WO2017092120A1

WO2017092120A1 - Method for determining puncture position of probe during measurement of concentration of ions in leaf

Info

Publication number: WO2017092120A1
Application number: PCT/CN2015/099356
Authority: WO
Inventors: 李青林; 毛罕平; 左志宇; 倪纪恒; 张晓东; 孙俊
Original assignee: 江苏大学
Priority date: 2015-11-30
Filing date: 2015-12-29
Publication date: 2017-06-08
Also published as: CN105466958A

Abstract

A method for determining a puncture position of a probe during the measurement of the concentration of ions in a leaf. The method comprises: firstly, acquiring microstructure images of a surface and a cross section of a tomato leaf with the aid of a scanning electron micrograph system; obtaining the geometric dimensions and distribution density of villi, vascular bundles and stomas of the front and back surfaces of the leaf through observation and statistical analysis of surface microstructures; determining a puncture position of a probe on the leaf surface according to reliable and accurate measurement principles; observing structural features of the cross section of the leaf in the scanning electron micrograph system, and analysing the distribution of various elements in the thickness direction of the leaf with the aid of energy spectrum measurement technology; and finally, according to distribution features of the elements, selecting a tissue with high distribution concentration and taking same as the measurement position of the probe. By means of the method of a scanning electron microscope technology combined with an energy spectrum measurement technology, the best puncture position and puncture depth of a probe during measurement of the concentration of ions in a leaf are determined to improve the measurement accuracy; and the method can be applied to the measurement of the concentration of the ions in the leaf.

Description

Method for determining probe puncture position when measuring ion concentration inside blade

Technical field

The invention relates to a plant physiological detection method, in particular to a method for determining a puncture position of an ion concentration measurement probe inside a blade based on a combination of scanning electron microscopy (SEM) technology and X-ray energy spectrum (EDX) technology. , belongs to the field of testing technology.

Background technique

Accurate and rapid detection of crop nutrition is the basis of precise agricultural development. Microscale detection has attracted the attention of researchers because of its early nature and accuracy.

From the physiological point of view of plants, nutrient stress causes a series of changes in plant body fluid concentration, physicochemical properties and leaf water potential. These changes will lead to changes in ion concentration in crop leaves. Therefore, it can be realized by detecting changes in ion concentration in leaves. Diagnosis of plant nutritional status.

The most commonly used microscale nutritional assays are ion selective microelectrodes.

The method of using microelectrodes to measure the nutritional status of crops is to measure the nutritional status by measuring the difference in ion concentration inside the mesophyll tissue by inserting the probe into the crop leaves. Because the arrangement of cells inside the blade differs in the thickness direction of the blade, the insertion position and insertion depth of the probe directly determine the measurement result.

The surface of tomato leaves is widely distributed with villus, vascular bundles and other structures. The thickness of leaves is generally about 100um. The mesophyll tissue is composed of upper epidermis, palisade tissue, sponge tissue and epidermis in the thickness direction. In the existing measurement method, the insertion of the microelectrode is basically based on the empirical method, and the position and depth of the puncture are randomly selected, which directly affects the test result, resulting in inaccurate measurement.

The invention starts from the microscopic scale, analyzes the structural composition of the blade, the distribution characteristics of each element in the thickness direction of the blade, and combines the shape parameters of the microelectrode to determine the optimal puncture position and the optimal puncture depth.

Summary of the invention

The object of the present invention is to provide a method for determining the puncture position of a probe when measuring the ion concentration inside the blade, so as to improve the accuracy of the measurement.

In order to solve the above technical problems, the present invention is directed to the randomness problem of the current probe penetration position and the penetration depth, and the combination of the scanning electron microscopy technique and the energy spectrum technique is adopted by the blade microstructure and the blade constituent elements. The statistical analysis of the distribution within the blade is based on the blade microstructure and energy spectrum analysis to determine the optimal puncture position and puncture depth of the probe during ion concentration measurement. The specific technical solutions are as follows:

A method for determining a probe puncture position when measuring an ion concentration inside a blade, comprising the steps of:

Step 1: Avoid the main vein and cut the leaves to obtain the leaf samples. The leaf sample size is 2cm*2cm, and the fixed solution is used. The sample of the leaves taken is fixed;

Step 2: Prepare a sample of the scanning electron microscope, set the instrument parameters and observe;

Step 3: Observe and analyze the surface structure of the leaf sample;

Step 4: Determine the optimal puncture position of the probe;

Step 5: Observe the cross-sectional structure of the blade sample;

Step 6: Obtain the longitudinal distribution of magnesium, potassium and calcium elements in the leaf sample tissue;

Step 7: Determine the optimal puncture location and puncture depth.

In the first step, the fixing solution is a glutaraldehyde fixing solution having a concentration of 4% to fix the taken leaf sample.

Step 2 is specifically as follows: the leaf sample obtained in the first step is dehydrated by ethanol step, and then placed in a K850 critical point dryer manufactured by Quorum, UK for drying, and the prepared blade sample is adhered to the conductive adhesive, and placed. The coating was carried out in an MSP-1S type ion sputtering apparatus manufactured by Japan SHINKKU VD Co., Ltd., and the thickness of the sputtered metal was 20 nm, and then the processed blade sample was attached to a sample stage of a Quanta 200 type scanning electron microscope manufactured by American fei company. Observe and take pictures under the acceleration voltage of 15kV.

The third step is specifically: under the scanning electron microscope system, 10 fields in different regions are selected to observe the surface microstructure of the leaf sample, including the distribution density and geometric size of the villi, vascular bundles, stomata, and epidermal cells, for measurement and statistics. Analysis shows the distribution density and geometric size of the surface fluff, vascular bundle, stomatal and epidermal cells of the leaf sample.

The step 4 is specifically: according to the surface microstructure of the blade sample observed in the third step, according to the density of the fluff and the distribution characteristics of the vascular bundle, combined with the geometrical dimensions of the probe and the characteristics of the material parameters, the position of the back of the blade to avoid the vascular bundle is selected as the probe. Puncture position.

The step 5 is specifically: observing the section layer of the leaf sample under the scanning electron microscope, measuring the thickness of the epidermis, the palisade tissue, the sponge tissue and the whole blade; taking a typical field of view to take a photograph; each leaf sample is subjected to electron microscopic observation and at least 10 fields of view, taking an average value.

The step 6 is specifically: by means of the energy spectrum measuring system, the longitudinal distribution of magnesium, potassium and calcium elements in the leaf sample tissue is obtained after full-spectral intelligent surface scanning of the palisade tissue and the sponge tissue in the section of the blade sample section. .

The step 7 is specifically: determining the position of the probe from the opposite side of the blade into the blade according to the geometrical size and distribution density of the front and back surface fluff, vascular bundle, and pore of the blade sample; according to the sample of the magnesium, potassium and calcium in the leaf sample The longitudinal distribution position in the tissue determines the corresponding probe penetration depth when measuring the Mg2+, K+, and Ca2+ ion concentrations.

The invention has the following advantages: the invention observes and analyzes the structural features of the blade from a microscopic scale, and The analysis of structural parameters determines the optimal puncture position of the probe on the surface of the blade. The distribution characteristics of magnesium, potassium and calcium in the leaf are obtained by analyzing the energy spectrum of the blade section. The distribution characteristics of magnesium, potassium and calcium in the leaf are obtained. The analysis determines the optimal penetration depth of the probe, which improves the accuracy of the measurement.

DRAWINGS

Figure 1-a Blade front microstructure

Figure 1-b The reverse microstructure of the blade

Figure 2-a is the probe structure

Figure 2-b Blade surface microstructure

Figure 3-a Fence tissue energy spectrum scanning area

Figure 3-b The energy spectrum corresponding to each element in the scanning area of the fence

Figure 4-a Fence tissue energy spectrum scanning area

Figure 4-b shows the element distribution in the sponge tissue of the blade

detailed description

The invention will be further described in detail below with reference to the accompanying drawings.

The scanning electron microscope (SEM) used in the specific embodiment of the present invention is a quanta 200 type manufactured by American fei company, and the energy spectrum analysis system (EDS) is an Inca X-Act type electric refrigeration spectrometer manufactured by Oxford, England. The scanning electron microscopy system and the energy spectrum analysis system were used to collect and analyze the distribution of the micro-structure and internal elements of greenhouse tomato leaves, and based on this. 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 March 2015 to August 2015, and the tomato variety was selected from the powder. The invention adopts the soilless cultivation method for sample cultivation, and uses the Yamazaki culture nutrient solution for watering. In order to ensure the representativeness of the samples, the inverted seven leaves of each plant were sampled and the measurements were completed at the Microscopy Center of Nanjing Forestry University.

(1) The sample is fixed in vivo

In order to ensure the surface microstructure of the leaves when they were observed after sampling, the greenhouse was sampled and immediately placed in a 4% glutaraldehyde fixative prepared in advance.

(2) Preparation and observation of scanning electron microscope samples

After the sample was dehydrated by ethanol step, it was placed in a K850 critical point dryer manufactured by Quorum, UK, and dried. The prepared sample was adhered to a conductive paste and placed in MSP-produced by Japan SHINKKU VD Co., Ltd. The coating was carried out in a 1S type ion sputtering apparatus, and the thickness of the sputtered metal was 20 nm, which was then attached to a scanning electron microscope sample stage, and observed under an acceleration voltage of 15 kV to take a picture.

(3) Observation of the surface structure of the blade

The microstructure of the leaf surface was observed under a scanning electron microscope, and the typical field of view was taken (Fig. 1). At least 10 fields of view were observed for each sample and averaged.

Normal leaves have a soft epidermis with no obvious waxy layer. It is observed that the surface of the blade has pyramidal and mushroom-shaped micro-structures such as fluff, stomata and vascular bundle. The distribution density of these structures is quite different in the front and back of the blade; especially the porosity and villus density, the stomata and fluff density on the front of the blade. 79.52±2.51 num/mm ^-2 and 34.24±1.24 num/mm ^-2 , respectively, the porosity and fluff density on the reverse side of the blade were 235.35±5.41 num/mm ^-2 and 28.64±1.23 num/mm ^{-2 , respectively} ; The outer blade surface is also distributed with vascular bundles, which can be divided into three types according to geometrical dimensions: main vein, branch vein and micro vein. The size of the stomata is small relative to the villi and veins, so we are focusing more on vascular bundles and fluff in our analysis.

(4) Determine the optimal puncture position of the probe

The probes of the measuring elements are generally pierced into the interior of the plant blade, and the nutrient level is diagnosed by measuring the ion concentration inside the blade. Therefore, choosing a reasonable puncture position directly affects the measurement results. The observation results show that the distribution density of the villus on the reverse side of the blade is small, and the distribution of the vascular bundle on the opposite side of the blade is easy to distinguish. Because the measuring electrode is generally made of glass, the tip geometry is slender and easy to break. Therefore, it should be avoided to penetrate the hard tissue when measuring. Whether it is vascular bundle or fluff, its tissue is mainly cellulose, and its hardness is much greater than that of leaf epidermal cells. The probe should be inserted from the back of the blade to avoid the vascular bundle when inserted.

(5) Observing the blade section structure

Leaf sections were observed under a scanning electron microscope, and the epidermis, palisade tissue, sponge tissue, and whole leaf thickness, as well as cell size of each tissue, were measured. Take a typical view to take a picture (Figure 3a, Figure 3b). At least 10 fields of view were counted by electron microscopy of each sample and averaged.

(6) Microscopic observation of the blade section: at normal level, the tomato leaf thickness is 123.23±2.5um, of which

The thickness of the sponge tissue was 69.15±6.5um, and the thickness of the palisade tissue was 49.87±1.3um.

Obtain the distribution of each element in the blade section

Scanning electron microscopy and energy spectrum analysis showed that the main elements in the palisade tissue and sponge tissue of tomato leaves were C, O, Na, Mg, P, S, K, Ca, Cu, Fe, Cl (Table 1); There are certain similarities in the energy spectrum distribution of sponge tissue and palisade tissue. The content of C and O accounts for more than 90% of the total of all elements; the content of Na, Mg, K, S, Fe and Cu in palisade tissue is higher than that of sponge tissue. ; P, Ca, Cl content in sponge tissue is more than palisade tissue; N and H are not detected in the energy spectrum of palisade tissue and sponge tissue because they are very light.

Table 1 Percentage of weight of each element in the leaf tissue

(7) Determine the optimal penetration depth of the probe

The ion probe is used to measure the K ⁺ , Mg ²⁺ , Ca ²⁺ plasma of the plant leaf scale. Because the content of each element in the sponge tissue and the palisade tissue is different, in order to ensure the reliability of the measurement, the content of the measured element is selected. Organization as a measurement organization. Therefore, the penetration depth of K ⁺ and Mg ²⁺ should be between 9 and 52 um, and the penetration depth of Ca ²⁺ should be between 52 and 118 um.

Claims

A method for determining a probe puncture position when measuring an ion concentration inside a blade, comprising the steps of:

Step 1: Avoid the leaves of the main vein and take the blade samples. The sample size of the leaves is 2cm*2cm, and the sample of the leaves is fixed with the fixing solution;

Step 2: Prepare a sample of the scanning electron microscope, set the instrument parameters and observe;

Step 3: Observe and analyze the surface structure of the leaf sample;

Step 4: Determine the optimal puncture position of the probe;

Step 5: Observe the cross-sectional structure of the blade sample;

Step 6: Obtain the longitudinal distribution of magnesium, potassium and calcium elements in the leaf sample tissue;

Step 7: Determine the optimal puncture location and puncture depth.
A method for determining a probe puncture position when measuring an ion concentration inside a blade according to claim 1, wherein in the step 1, the fixing liquid is a glutaraldehyde fixing solution having a concentration of 4% to fix the taken leaf sample.
The method for determining a probe puncture position when measuring the ion concentration inside the blade according to claim 1, wherein the step 2 is specifically: the leaf sample obtained in the step 1 is dehydrated by the ethanol step, and then placed in the British Quorum company. The K850 type critical point dryer was dried, and the prepared blade sample was adhered to a conductive paste and placed in an MSP-1S type ion sputtering apparatus manufactured by Japan SHINKKU VD Co., Ltd. for sputtering. It was 20 nm, and then the processed blade sample was attached to a sample stage of a quanta 200 type scanning electron microscope manufactured by American fei company, and observed under an acceleration voltage of 15 kV, and photographed.
A method for determining a probe puncture position when measuring an ion concentration inside a blade according to claim 1, wherein the step 3 is specifically: selecting a field of view of the leaf sample by 10 fields in different regions under a scanning electron microscope system. The structure, including the distribution density and geometrical size of the villi, vascular bundles, stomata, and epidermal cells, was measured and statistically analyzed to obtain the distribution density and geometric size of the villi, vascular bundles, stomata, and epidermal cells on the surface of the leaf samples.
A method for determining a probe puncture position when measuring an ion concentration inside a blade according to claim 1, wherein the step 4 is specifically: according to the surface microstructure of the blade sample observed in the third step, according to the villus density and the vascular bundle The distribution characteristics, combined with probe geometry and material parameter characteristics, select the position of the back of the blade to avoid the vascular bundle as the probe puncture position.
The method for determining the position of the probe puncture when measuring the ion concentration inside the blade according to claim 1, wherein the step 5 is specifically: observing the section layer of the blade sample under the scanning electron microscope, measuring the epidermis, the palisade tissue, Sponge tissue and the thickness of the whole blade; the typical field of view was taken for photographing; at least 10 fields of view were observed by electron microscopy of each leaf sample, and the average value was taken.
The method for determining the position of the probe puncture when measuring the ion concentration inside the blade according to claim 1, wherein the step 6 is specifically: using the energy spectrum measuring system, respectively, in the blade sample section layer, respectively, the fence structure and the sponge The longitudinal distribution of magnesium, potassium and calcium elements in the leaf sample tissue was obtained after the whole spectrum intelligent surface scanning.
A method for determining a probe puncture position when measuring an ion concentration inside a blade according to claim 1, wherein the step 7 is specifically: according to the geometrical size and distribution density of the front and back surface fluff, the vascular bundle, and the pore of the blade sample. Determine the position of the probe from the opposite side of the blade into the blade; determine the corresponding probe penetration when measuring the concentration of Mg 2+ , K + , and Ca 2+ ions according to the longitudinal distribution of magnesium, potassium and calcium in the leaf sample tissue depth.