CN115326773A - Dark field-hyperspectral imaging-based plant root system soil nano colloid interaction visualization method - Google Patents
Dark field-hyperspectral imaging-based plant root system soil nano colloid interaction visualization method Download PDFInfo
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Abstract
The invention provides a method for observing interaction between a plant root system and soil nano-colloids by using a dark field-hyperspectral imaging technology. The method specifically comprises the following steps: the roots of plants cultured in nutrient solution (containing colloid) are washed by deionized water and 3-10 mol of potassium chloride solution in sequence and then placed on a glass slide. And respectively collecting hyperspectral data of the root system and the colloid in a range of 400-1000nm by using a dark field-hyperspectral nano fluorescence imaging system, and creating a standard reference hyperspectral library. The positions of the root system and the colloid are observed through a bright field of an inverted fluorescence microscope, then the dark field is switched to, hyperspectral information of a sample to be detected is collected by a hyperspectral nano fluorescence analysis system and stored in a spectrum library. And finally, matching a high-energy spectrum image of the root system cultured by the nutrient solution (containing the colloid) with a standard hyperspectral library by using a spectral angle mapper classification algorithm, coloring the same spectral distribution area, and superposing the colored same spectral distribution area on the original data to observe the position of a target substance in each sample, thereby visually observing the interaction of the root system and the colloid.
Description
Technical Field
The invention relates to an environmental micro-interface chemical dark field-hyperspectral imaging research, in particular to a method for visualizing the interaction of plant root system soil nano colloids.
Background
With the popularization of nano products, nano materials inevitably enter the soil environment to form nano colloids in the processes of production, use, waste treatment and the like. It is in a dispersed state in the environment and is a heterogeneous system with dimensions in the nanometer scale (1-100 nm). In general, the nanocolloids in soil are complex in composition and diverse in morphology, but mainly consist of low molecular weight biological decay decomposition products (such as humus), various fibrous and network organic compounds, and nanominerals formed by chemical weathering of soil. Wherein the nanominerals typically comprise aluminosilicate minerals (clays); oxides and hydroxides of aluminum, iron, manganese.
The surface of the nano colloid has rich functional groups and has the characteristic of large adsorption capacity. It can be enriched with heavy metals in soil systems and can migrate to various tissues of plants through plant root systems. Thus, long-term accumulation of soil nanocolloids can disturb the soil-plant system, leading to crop losses and creating food safety issues. The research on the interaction between the plant root system and the soil nano colloid is especially important. Due to the complexity of soil media, the influence of soil nanocolloids on plant roots is not clear, most researches focus on the separation, enrichment and characterization of the nanocolloids, and the researches on interaction mechanisms and toxicity of the nanocolloids and the plant roots are less.
At present, the method for researching the interaction between a plant root system and soil nano-colloids is mainly based on a chemical detection method to determine the concentration and magnetic signals of the nano-colloids in a plant body; the absorption, migration and conversion process of the soil nano colloid in the plant root system is proved by using a tracer atom method and a transmission electron microscope. The methods need to rely on corresponding detection instruments, consume a large amount of reagents, have high detection cost, and need complex sample preparation processes and detection steps, so that the problems of time consumption and labor consumption exist.
The hyperspectral imaging technology is the fusion of the traditional spectrum technology and the image technology, and has the characteristic of integrating maps by simultaneously acquiring hyperspectral information and image corresponding information of a sample. The hyperspectral characteristic change can reflect the change of plant physiological information, the image characteristic change can reflect the apparent characteristics of crop color, shape, texture and the like, and the method has a good application prospect in judging the rapid detection and visualization of physiological and biochemical indexes of crops under the stress of soil nano colloids. Therefore, the hyperspectral imaging technology has strong spatial analysis capability, and the internal and external information of the sample can be simultaneously expressed.
The dark field-hyperspectral imaging technology is combined with a dark field imaging mode to determine the positions and components of nanoparticles in biological tissues. Under conditions of visible and near infrared wavelengths (400-1000 nm) a dozen different nanoparticles can be characterized, the spectral data can be generated at high spectral resolution of about 2nm, and the pixel size can be as small as 100nm providing the ability to identify nanoscale materials in complex environments (e.g., plant tissue) in a semi-quantitative manner. Each pixel of the hyperspectral image contains the spectral response of the spatial region of the pixel. Using integrated hyperspectral image analysis software, the unique spectral response of the nanomaterial can be identified and mapped across the entire sample. Therefore, dark field-hyperspectral imaging analysis is an effective tool for detecting and characterizing nano materials in an environmental system, so that the interaction of soil nano colloid and a plant root system can be researched. The technology does not need fluorescent labeling and a special sample preparation process, and is an in-situ visualization method. However, a visual research method for the interaction of the plant root system soil nano colloid by using a dark field-hyperspectral imaging technology does not exist at present.
Disclosure of Invention
In order to overcome the defects and shortcomings in the prior art, the invention aims to provide a method for visualizing the interaction of plant root system soil nano colloid based on dark field-hyperspectral imaging.
The purpose of the invention is realized by the following technical scheme:
plant root system soil nano colloid interaction visualization based on dark field-hyperspectral imaging
The method comprises the following steps:
(1) Germinating plant seeds for 3-5 days, then transferring the plant seeds into 1/4 intensity Hoagland nutrient solution for pre-culture for 7 days, and performing in a constant-temperature illumination incubator;
(2) Transferring the model plant seedlings into a 1/4 strength Hoagland nutrient solution containing soil nano colloid after the pre-culture is finished;
(3) After the culture is finished, washing the roots of the model plants by deionized water and 3-10 mol of potassium chloride solution in sequence to remove impurities;
(4) Then, placing the plant root system sample on a glass slide, and keeping the surface smooth;
(5) A dark field-hyperspectral nano fluorescence imaging system respectively collects hyperspectral signals of a blank root system sample and a standard soil nano colloidal solution, and a standard reference hyperspectral library is created by collecting spectrum results of the blank plant root system and the soil nano colloidal solution;
(6) And (3) searching the positions of the plant root system and the soil nano colloid by utilizing the bright field of the inverted fluorescence microscope, then converting the positions to the dark field, and acquiring hyperspectral information of the sample to be detected by utilizing a hyperspectral nano fluorescence analysis system. Storing the hyperspectral information into a spectrum library through system self-contained software for subsequent spectrum mapping of hyperspectral images of other samples;
(7) And then matching the high-energy spectrum image of the plant root system cultured by the soil nano colloid nutrient solution with a spectrum library according to a Spectrum Angle Mapper (SAM) classification algorithm in the hyperspectral nano fluorescence analysis system software, coloring the region with the same spectrum distribution, and superposing the region on the original data to observe the position of the target substance in each sample.
Further limited, the extraction method of the soil colloid comprises the following steps:
(1) Air-drying the soil to be extracted, grinding and sieving, and uniformly mixing the sieved soil;
(2) Adding ultrapure water into the uniformly mixed soil and stirring by using a glass rod to obtain a soil suspension;
(3) Carrying out ultrasonic dispersion treatment on the soil suspension, and transferring the upper-layer liquid into a centrifugal tube;
(4) Centrifuging the components transferred into the centrifugal tube to obtain a soil nano colloidal suspension;
(5) Concentrating the soil nano colloid suspension by using a tangential flow ultrafiltration method;
(6) Dialyzing the concentrated solution by using ultrapure water to remove redundant ions and small molecular substances in the concentrated solution;
(7) Placing the dialyzed soil nano colloid suspension into a centrifugal concentrator for centrifugal concentration to obtain soil nano colloid;
further defined, the (pre) culture conditions are: the illumination intensity is 560 mu mol/m 2/ s, performing light-dark circulation culture for 14h;
further limiting, the concentration of the soil nano colloid is 1mg/L;
the invention has the technical effects that:
the invention collects the hyperspectral data of the plant root system and the soil nano colloid in the range of 400-1000nm by utilizing a dark field-hyperspectral nano fluorescence imaging system. The spectral results are then used to create a standard reference hyperspectral library. The positions of the root system and the soil nano colloid are observed through a bright field of an inverted fluorescence microscope, then the positions are converted to a dark field, a hyperspectral nano fluorescence analysis system is used for collecting hyperspectral information of a sample to be detected, the hyperspectral information is stored in a spectrum library through analysis software carried by the system, and the hyperspectral information is used for subsequent spectrum mapping of a hyperspectral image of the sample. And then matching the high-energy spectrum image of the plant root system cultured by the nutrient solution containing the soil nano colloid with a spectrum library by utilizing a spectrum angle mapper classification algorithm (SAM) in the hyperspectral nano fluorescence analysis system software, coloring the region with the same spectrum distribution, and superposing the region on the original data to observe the position of the target substance in each sample, thereby performing two-dimensional visual observation on the interaction of the plant root system and the soil nano colloid, realizing the resolution ratio from micron level to nanometer level, and having the characteristics of high selectivity and high sensitivity.
Other techniques such as: electron microscope and Raman integrated electronMicroscopy (RISE) can also provide both morphological and chemical information, but these techniques have high detection limits (10) 10 -10 17 particles/L). In addition, the thermal cracking gas chromatography-mass spectrometry (Py-GC-MS) requires the destruction of the sample to be tested to obtain the chemical information of the sample. The dark field-hyperspectral imaging technology is a non-destructive method, can identify nanoparticles with the particle size of more than 10nm, does not need fluorescent labeling, is used in a special sample preparation process, and can be used for other characterization technologies. Meanwhile, the technology is a method for quickly positioning particles, is much faster than other drawing technologies such as Raman and the like, can image a region of 90 mu m multiplied by 90 mu m within a few minutes, and has low detection limit (10) 8 -10 9 particles/L). This is to be distinguished from existing characterization techniques. Dark field-hyperspectral imaging is also a pixel-level hyperspectral scanning technology, and the spectral resolution is 1.5nm. The spectral imaging system can perform Mapping analysis on nanomaterials in biological samples or other samples.
Drawings
FIG. 1 is a standard hyperspectral image of rice root and soil nano-colloids.
FIG. 2 is a dark field-hyperspectral nanometer fluorescence imaging image of a plant root after the action of a soil nanometer colloid A.
FIG. 3 is a dark field-hyperspectral nanometer fluorescence imaging image of a plant root after the action of a soil nanometer colloid B.
FIG. 4 is a dark field-hyperspectral nanometer fluorescence imaging image of a plant root after the action of a soil nanometer colloid C.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be purely exemplary and are not intended to limit the scope of the invention, which is defined in the appended claims, as may be amended by those skilled in the art upon reading the present invention.
Example 1:
the method comprises the following steps: and (4) extracting soil nano colloid.
Air drying soil sample A (yellow loam, paddy field), grinding, sieving (10 mesh), and mixing. 1L of ultrapure water was added to the uniformly mixed soil (50 g) and stirred with a glass rod for 5 to 10min to prepare a suspension, which was then subjected to ultrasonic dispersion treatment for 1 hour. And after the ultrasonic treatment is finished, centrifuging the upper layer liquid for three times (6030 rpm/min,4 ℃ and 20 min) to obtain the soil nano colloid suspension. Concentrating the soil nano colloid suspension (600 mL) by using a tangential flow ultrafiltration method, and finally concentrating to 30mL, wherein the molecular weight cutoff of a membrane is 5kDa, the material is polyether sulfone resin, and the rotating speed of a peristaltic pump is set to be 120rpm/min. And dialyzing the concentrated solution by using ultrapure water to remove redundant ions and small molecular substances in the concentrated solution, wherein the molecular weight cut-off of a dialysis bag is 5kDa, placing the dialysis bag in a 1L beaker containing the ultrapure water, stirring for 12h on a magnetic stirrer at the stirring speed of 150rpm/min, and replacing the ultrapure water every 4h. And (4) placing the dialyzed soil nano colloid suspension into a centrifugal concentrator for centrifugal concentration (-20 ℃,2000rpm/min,24 h.) to obtain the soil nano colloid A.
Step two: and (4) germinating and culturing the plant.
The plant seeds were washed, soaked with deionized water at 28 ℃ for 24h, sterilized with 1.25% NaClO for 10min, and then rinsed with deionized water. Selecting healthy seeds, germinating on wet gauze for 3-5 days, and pre-culturing in Hoagland solution with 1/4 strength. To simulate a dark soil environment, all containers were wrapped in black plastic bags and grown in a greenhouse with a light intensity of 5000lx and a light-to-dark ratio of 14h. The preculture was carried out at 28 ℃ in the daytime, 25 ℃ in the dark and 70% humidity. Selecting seedlings with the same growth conditions, pre-culturing for 7 days, treating the plants with 1/4 strength Hoagland nutrient solution to expose soil nano colloid A (1 mg/L), setting 6 parallel treatments in each group, and replacing the culture medium once every 2 days. The position of the culture vessel is randomly shifted to reduce the influence of the position on the results.
Step three: the interaction of the plant root system soil nano colloid can be visually analyzed.
After the culture is finished, washing the roots of the model plants by deionized water and 3-10 mol of potassium chloride in sequence to remove impurities; then putting the plant root system sample on a glass slide, and keeping the surface smooth; respectively acquiring hyperspectral signals of a blank root system sample and a standard soil nano colloidal solution by using a dark field-hyperspectral nano fluorescence imaging system, and creating a standard reference hyperspectral library by collecting the spectrum results of the blank plant root system and the soil nano colloidal solution; and matching the hyperspectral image information of the plant root system cultured by the soil nano colloid nutrient solution with a hyperspectral library of a standard sample according to a Spectrum Angle Mapper (SAM) classification algorithm in hyperspectral nano fluorescence analysis system software, coloring the regions with the same hyperspectral distribution, and superposing the regions on the original data to observe the position of the target substance in each sample.
In the invention, a Spectrum Angle Mapper (SAM) classification algorithm in hyperspectral nano-fluorescence analysis system software is an automatic spectrum classification method based on mathematics, and an n-D angle matching technology is used. The spectrum of each pixel in the image is regarded as a high-dimensional vector, the similarity between the spectra is measured by calculating the included angle between the two vectors, the smaller the included angle is, the more similar the two spectra are, the higher the possibility of belonging to the same ground object is, and therefore the category of unknown data can be distinguished according to the size of the spectrum angle. And when classifying, calculating the spectrum angle between the unknown data and the known data, and classifying the class of the unknown data into the class corresponding to the minimum spectrum angle. Calculating the distance between two vectors (X) according to the cosine between two vectors * Unknown vector, X i Known vector), i.e.:
and calculating the included angle between the unknown vector and all known vectors, and considering the included angle as the same category with the vector with the minimum included angle.
Example 2:
the method comprises the following steps: and (4) extracting soil nano colloid.
The procedure was substantially the same as in the first step of example 1, except that soil sample B (black soil, paddy field) was subjected to soil nanocolloid extraction.
Step two: and (4) germinating and culturing the plant.
The process is basically the same as the second step in example 1, except that soil nano-colloid B is added during the cultivation process.
Step three: and (4) performing visual analysis on the interaction of the plant root system soil nano colloid.
Same as step three in example 1.
Example 3:
the method comprises the following steps: and (4) extracting soil nano colloid.
The procedure was essentially the same as in example 1, except that soil sample C (medium loam, rice field) was subjected to soil nanocolloid extraction.
Step two: and (4) germinating and culturing the plant.
The process is substantially the same as the second step in example 1, except that soil nanocolloid C is added during the cultivation.
Step three: the interaction of the plant root system soil nano colloid can be visually analyzed.
Same as step three in example 1.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
FIG. 1 shows the standard hyperspectral images of rice roots and soil nano-colloids. Fig. 2-4 are dark field-hyperspectral nano fluorescence imaging images of plant roots after being acted by the soil nano colloid a, the soil nano colloid B and the soil nano colloid C of examples 1-3, respectively. The drawings illustrate:
FIG. 1 (a) is a hyperspectral image of a plant root sample (blank sample) cultured in a nutrient solution, and the image only contains the root sample. FIGS. 1 (b) - (d) are hyperspectral images of soil nanocolloid A, B, C, respectively, which illustrate that the soil nanocolloid is in the form of dispersed particles with a particle size of about 55-139nm. Fig. 1 (e) is hyperspectral data for blank and soil nanocolloid A, B, C. Under a dark field microscope, characteristic absorption peaks of blank sample soil nano colloid A, B, C are 567nm, 618nm, 614nm and 611nm respectively, and are obtained by self-contained software of a scattering spectrum. The hyperspectral spectrogram data shows that the characteristic wavelengths of the root system and the soil nano colloid sample are obviously different, so that the root system and the soil nano colloid sample can be accurately distinguished.
Fig. 2 shows that after the interaction between the root system and the soil nano-colloid a, the colloid particles a are mainly gathered in the root tip mucus of the root system and do not enter the plant body.
FIG. 3 shows that after interaction between the root system and the soil nano-colloid B, the colloid B is mainly gathered at the root hair position of the root system by the absorption of the root system.
Fig. 4 shows that after interaction between the root system and the soil nano-colloid B, the colloid particles C are mainly concentrated at the root tip part by absorption of the root system.
Claims (8)
1. A dark field-hyperspectral imaging-based plant root system soil nano colloid interaction visualization method is characterized by comprising the following steps:
(1) The plant roots cultured by the nutrient solution containing the soil nano colloid are sequentially washed by deionized water and a potassium chloride solution to remove impurities, and then the plant root system sample is placed on a glass slide to keep the surface smooth;
(2) Collecting hyperspectral data of the plant root system by using a dark field-hyperspectral imaging system, creating a reference hyperspectral library by collecting the spectrum results of a blank plant root system and a soil nano colloid solution, and then determining the characteristic hyperspectral information of the plant root system and the soil nano colloid;
(3) And matching standard hyperspectral characteristic information of the plant root system cultured by the soil nano colloid nutrient solution with hyperspectral information of the test sample image by using an SAM classification algorithm to observe the position of the soil nano colloid in each root system sample and realize two-dimensional visual observation on the interaction of the plant root system and the soil nano colloid.
2. The dark field-hyperspectral imaging-based plant root system soil nanocolloid interaction visualization method according to claim 1, wherein the dark field-hyperspectral imaging detection conditions are as follows:
using 75% light source intensity and 0.25s exposure time; each pixel of the dark-field hyperspectral image contains its light reflection spectrum in the range of 400-1000 nm.
3. The dark field-hyperspectral imaging-based visualization method for the soil nanocolloid interaction of the plant root system according to claim 1, wherein the model plant in the step (1) is obtained by germinating plant seeds for 3-5 days, pre-culturing the plant seeds for 7 days by using 1/4 strength Hoagland nutrient solution, and culturing the plant seeds for 7 days after adding soil nanocolloid.
4. The dark field-hyperspectral imaging-based plant root system soil nanocolloid interaction visualization method according to claim 1, wherein the step (2) of determining the characteristic hyperspectral information of the plant root system and the soil nanocolloid is to use an inverted fluorescence microscope bright field to find the positions of the plant root system and the soil nanocolloid, then to convert the positions into a dark field, use a hyperspectral nanometer fluorescence analysis system to acquire the hyperspectral information of a sample to be tested, and store the hyperspectral information into a spectral library through software carried by the system for subsequent spectral mapping of hyperspectral images of other samples.
5. The dark field-hyperspectral imaging-based visualization method for the interaction of the soil nanocolloids of the plant root system according to claim 1, wherein the soil nanocolloids are obtained by the following method:
(1) Air-drying the soil to be extracted, grinding and sieving, and uniformly mixing the sieved soil;
(2) Adding ultrapure water into the uniformly mixed soil, and stirring to obtain a soil suspension;
(3) Carrying out ultrasonic dispersion treatment on the soil suspension, and taking the upper layer liquid;
(4) Centrifuging the upper layer liquid component to obtain a soil nano colloid suspension;
(5) Concentrating the soil nano colloid suspension by using a tangential flow ultrafiltration method;
(6) Dialyzing the concentrated solution by using ultrapure water to remove redundant ions and small molecular substances in the concentrated solution;
(7) And (4) carrying out centrifugal concentration on the dialyzed soil nano colloid suspension to obtain the soil nano colloid.
6. The dark field-hyperspectral imaging-based method for visualizing the soil nanocolloid interaction of a plant root system according to claim 1, wherein the culture is nutrient solution culture.
7. The dark field-hyperspectral imaging-based visualization method for the soil nano-colloid interaction of the plant root system according to claim 1, wherein the culture solution contains a soil nano-colloid concentration of 1mg/L.
8. The dark field-hyperspectral imaging-based plant root system soil nanocolloid interaction visualization method according to claim 3, wherein the pre-culture is performed in a constant temperature illumination incubator. Wherein the illumination intensity is 560 mu mol/m 2 And/s, culturing in a dark-dark cycle culture medium with the humidity of 80% in a climatic incubator at the temperature of 28.0 +/-0.5 ℃ for 14h.
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