosphorus and potassium in crop leaves, and provides the spatial distribution characteristics of nitrogen, phosphorus and potassium in the leaf tissue, which solves the problem that nitrogen can not be realized at present. The problem of the distribution characteristics of phosphorus and potassium in the leaves.
发明内容Summary 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 object of the present invention is to provide a SEM-EDX detection method for the distribution characteristics of nitrogen, phosphorus and potassium in crop leaves, to obtain the distribution characteristics of nitrogen, phosphorus and potassium in the mesophyll tissue, and to provide the distribution of nitrogen, phosphorus and potassium in the leaves based on energy spectrum technology. The method of determining the characteristics of the distribution 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：In view of the fact that the current micro-scale nutrient detection is mostly 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 in the leaves based on the energy spectrum technology, and can obtain nitrogen and phosphorus in the mesophyll tissue range. The distribution characteristics of potassium elements; the method for determining the distribution characteristics of nitrogen, phosphorus and potassium in the leaves based on the energy spectrum technology. In order to solve the above technical problems, the present invention combines scanning electron microscopy (SEM) and X-ray energy spectroscopy (EDX). In this way, the surface cross-section of the blade section and the line scan in the thickness direction were obtained. The energy spectrum of NPK in the blade was obtained, and the energy spectrum image was analyzed to obtain the spatial distribution characteristics of nitrogen, phosphorus and potassium in the mesophyll tissue. . The specific technical solutions are 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 in crop leaves, which 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: Avoid the leaves of the main vein and take the blade samples. The sample size of the leaves is 1.0cm*1.0cm. The leaf samples are laid in an aluminum box for quick-freezing fixation. Then the sample is taken out and vacuum-dried immediately to obtain a dried sample.
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 the dried blade sample is cut by a blade, the section is fixed upward on the sample stage, and the surface of the section is sprayed with gold by an ion sputtering apparatus to obtain a leaf sample subjected to gold spray treatment;
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: The sample of the sprayed gold leaf is sprayed with gold facing up, and is fixed on the sample stage of the scanning electron microscope with a conductive tape, and vacuum is applied to obtain a vacuum sample of the blade;
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: setting the working parameters of the spectrometer such that the incident electron beam of the X-ray 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 blade section micro-area within the blade thickness range, and obtain the distribution energy spectrum images of nitrogen, phosphorus and potassium in the whole section of the blade 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: Perform line scanning in the thickness direction of the blade to obtain the energy spectrum lines of NPK 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 in the blade section, and determine the distribution characteristics of nitrogen, phosphorus and potassium in the leaf section, including the distribution location characteristics and distribution content characteristics, so as to detect the distribution 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。In the first step, the liquid nitrogen is fixed, and the fresh leaf sample is placed in an aluminum box and quickly put into liquid nitrogen for quick freezing and fixing, the fixing time is about 1.5 min, and then vacuum freeze-drying is immediately performed.
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 is specifically: 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 step 3 is specifically: using a carbon conductive adhesive having a resistivity of less than 5 ohms/mm 2 , a base material is an insulating nonwoven fabric, and a conductive material is carbon powder; and a scanning electron microscope is a quanta 200 type environmental scanning electron microscope produced by the American fei company. On the sample stage, observation was carried out at an accelerating voltage of 15 kV.
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。The step 4 is specifically: using the Inca X-Act type electric refrigeration spectrometer produced by the Oxford Company of the United Kingdom to analyze the distribution of each element of the blade section, the acceleration voltage is 20Kv, the count rate of the spectrometer is 1500 cps, and the sampling time is 600s. The spectrometer detector is inserted into the sample chamber, and the characteristic X-rays emitted by the sample are detected, and the energy spectrum image is obtained by feeding the cable to the pulse processor and the computer on the right side of the electron microscope.
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 step 5 is specifically: the electron beam is repeatedly subjected to raster scanning analysis in the micro-area of the blade 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 step 6 is specifically: the electron beam is scanned in the thickness of the blade section, and the line profiles of the nitrogen, phosphorus and potassium elements are respectively collected.
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 7 is specifically: analyzing the spatial distribution characteristics of nitrogen, phosphorus and potassium in the blade according to the element distribution map obtained in steps 5 and 6. The brighter the color in the surface distribution image, the more the element distribution content is online. The higher the peak value 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 present invention has a beneficial effect. The invention combines the scanning electron microscopy technique and the energy spectrum technology to obtain the internal structural features of the blade and the distribution characteristics of the nitrogen, phosphorus and potassium elements; the present invention explores the distribution characteristics of the nitrogen, phosphorus and potassium elements in the inner cell scale of the blade from the microscopic scale. It can qualitatively describe their spatial distribution characteristics inside the blade, and provide a basis for micro-scale measurement of crop nutrient information; the present invention provides a method for measuring the distribution characteristics of nitrogen, phosphorus and potassium elements in the blade.
附图说明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 drawings and specific embodiments.
图1是本发明测量原理示意图；Figure 1 is a schematic view of the measurement principle of the present invention;
图2是叶片断面的扫描电镜图片；Figure 2 is a scanning electron microscope image of a blade section;
图3是叶片断面氮元素面扫描结果图；Figure 3 is a diagram showing the result of scanning the nitrogen element surface of the blade section;
图4是叶片断面磷元素面扫描结果图；Figure 4 is a diagram showing the result of scanning the phosphorus surface of the blade section;
图5是叶片断面钾元素面扫描结果图；Figure 5 is a graph showing the results of the potassium element surface scanning of the blade section;
图6是叶片断面线扫描位置图；Figure 6 is a scanning position diagram of a blade section line;
图7是叶片断面氮元素线扫描结果图；Figure 7 is a diagram showing the result of scanning the nitrogen element line of the blade section;
图8是叶片断面磷元素线扫描结果图；Figure 8 is a diagram showing the result of scanning the phosphorus element line of the blade section;
图9是叶片断面钾元素线扫描结果图。Fig. 9 is a view showing the result of scanning the potassium element line of the blade 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 are further described in detail below with reference to 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INCAX-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 used in the specific embodiment of the present invention is a quanta 200 type manufactured by Fei Company of the United States, and the cross-sectional microstructure of greenhouse tomato leaves is collected by a microscopic image acquisition system in a scanning electron microscope system. The energy spectrum measuring system used in the specific embodiment of the present invention is an INCAX-Act type of OXFORD, UK, which uses the energy spectrum measuring system to collect the distribution and content information of each element of the tomato leaf section. The invention was carried out 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 selected from the powder. In order to ensure that the basic research in the early stage can accurately extract the effective characteristics of tomato nutrients, the present invention adopts the soilless cultivation 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 five 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。Fresh leaf samples were placed in an appropriately sized aluminum box and quickly placed in liquid nitrogen for quick freezing. The fixing time was about 1.5 min, and then immediately placed in an Alpha‐2‐4 type produced by Christ, Germany for vacuum freeze drying and 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。The lyophilized blade sample was cut with a blade in the thickness direction, and the cross section was fixed upward on the conductive paste, and the gold was sprayed by a Japanese HITACHI E1010 ion sputtering apparatus. The spraying time was 1 min, and the current was 15 mA. . Then, the processed blade samples were attached to a sample stage of a quanta 200 type environmental scanning electron microscope manufactured by American fei, and observed under an accelerating voltage of 15 kV, and were made of Inca X-Act type produced by Oxford, England. The cold energy spectrometer analyzes the distribution of each element of the blade section. The accelerating voltage is 20Kv, the counting rate of the spectrometer is 1500 cps, and the sampling time is 600 s. Figure 1 is a schematic diagram of the measurement principle.
(3)观察叶片断面微结构(3) Observing the blade cross-section microstructure
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 blade section was observed under a scanning electron microscope, and the thickness of the epidermis, the thickness of the palisade tissue, the thickness of the sponge tissue, and the thickness of the entire blade were measured. Take a typical view to take a photo. At least 10 fields of view were counted by electron microscopy of each sample and averaged.
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。The microscopic observation of the blade section showed that the thickness of the tomato leaf was 123.23±2.5um, and the thickness of the sponge 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) Observing the distribution of leaf section elements
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。The detector of the spectrometer is inserted into the sample chamber, the characteristic X-rays emitted by the sample are detected, the surface scan of the blade section and the line scan of the thickness direction are respectively performed, and the scan result is fed through the cable to the pulse processor and the computer on the right side of the electron microscope. Obtain an energy spectrum image of the distribution of NPK.
(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：Figure 3 to Figure 5 show the distribution of nitrogen, phosphorus and potassium in the face of the blade by SEM-EDX. The greater the brightness of the image, the more the element is distributed. The smaller the brightness, the less the element is distributed. . Figure 6 shows the position of the blade cross-sectional line scan. Figures 7 to 9 show the distribution of nitrogen, phosphorus and potassium in the thickness direction of the blade section. The higher the peak value in the line, the more the element content is. The lower the peak, the less the element content is. According to the above principles, the spatial distribution characteristics of NPK 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；The distribution signal of nitrogen element can be detected in the whole blade section, but the distribution has certain regional characteristics. Whether it is epidermal cells, palisade tissue cells or sponge tissue cells, the distribution density around the contour of the cell wall is significantly higher than other parts. ;
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 and the distribution signal of nitrogen have greater consistency in regional characteristics, and also exhibit more features along the contour of the cell, but less in the interior of 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。 The distribution signal of potassium element is also visible in the whole blade section, but the distribution characteristics of nitrogen and phosphorus are more evenly distributed throughout the blade section, but lower than the other areas.

Claims (6)

  1. 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 in crop leaves, which is characterized by the following steps
    步骤一：避开主脉切取新鲜叶片样品，大小为1.0cm*1.0cm，得新鲜叶片样本；将新鲜叶片样本平铺在铝盒里进行速冻固定，然后取出叶片样本立即进行真空冷冻干燥，得干燥后的叶片样本；Step 1: Avoid the main vein and cut the fresh leaf sample, the size is 1.0cm*1.0cm, and obtain the fresh leaf sample; the fresh leaf sample is tiled in the aluminum box for quick freezing, then the leaf sample is taken out and vacuum freeze-dried immediately. Dried leaf 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 the dried blade sample is cut by a blade, the section is fixed upward on the sample stage, and the surface of the section is sprayed with gold by an ion sputtering apparatus to obtain a blade sample subjected to gold spray treatment;
    步骤三：将所述经过喷金处理的叶片样本的喷金处理断面朝上，并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: spraying the gold-treated blade sample with the gold-plated processing section facing upward, and fixing it on the sample stage of the scanning electron microscope with a conductive tape, and vacuuming to obtain a vacuum-treated 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: setting the working parameters of the spectrometer such that the incident electron beam of the X-ray 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，分别获得叶片断面厚度方向微区内的氮、磷、钾元素的分布能谱图像；Step 5: Set the scanning area, and scan the leaf section micro-area in the thickness range of the blade to obtain the distribution spectrum images of nitrogen, phosphorus and potassium in the micro-region of the blade section thickness direction;
    步骤六：在叶片断面厚度方向，进行线扫描，分别获得叶片断面厚度方向的氮、磷、钾的能谱线；Step 6: Perform line scanning in the thickness direction of the blade section to 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、钾元素在叶片样本断面的分布特征，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 in the blade section, and determine the distribution characteristics of nitrogen, phosphorus and potassium in the leaf sample section, including the distribution location characteristics and distribution content characteristics, so as to detect the distribution of nitrogen, phosphorus and potassium in the crop leaves. feature.
  2. 根据权利要求1所述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:步骤一中所述的速冻固定为液氮冷冻固定，具体为将所述新鲜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，干燥时间为36h。The SEM-EDX detection method for the distribution characteristics of nitrogen, phosphorus and potassium in a crop blade according to claim 1, wherein the quick freezing fixation in the first step is liquid nitrogen freezing and fixing, specifically, the fresh leaf sample is flat. It was placed in an aluminum box and quickly placed in liquid nitrogen for quick freezing. The fixing time was about 1.5 min, and then vacuum freeze-drying was carried out immediately, and the drying time was 36 h.
  3. 根据权利要求1所述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：步骤二中所述喷镀黄金的喷镀时间为1min，电流为15mA。The method of claim 1, wherein the spraying time of the gold plating is 1 min and the current is 15 mA.
  4. 根据权利要求1所述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：所述导电胶为电阻率小于5ohms/mm2、基底材料为绝缘无纺布，导电物质为碳粉的碳导电胶；所述扫描电镜的加速电压为15kV。The SEM-EDX detecting method for the distribution characteristics of nitrogen, phosphorus and potassium in a crop blade according to claim 1, wherein the conductive adhesive has a resistivity of less than 5 ohms/mm 2 , the base material is an insulating nonwoven fabric, and the conductive material It is a carbon conductive paste of carbon powder; the scanning electron microscope has an accelerating voltage of 15 kV.
  5. 根据权利要求1所述一种作物叶片内部氮磷钾分布特征的SEM-EDX检测方法，其 特征在于所述步骤四具体为：用Inca X-Act型电制冷能谱仪对所述抽真空处理叶片样本断面的各元素分布进行分析，加速电压为20Kv；所述能谱仪的计数率为1500cps，采谱时间为600s。The SEM-EDX detecting method for the distribution characteristics of nitrogen, phosphorus and potassium in a crop blade according to claim 1, The step 4 is characterized in that: the distribution of each element of the sample section of the vacuum-treated blade is analyzed by an Inca X-Act type electric refrigeration spectrometer, and the acceleration voltage is 20 Kv; the count rate of the spectrometer is 1500 cps, the sampling time is 600 s.
  6. 根据权利要求1所述一种作物叶片内部氮磷钾分布特征的SEM-EDX检测方法，其特征在于所述步骤七具体为：确定氮、磷、钾在叶片样本断面的空间分布特征，氮、磷、钾在整个叶片样本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；钾在整个叶片样本断面范围内呈现较均匀分布。 The SEM-EDX detection method for the distribution characteristics of nitrogen, phosphorus and potassium in a crop blade according to claim 1, wherein the step 7 is specifically: determining the spatial distribution characteristics of nitrogen, phosphorus and potassium in the sample section of the blade, nitrogen, Phosphorus and potassium were distributed throughout the blade sample section. The distribution characteristics of nitrogen and phosphorus were similar. The distribution density around the cell wall contour was significantly higher than other parts. Potassium was evenly distributed throughout the blade sample section.
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