A method for subcellular self-indication tracing of metal elements by ion imaging positioning

Abstract

The application provides a method for subcellular self-tracing ion imaging positioning of metal elements. The method comprises the following steps: subcellular structure development and metal ion imaging are performed on a sample slice by using a SEM-FIB-TOF-SIMS system, wherein, in the SEM-FIB-TOF-SIMS system, the subcellular structure development is performed on the sample slice by using SEM; the surface of the subcellular structure is bombarded by FIB, the secondary ions excited by the bombardment are detected by TOF-SIMS, and the ion information of an analysis region is obtained and imaged. The method can solve the problem that the subcellular positioning method of metal elements in the prior art cannot meet the actual application requirements, and is suitable for the technical field of ion imaging.

Description

Technical Field

[0001] This invention relates to the field of ion imaging technology, and more specifically, to a method for subcellular self-tracing ion imaging and localization of metal elements. Background Technology

[0002] Currently, research on the subcellular localization of metal elements is mainly divided into in vivo in situ visualization studies and in vitro quantitative studies after extracting different cell components. However, in vivo in situ visualization studies have the following problems: (1) For most elements, such as transition elements, there are no commercially available probes for indirect tracing using probe labeling; (2) Radioactive isotope self-tracing imaging methods are complex to operate and harmful to the human body; (3) Non-radioactive isotope self-tracing imaging methods are difficult to accurately determine subcellular localization due to poor repeatability and unclear images. Summary of the Invention

[0003] The main objective of this invention is to provide a method for subcellular self-tracing ion imaging localization of metal elements, so as to solve the problem that the existing subcellular localization methods for metal elements are difficult to meet the needs of practical applications.

[0004] To achieve the above objectives, according to a first aspect of the present invention, a method for subcellular self-tracing ion imaging localization of metal elements is provided. The method includes: using a SEM-FIB-TOF-SIMS system to perform subcellular structure development and metal ion imaging on a sample slice, wherein, in the SEM-FIB-TOF-SIMS system, SEM is used to develop the subcellular structure of the sample slice; FIB is used to bombard the surface of the subcellular structure, and the excited secondary ions are detected by TOF-SIMS to obtain ion information of the analysis area and image it.

[0005] Furthermore, the sample sections include plant sections, animal tissue sections, bacterial samples, or fungal samples, and correspondingly, the subcellular structures include plant subcellular structures, animal subcellular structures, bacterial subcellular structures, or fungal subcellular structures.

[0006] Furthermore, metal ions include transition metal ions or non-transition metal ions.

[0007] Furthermore, transition metal ions include rare earth element ions.

[0008] Furthermore, the method for preparing plant sections includes: cutting plant tissue into small pieces and fixing them to obtain fixed samples; and sequentially performing dehydration treatment, infiltration embedding treatment, and sectioning treatment on the fixed samples to obtain plant sections.

[0009] Furthermore, the fixation process includes pre-fixation and post-fixation. Pre-fixation includes using paraformaldehyde and glutaraldehyde to pre-fix the small pieces, obtaining pre-fixed samples. Post-fixation includes using osmium tetroxide solution to post-fix the pre-fixed samples, obtaining fixed samples. Dehydration includes using ethanol solutions with gradually increasing concentrations to dehydrate the fixed samples, obtaining dehydrated samples. Infiltration embedding includes using propylene oxide and embedding agents to infiltrate and embed the dehydrated samples, obtaining infiltration embedded samples. Sectioning includes slicing the infiltration embedded samples to obtain plant sections.

[0010] Furthermore, the size of the small pieces is 1-2mm. 3 The pre-fixation treatment includes: placing the small piece in a solution containing 1-4 wt% paraformaldehyde and 0.5-5 wt% glutaraldehyde, fixing at 15-25°C in the dark for 2-3 hours or at 4°C in the dark overnight to obtain the pre-fixed sample; the post-fixation treatment includes: placing the pre-fixed sample in a 1-2 wt% osmium tetroxide solution, fixing at 15-25°C in the dark for 4 hours to obtain the fixed sample; the dehydration treatment includes: sequentially applying 30% (v / v), 50% (v / v), 70% (v / v), 90% (v / v), 100% (v / v), and 100% (v / v) ethyl acetate. The fixed samples were dehydrated using alcohol solutions. Treatment time was 15 min for 30% (v / v)-90% (v / v) ethanol solutions and 20 min for 100% (v / v) ethanol solutions to obtain dehydrated samples. The infiltration and embedding treatment included: for the dehydrated samples, sequential treatment with propylene oxide at 15-25℃ for 1 h, treatment with a gradient mixture of propylene oxide and embedding agent at 15-25℃ for 4 h each, treatment with the embedding agent at 15-25℃ for 8 h, followed by polymerization at 60℃ for 48 h to obtain infiltration and embedding samples. The thickness of the plant slices was 80-150 nm.

[0011] Furthermore, before preparing sample sections, the plants are treated with metal ions, including: spraying a solution containing metal ions onto the plant leaves until droplets begin to fall; the concentration of metal ions in the solution is 1 nmol / L-1 mmol / L.

[0012] Furthermore, when using SEM to visualize subcellular structures, the electron beam current is 21pA-1.4nA, the accelerating voltage is 3-10kV, the scan rate is 1-100μs, and the magnification is 2500-5000×.

[0013] Furthermore, when using FIB-TOF-SIMS to bombard the surface of subcellular structures, detect secondary ion signals, and perform ion imaging, the ion beam current was 24-80 pA, the accelerating voltage was 30 kV, and a total of 50-200 images were collected.

[0014] Furthermore, after performing subcellular structure imaging and ion imaging, the image region and the image region data table are output, and a mass spectrometry image that can characterize the relative abundance of metal ions in the image region is drawn based on the data table.

[0015] By applying the technical solution of this invention, a SEM-FIB-TOF-SIMS system is used to achieve self-tracing ion imaging and localization of subcellular structures. First, SEM is used to develop subcellular structures in sample slices; then, FIB is used to bombard the surface of the subcellular structures, and the excited secondary ions are detected by TOF-SIMS, thereby obtaining ion information of the analysis area and imaging it. This achieves ion imaging and localization at the subcellular structural level, with clear images, accurate localization, and simple operation, without the need for radioactive isotopes. Attached Figure Description

[0016] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0017] Figure 1 The image shows the subcellular self-tracing ion imaging results of rare earth elements according to Embodiment 1 of the present invention, wherein PM represents the plasma membrane, V represents the vacuole, CP represents the chloroplast, and Ce represents the cerium ion.

[0018] Figure 2 The mass spectrum of the relative abundance of rare earth elements in chloroplasts in Example 1 of the present invention is shown.

[0019] Figure 3 The diagram shows the results of rare earth radioisotope self-tracing development according to Embodiment 1 of the present invention, wherein PM represents the plasma membrane, V represents the vacuole, T represents the vacuolar membrane, ChM represents the chloroplast membrane, and small arrows without abbreviations represent rare earth elements.

[0020] Figure 4 The diagram shows the results of rare earth radioisotope self-tracing development according to Embodiment 1 of the present invention, where T represents vacuolar membrane, ChM represents chloroplast membrane, Chl represents chloroplast, and small arrows without abbreviations represent rare earth elements.

[0021] Figure 5 The image shows the results of rare earth non-radioactive isotope self-tracing imaging according to Example 1 of the present invention, where CW represents the cell wall and PM represents the plasma membrane.

[0022] Figure 6 The image shows the subcellular self-tracing ion imaging results of transition metal elements (iron, Fe) according to Embodiment 1 of the present invention.

[0023] Figure 7The image shows the subcellular self-tracing ion imaging results of transition metal elements (zinc, Zn) according to Embodiment 1 of the present invention.

[0024] Figure 8 The image shows the subcellular self-tracing ion imaging results of a non-transition metal element (calcium, Ca) according to Embodiment 1 of the present invention.

[0025] Figure 9 The image shows the subcellular self-tracing ion imaging results of a non-transition metal element (magnesium, Mg) according to Embodiment 1 of the present invention.

[0026] Figure 10 The image shows the subcellular self-tracing ion imaging localization results of rare earth elements in corn according to Embodiment 2 of the present invention, wherein Ce represents cerium ions.

[0027] Figure 11 The image shows the subcellular self-tracing ion imaging localization results of rare earth elements according to Embodiment 3 of the present invention, wherein Ce represents cerium ions.

[0028] Figure 12 The image shows the subcellular self-tracing ion imaging localization results of rare earth elements according to Embodiment 4 of the present invention, wherein Ce represents cerium ions.

[0029] Figure 13 The image shows the subcellular self-tracing ion imaging localization results of rare earth elements according to Embodiment 5 of the present invention, wherein Ce represents cerium ions. Detailed Implementation

[0030] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0031] Terminology Explanation:

[0032] SEM: Scanning electron microscope.

[0033] FIB: Focused-ion-beam.

[0034] TOF-SIMS: Time-of-flight-secondary ion mass spectrometry.

[0035] Subcellular: Subcellular structures are structures that are more refined than cellular structures.

[0036] Subcellular structures: These are cellular structures that are only visible under an electron microscope, such as mitochondria, centrosomes, Golgi apparatus, and pits in the cell wall.

[0037] Subcellular localization: refers to the specific location of a protein, gene expression product, or compound within a cell, such as in the nucleus, cytoplasm, cell membrane, or other specific subcellular structures.

[0038] As mentioned in the background section, existing research on subcellular visualization and localization of metal elements is hampered by limitations such as a limited variety of probes, high cost and hazard of radioactive isotope reagents, and unclear imaging, making it difficult to accurately locate metal elements at the subcellular level. Therefore, in this application, the inventors attempt to develop a novel method for subcellular self-tracing ion imaging localization of metal elements, and propose the protection scheme of this application.

[0039] In a typical embodiment of this application, a method for subcellular self-tracing ion imaging and localization of metal elements is provided. The method includes: using a SEM-FIB-TOF-SIMS system to perform subcellular structure development and metal ion imaging on sample slices; wherein, in the SEM-FIB-TOF-SIMS system, SEM is used to develop the subcellular structure of the sample slices; FIB is used to bombard the surface of the subcellular structure, and the excited secondary ions are detected by TOF-SIMS to obtain ion information of the analysis area and image it.

[0040] The SEM-FIB-TOF-SIMS system used in this application employs a Helios 5CX SEM-FIB (ThermoFisher Scientific, the United States of America) and a C-TOF TOF (Tofwerk, Switzerland) TOF-SIMS system. Connecting these devices establishes the system. The method can also be implemented using other devices with similar functions; therefore, the subcellular self-tracing ion imaging and localization method for metal elements is not limited to detection using specific device models. In this method, the subcellular structure is first determined using SEM, and then the specific subcellular structure is bombarded to achieve imaging and localization of metal ions within the subcellular structure. Using this method, one or more metal ions within a subcellular structure can be simultaneously imaged and localized.

[0041] In a preferred embodiment, the sample or sample section includes, but is not limited to, plant sections, animal tissue sections, bacterial samples, or fungal samples. Correspondingly, the subcellular structure includes plant subcellular structures, animal subcellular structures, bacterial subcellular structures, or fungal subcellular structures. Preferably, the plant can be a model plant or a non-model plant; it can be a non-crop plant or a crop plant; the sample source can be a leaf, root, or other part; specifically, it includes, but is not limited to, Arabidopsis thaliana or maize. The method of this application is applicable to all cellular structures.

[0042] In a preferred embodiment, the metal ions include transition metal ions or non-transition metal ions;

[0043] Transition metals are the 68 metallic elements whose valence electron configurations are located in the d, ds, or f blocks of the periodic table, including but not limited to rare earth elements. Non-transition metals are metallic elements that do not belong to the transition metal group, including but not limited to metals in Groups I, II, III, IV, V, and VI. Throughout the plant's life cycle, except for artificial elements, these elements are continuously absorbed and transported by plant cells to different subcellular regions, accumulating at these sites and thus affecting plant growth and development. For a long time, revealing the mechanisms of absorption, transport, and accumulation of metal elements (especially transition metals) in plant cells has been a hot topic of research for researchers in biology, chemistry, environment, and agronomy worldwide. Among these, revealing the subcellular localization of metal elements in plants is widely recognized as one of the important means to answer these key questions.

[0044] In the aforementioned method for subcellular self-tracing ion imaging and localization of metal elements, a scanning electron microscope (SEM) and a focused ion beam-time-of-flight (SEM-FIB-TOF-SIMS) system are used. First, SEM is used to visualize the subcellular structure of individual cells in a sample slice. Then, the obtained subcellular structure is bombarded with an ion beam emitted from an FIB, and the secondary ions excited after bombardment are collected using TOF-SIMS. Ion information of the bombarded region is obtained and imaged in the corresponding analysis software, thereby achieving the imaging and localization of metal ions in the subcellular structure. This method has the advantages of simple operation, speed, clear images, and good reproducibility, laying a technical foundation for studying the cellular and molecular mechanisms by which transition metal elements regulate plant growth.

[0045] The aforementioned metal ions are all compounds (including substances defined in professional English terms, such as Compound), coordination compounds (including substances defined in professional English terms, such as Coordination Compound or Coordination Complex), or complexes (including substances defined in professional English terms, such as Complex) of metal ions in stable valence states.

[0046] In a preferred embodiment, the transition metal ions include rare earth element (REE) ions. The working principle of FIB-TOF-SIMS is that a high-energy focused ion beam irradiates the sample surface, exciting secondary ions from the atomic layers of the sample surface. Then, based on the different times it takes for secondary ions of different masses to reach the detector, the corresponding ions are detected and imaged. The ion sputtering in FIB and the ion detection in TOF-SIMS are both related to the properties of the analyte ions. For example, high ion beam current and accelerating voltage are required to sputter REE secondary ions. Under these conditions, other metal ions can be detected and imaged.

[0047] Rare earth elements refer to scandium, yttrium, and 15 lanthanide elements. Specifically, they include one or more of scandium, yttrium, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.

[0048] In a preferred embodiment, the method for preparing plant sections includes: cutting plant tissue into small pieces and fixing them to obtain fixed samples; and sequentially performing dehydration, infiltration embedding, and sectioning on the fixed samples to obtain plant sections. More preferably, for leaf samples, cutting them into pieces along the direction parallel to the main vein and performing subsequent fixation can obtain plant leaf samples suitable for the above method.

[0049] In a preferred embodiment, the fixation process includes a pre-fixation process and a post-fixation process.

[0050] In a preferred embodiment, the pre-fixation treatment includes: pre-fixing the small piece with paraformaldehyde and glutaraldehyde to obtain a pre-fixed sample.

[0051] In a preferred embodiment, the post-fixation process includes: using an osmium tetroxide solution to perform post-fixation on the pre-fixed sample to obtain a fixed sample.

[0052] In a preferred embodiment, the dehydration process includes: sequentially dehydrating the fixed sample using an ethanol solution with gradually increasing concentration to obtain a dehydrated sample.

[0053] In a preferred embodiment, the permeation embedding process includes: permeating and embedding a dehydrated sample with propylene oxide and an embedding agent to obtain a permeation-embedded sample.

[0054] In a preferred embodiment, the slicing process includes: slicing the infiltration-embedded sample to obtain sample slices.

[0055] In a preferred embodiment, the size of the small piece is 1-2 mm. 3 .

[0056] In a preferred embodiment, the pre-fixation treatment includes: placing the small piece in a solution containing 1-4 wt% paraformaldehyde and 0.5-5 wt% glutaraldehyde, fixing it at 15-25°C in the dark for 2-3 hours or at 4°C in the dark overnight to obtain a pre-fixed sample.

[0057] In a preferred embodiment, the post-fixation treatment includes: placing the pre-fixed sample in a 1-2 wt% osmium tetroxide solution and fixing it at 15-25°C in the dark for 4 hours to obtain the fixed sample.

[0058] In a preferred embodiment, the dehydration treatment includes: sequentially treating the fixed sample with 30% (v / v), 50% (v / v), 70% (v / v), 90% (v / v), 100% (v / v) and 100% (v / v) ethanol solutions, wherein the treatment time with 30%-90% (v / v) ethanol solution is 15 min and the treatment time with 100% (v / v) ethanol solution is 20 min, to obtain a dehydrated sample.

[0059] In a preferred embodiment, the permeation embedding treatment includes: for a dehydrated sample, sequentially treating it with propylene oxide at 15-25°C for 1 hour, treating it with a gradient mixture of propylene oxide and embedding agent at 15-25°C for 4 hours each, treating it with the embedding agent at 15-25°C for 8 hours, and then polymerizing it at 60°C for 48 hours to obtain a permeation embedded sample.

[0060] The specific components of the embedding agent used in this application include: aliphatic epoxy resin (3,4-epoxycyclohexenemethyl, ERL-4221), curing agent (nonylsuccinic anhydride, NSA), epoxy plasticizer (polymer containing α-hydro-ω-hydroxy poly[oxy(methyl-1,2-ethylidene)](chloromethyl)ethylene oxide, DER-736), and catalyst (N,N-dimethylethanolamine, DEMA); in every 10g of embedding agent, the mass ratio of ERL-4221, NSA, and DER-736 is 1:2:1, and DEMA is 100μL. Other embedding agents in the prior art can also be used, or the components in the above embedding agent can be flexibly adjusted, as long as the embedding effect can be achieved.

[0061] In a preferred embodiment, the propylene oxide and embedding agent gradient mixture includes mixtures in which the volume ratios of propylene oxide and embedding agent are 2:1, 1:1 and 1:2, or 3:1, 1:1 and 1:3, respectively. The sample is treated using the above-mentioned mixture with a concentration gradient, and the concentration of the embedding agent in the mixture continuously increases to ensure that the embedding agent completely penetrates into the sample tissue.

[0062] In a preferred embodiment, the thickness of the sample slice is 80-150 nm.

[0063] Using the above methods, through fixation, dehydration, infiltration embedding, and sectioning, suitable section samples for electron microscopy imaging and ion beam bombardment can be obtained.

[0064] In a preferred embodiment, before preparing sample slices, the plant is treated with metal ions, including spraying a solution containing metal ions onto the plant leaves until droplets begin to fall.

[0065] The above method allows for the analysis of metal ions in all cell samples, enabling research into the mechanisms of metal ion transport and accumulation within cells. Pretreatment of cells with metal ions is not necessary. Using this treatment method, spraying a solution containing metal ions onto the leaf surface allows for the rapid entry of metal ions, especially rare earth metals, into cells, simulating the absorption and transport processes of metal ions during actual plant growth.

[0066] In a preferred embodiment, the concentration of metal ions in the solution is 1 nmol / L to 1 mmol / L.

[0067] Using the above treatment method, a solution containing specific metal ions is sprayed onto blank plant leaves to provide these metal ions to the cells of the plant leaves. This allows the plant cells to spontaneously absorb, transport, and accumulate the metal ions, resulting in the distribution of metal ions in different subcellular structures within the plant cells. This facilitates subsequent research on the localization of metals in subcellular structures and the mechanisms of metal absorption, transport, and accumulation in plant cells.

[0068] For other types of samples, by flexibly selecting existing methods for preparing electron microscope samples, it is also possible to obtain samples or sample slices for the aforementioned method of subcellular self-tracing ion imaging and localization of metal elements.

[0069] In a preferred embodiment, when using SEM to visualize subcellular structures, the electron beam current is 21pA-1.4nA, the accelerating voltage is 3-10kV, the scan rate is 1-100μs, and the magnification is 2500-5000×.

[0070] In a preferred embodiment, when using FIB-TOF-SIMS to perform surface bombardment, secondary ion signal detection, and ion imaging on subcellular structures, the ion beam current is 24-80 pA, the accelerating voltage is 30 kV, and a total of 50-200 images are acquired.

[0071] Using the aforementioned SEM parameters, the subcellular structures of plant cells can be clearly visualized, and the influence of the electron beam emitted by the SEM on cells and subcellular structures can be reduced, providing a reference for subsequent accurate ion beam bombardment and the localization of metal ions in subcellular structures. In using FIB-TOF-SIMS, the imaging image, pixels, and number of acquisitions can be flexibly selected as needed. In this application, the imaging image resolution is 512×442, with a pixel binning of 4, and a total of 50-200 images are acquired.

[0072] Using the parameters of FIB and TOF-SIMS mentioned above, it is possible to achieve precise surface bombardment of subcellular structures and capture and detect the secondary ion signals generated when bombarding metal ions, thereby accurately locating the position of metal ions on subcellular structures and realizing ion imaging localization at the subcellular level.

[0073] While existing technologies such as SEM, FIB, and TOF-SIMS are widely used for correlation ion imaging of solid materials such as soil, graphite, ceramics, and metals, those skilled in the art have not considered how to cascade these devices to obtain the localization and imaging of metal ions in biological cells, especially plant cells and subcellular structures. Furthermore, it is difficult to predict what kind of technical effect an assembled device with a particular cascade structure could achieve. That is, in the above methods, if the parameters of the SEM-FIB-TOF-SIMS system of the assembled device exceed the preferred parameter range, or if the steps used in the preparation of sample slices (especially plant slices) exceed the preferred range, the localization and imaging effect of subcellular self-tracing ions of metal elements in plants will be poor, or even impossible to achieve the localization and imaging of ions in subcellular structures.

[0074] In a preferred embodiment, after subcellular structure imaging and ion imaging, an image region and a data table of the image region are output, and a mass spectrometry image of the relative abundance of metal ions in the image region is drawn based on the data table.

[0075] Preferably, the region of interest is captured using TOF-SIMS Explorer software, and a data table of all parameters for that region is exported. Then, a relative abundance mass spectrum image of the target metal ion in the region of interest is plotted using plotting software such as Origin 2022. The peak height in the mass spectrum image can trace the relative content or abundance of the ion to be detected in the target region. The output image is then overlaid with the SEM image and the FIB-TOF-SIM ion image using plotting software such as Adobe Photoshop, resulting in an image as shown below. Figure 1 The image shown demonstrates ion imaging localization at the subcellular level.

[0076] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0077] Example 1:

[0078] Specific steps of the subcellular self-tracing ion imaging localization method for rare earth element cerium (Ce) in Arabidopsis leaves:

[0079] (1) Ce treatment of plants: spray 80 μmol·L⁻¹ evenly on the leaves of Arabidopsis thaliana. -1 Cerium chloride (CeCl3) was added until the droplets began to fall;

[0080] (2) Sampling: Arabidopsis leaves were cut into 2mm sections along the direction parallel to the main vein. 3 Small pieces;

[0081] (3) Pre-fixation: The sample was placed in a solution containing 4 wt% paraformaldehyde (Shanghai Jizhi Biochemical Technology Co., Ltd., P39200) and 5 wt% glutaraldehyde and fixed overnight at 4°C in the dark.

[0082] (4) Post-fixation: The sample was placed in a 1 wt% osmium tetroxide solution and fixed at 25°C in the dark for 4 h;

[0083] (5) Dehydration: The fixed samples were dehydrated sequentially with ethanol solutions of volume ratios of 30%, 50%, 70%, 90%, 100% and 100%, with the treatment time of 30%-90% ethanol solution being 15 min and the treatment time of 100% ethanol solution being 20 min.

[0084] (6) Infiltration and embedding: The dehydrated sample was treated sequentially with propylene oxide at 25°C for 1 h, with a gradient mixture of propylene oxide and embedding agent (v:v = 2:1, 1:1, 1:2) at 25°C for 4 h each, and with the embedding agent at 25°C for 8 h, followed by polymerization at 60°C for 48 h. The specific components of the embedding agent included: aliphatic epoxy resin (3,4-epoxycyclohexenemethyl, ERL-4221), curing agent (nonylsuccinic anhydride, NSA), epoxy plasticizer (polymer containing α-hydro-ω-hydroxy poly[oxy(methyl-1,2-ethylene)](chloromethyl)ethylene oxide, DER-736), and catalyst (N,N-dimethylethanolamine, DEMA). In every 10 g of embedding agent, the mass ratio of ERL-4221, NSA, and DER-736 was 1:2:1, and DEMA was 100 μL.

[0085] (7) Slicing: Cut the embedded sample into ultrathin slices with a thickness of 120 nm;

[0086] (8) Development of plant subcellular structures: Plant subcellular structures were developed using SEM (Helios 5CX, Thermo Fisher Scientific, the United States of America) with an electron beam current of 0.35 nA, an accelerating voltage of 5 kV, a scan rate of 15 μs, and a magnification of 3500 × 1000.

[0087] (9) Ion imaging: The sample surface was bombarded using a FIB (Helios 5CX, Thermo Fisher Scientific, the United States of America) with an ion beam current of 40 pA and an accelerating voltage of 30 kV. The excited secondary ions were detected by TOF-SIMS to obtain ion information of the analysis area and image it. The image resolution was 512×442 with 4 pixels merged, and a total of 100 images were acquired.

[0088] (10) Use the TOF-SIMS explorer software to capture the region of interest in the image and export a data table of all parameters of the region of interest in the image.

[0089] (11) Use Origin 2022 plotting software to plot the mass spectrum image of the target metal ions in the region of interest.

[0090] Example 1 of this invention obtained subcellular self-tracing ion imaging of Ce in Arabidopsis thaliana leaves, and the results are as follows: Figure 1 As shown. Figure 1 As shown, 80 μmol·L -1After treatment of Arabidopsis leaves with CeCl3, Ce localized to the plasma membrane (PM), vacuoles (V), and chloroplasts (CP) of Arabidopsis leaves. The localization of Ce in chloroplasts (P) was also obtained. Figure 1 The mass spectrum of relative abundance (within the rectangular bounding box region) is shown in the following figure. Figure 2 As shown.

[0091] Combining existing technologies, rare earth radioisotope self-tracing and development ( Figure 3 and Figure 4 , from Wang L, Li J, Zhou Q, et al. Rare earth elements activate endocytosis in plant cells[J]. Proceedings of the National Academy of Sciences, 2014, 111(35): 12936-12941.) and rare earth non-radioactive isotope self-tracing imaging ( Figure 5 According to the results obtained from the imaging method of Cheng M, Wang L, Zhou Q, et al. Lanthanum (III)triggers Atrboh D- and jasmonic acid-dependent systemic endocytosis in plants[J]. Nature Communications, 2021, 12(1): 4327., the method of the present invention can quickly obtain subcellular localization information of rare earth elements in Arabidopsis thaliana with clear images.

[0092] Example 1 of this invention also obtained subcellular self-tracing ion imaging of other transition metal elements [iron (Fe), zinc (Zn)] and non-transition metal elements [calcium (Ca), magnesium (Mg)] in Arabidopsis leaves, and the results are described in [link to example]. Figure 6 , Figure 7 , Figure 8 and Figure 9 .

[0093] Example 2

[0094] Subcellular self-tracing ion imaging localization of Ce in maize leaves. This embodiment follows the same steps as Example 1, and the results are consistent with Example 1. See [link to example]. Figure 10 .

[0095] Example 3

[0096] Subcellular self-tracing ion imaging localization of Ce in Arabidopsis leaves. In this example, 0.01 nmol·L⁻¹ ions were uniformly sprayed onto the Arabidopsis leaves. -1CeCl3. Apart from this, the other steps are the same as in Example 1, and the effects are as follows: Figure 11 The results showed that, compared with Example 1, subcellular self-tracing ion imaging of Ce in Arabidopsis leaves could not be obtained.

[0097] Example 4

[0098] Subcellular self-tracing ion imaging localization of Ce in Arabidopsis leaves. In this embodiment, the scan rate during SEM imaging was 500 ns. Apart from this, the other steps were the same as in Example 1, and the results are as follows: Figure 12 The results showed that, compared with Example 1, clear subcellular self-tracing ion imaging of Ce in Arabidopsis leaves could not be obtained.

[0099] Example 5

[0100] Subcellular self-tracing ion imaging localization of Ce in Arabidopsis thaliana leaves. In this embodiment, 25 images were acquired during FIB-TOF-SIMS bombardment of the sample surface, secondary ion signal detection, and ion imaging. Apart from this, the other steps are the same as in Example 1, and the results are as follows: Figure 13 The results showed that, compared with Example 1, subcellular self-tracing ion imaging of Ce(III) in Arabidopsis leaves could not be obtained.

[0101] Example 6

[0102] Subcellular self-tracing ion imaging localization of Ce in Arabidopsis thaliana leaves. In this embodiment, the accelerating voltage was 2 kV during SEM imaging. Other than that, the steps were the same as in Example 1. Results showed that, compared to Example 1, clear subcellular self-tracing ion imaging of Ce in Arabidopsis thaliana leaves could not be obtained.

[0103] Example 7

[0104] Subcellular self-tracing ion imaging localization of Ce in Arabidopsis thaliana leaves. In this embodiment, the magnification was 1000× during SEM imaging. Apart from this, the other steps were the same as in Example 1. The results showed that, compared to Example 1, clear subcellular self-tracing ion imaging of Ce in Arabidopsis thaliana leaves could not be obtained.

[0105] Example 8

[0106] Subcellular self-tracing ion imaging localization of Ce in Arabidopsis thaliana leaves. In this embodiment, the ion beam current was 7.7 pA during FIB-TOF-SIMS bombardment of the sample surface, secondary ion signal detection, and ion imaging. Other than this, the steps were the same as in Example 1. The results showed that, compared to Example 1, subcellular self-tracing ion imaging of Ce in Arabidopsis thaliana leaves could not be obtained.

[0107] As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects: The method for subcellular self-tracing ion imaging and localization of metal elements disclosed in this application has the advantages of simple operation, speed, clear images, good repeatability, and high safety. It is applicable to cells of various biological sources and various metal ions, and can realize the imaging and localization of metal ions at the subcellular structural level. In particular, it can be applied not only to the subcellular localization of various stable valence transition metal ions in plants, but also to the subcellular localization of various stable valence non-transition metal ions in plants.

[0108] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for subcellular self-tracing ion imaging and localization of metal elements, characterized in that, The method includes: The SEM-FIB-TOF-SIMS system was used to perform subcellular structure imaging and metal ion imaging on sample sections. In the SEM-FIB-TOF-SIMS system, the following steps are performed sequentially: (i) The subcellular structures of the sample slices were visualized using SEM; (ii) Surface bombardment of the subcellular structure using FIB; (iii) The secondary ions excited by the surface bombardment are detected by TOF-SIMS to obtain ion information of the analysis area and image it.

2. The method according to claim 1, characterized in that, The sample sections include plant sections, animal tissue sections, bacterial samples, or fungal samples. Correspondingly, the subcellular structures include plant subcellular structures, animal subcellular structures, bacterial subcellular structures, or fungal subcellular structures.

3. The method according to claim 2, characterized in that, The metal ions include transition metal ions or non-transition metal ions.

4. The method according to claim 3, characterized in that, The transition metal ions include rare earth element ions.

5. The method according to claim 2, characterized in that, The method for preparing the plant slices includes: Plant tissue was cut into small pieces and fixed to obtain fixed samples; The fixed sample was subjected to dehydration, infiltration embedding, and sectioning in sequence to obtain the plant section.

6. The method according to claim 5, characterized in that, The fixation process includes pre-fixation and post-fixation.

7. The method according to claim 6, characterized in that, The pre-fixation treatment includes: pre-fixing the small block with paraformaldehyde and glutaraldehyde to obtain a pre-fixed sample.

8. The method according to claim 7, characterized in that, The post-fixation process includes: using osmium tetroxide solution to perform post-fixation on the pre-fixed sample to obtain the fixed sample.

9. The method according to claim 8, characterized in that, The dehydration process includes: sequentially dehydrating the fixed sample using an ethanol solution with gradually increasing concentration to obtain a dehydrated sample.

10. The method according to claim 9, characterized in that, The permeation embedding process includes: permeating and embedding the dehydrated sample with propylene oxide and an embedding agent to obtain a permeation-embedded sample.

11. The method according to claim 10, characterized in that, The slicing process includes: slicing the infiltration-embedded sample to obtain the plant slices.

12. The method according to claim 3, characterized in that, Before preparing the plant slices, the plant is treated with the metal ions, including: spraying a solution containing the metal ions onto the leaves of the plant until droplets begin to fall.

13. The method according to claim 12, characterized in that, The concentration of the metal ions in the solution is 1 nmol / L - 1 mmol / L.

14. The method according to claim 6, characterized in that, The size of the small piece is 1-2 mm. 3 .

15. The method according to claim 7, characterized in that, The pre-fixation treatment includes: placing the small piece in a solution containing 1-4 wt% paraformaldehyde and 0.5-5 wt% glutaraldehyde, fixing it at 15-25 ℃ in the dark for 2-3 h or at 4 ℃ in the dark overnight to obtain the pre-fixed sample.

16. The method according to claim 8, characterized in that, The post-fixation treatment includes: placing the pre-fixed sample in a 1-2 wt% osmium tetroxide solution and fixing it at 15-25 ℃ in the dark for 4 h to obtain the fixed sample.

17. The method according to claim 9, characterized in that, The dehydration treatment includes sequentially applying 30% (v / v), 50% (v / v), 70% (v / v), 90% (v / v), 100% (v / v), and 100% (v / v) ethanol solutions to the fixed sample, wherein the treatment time for the 30% (v / v)-90% (v / v) ethanol solution is 15 min, and the treatment time for the 100% (v / v) ethanol solution is 20 min, to obtain the dehydrated sample.

18. The method according to claim 10, characterized in that, The permeation and embedding treatment includes: treating the dehydrated sample sequentially with propylene oxide at 15-25 ℃ for 1 h, treating with a gradient mixture of propylene oxide and embedding agent at 15-25 ℃ for 4 h each, treating with the embedding agent at 15-25 ℃ for 8 h, and then polymerizing at 60 ℃ for 48 h to obtain the permeation and embedding sample.

19. The method according to claim 6, characterized in that, The thickness of the plant slices is 80-150 nm.

20. The method according to claim 1 or 2, characterized in that, When using the SEM to visualize subcellular structures, the electron beam current is 21 pA-1.4 nA, the accelerating voltage is 3-10 kV, the scan rate is 1-100 μs, and the magnification is 2500-5000 ×.

21. The method according to claim 1 or 2, characterized in that, When using FIB-TOF-SIMS to bombard the subcellular structure, perform secondary ion signal detection, and ion imaging, the ion beam current is 24-80 pA, the accelerating voltage is 30 kV, and a total of 50-200 images are collected.

22. The method according to claim 1 or 2, characterized in that, After performing the subcellular structure imaging and ion imaging, an image region and a data table of the image region are output. Based on the data table, a mass spectrometry image representing the relative abundance of the metal ions in the image region is drawn.

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