Method for rapidly preparing cell scanning electron microscope sample through cell surface metal deposition

Abstract

The invention relates to a method for rapidly preparing a cell scanning electron microscope sample through cell surface metal deposition. The method comprises immobilizing a cell sample with an immobilizing agent; incubating in a catalyst solution; placing in a metal deposition solution for cell metal deposition; the cell sample is obtained. The prepared cell sample can be directly used for scanning electron microscope imaging after being dried. According to the method, the micro-nano structure on the surface of the cell is directly mechanically enhanced in the solution through electroless metal deposition, the cell sample is endowed with electrical conductivity, additional chemical modification on the cell is not needed, gradient dehydration on the cell is not needed, and metal spraying treatment on the cell is not needed; and cell scanning electron microscope sample preparation and imaging can be conveniently and quickly carried out.

Description

Technical Field

[0001] This application relates to the field of biological sample preparation technology, specifically to a method for rapidly preparing cell scanning electron microscopy samples by metal deposition on the cell surface. Background Technology

[0002] As the fundamental building blocks of living organisms, cells utilize intricate micro- and nanostructures for molecular recognition, signal transduction, and energy exchange between the organism and its environment. For example, immune cells can rapidly recognize cancer cell surface antigens, efficiently process invading bacteria, and significantly enhance adhesion to nanomaterials using their surface microvilli or pseudopodia. Hair cells in the cochlea can utilize their surface stenociliary structures for auditory perception, and intestinal cells can utilize their surface microvilli to enhance nutrient absorption. Revealing the characteristics of surface micro- and nanostructures during cellular activities is crucial for a deeper understanding of cellular biological functions and the mechanisms of cell-cell and cell-material interactions. Therefore, developing cell sample preparation techniques for the preservation and characterization of surface micro- and nanostructures is essential.

[0003] Characterization techniques for cell surface micro- and nanostructures mainly include fluorescence microscopy, atomic force microscopy, and scanning electron microscopy. Fluorescence microscopy allows for real-time and dynamic observation of cells, but it suffers from limitations such as limited resolution, complex fluorescent labeling operations, and phototoxicity. Atomic force microscopy can provide three-dimensional morphology of cell surfaces, but it is hampered by slow scanning speed and difficulty in characterizing complex structures. Scanning electron microscopy (SEM) offers advantages such as high resolution, fast imaging speed, and strong three-dimensional morphology representation, playing a crucial role in characterizing the fine micro- and nanostructures of cell samples. However, traditional SEM sample preparation processes involve steps such as chemical fixation with aldehyde cross-linking agents, gradient dehydration, critical carbon dioxide drying, and gold sputtering, which are cumbersome, time-consuming, and dependent on specialized equipment, limiting its wider application. Furthermore, chemical fixation using aldehyde cross-linking agents alone can damage the sample structure due to dehumidification and other drying stresses. Therefore, there is an urgent need for a simple, efficient method for preparing SEM samples that maintains the integrity of the sample structure. Summary of the Invention

[0004] This application addresses at least one of the problems of the related technology in the following aspects.

[0005] Therefore, this application proposes a method for rapidly preparing cell samples, comprising the following steps: fixing the cell sample with a fixative; incubating it in a catalyst solution; depositing metal onto the cells in a metal deposition solution; and obtaining the cell sample.

[0006] In some embodiments, the cells are animal cells.

[0007] In some embodiments, the cell is a mammalian cell.

[0008] In some embodiments, the fixative is any one or more of formaldehyde, paraformaldehyde, and glutaraldehyde.

[0009] In some embodiments, the fixative is a mixed solution of paraformaldehyde and glutaraldehyde.

[0010] In some embodiments, the fixative is a mixed solution of 1-5% paraformaldehyde and 0.1-0.5% glutaraldehyde.

[0011] In some embodiments, the fixed time is 10 to 20 minutes.

[0012] In some embodiments, the catalyst is a palladium-containing compound.

[0013] In some embodiments, the catalyst is ammonium tetrachloropalladium, more specifically 2-15 mM ammonium tetrachloropalladium.

[0014] In some embodiments, the incubation time is not less than 5 minutes.

[0015] In some embodiments, the incubator is washed 1 to 5 times with deionized water or PBS buffer.

[0016] In some embodiments, the metal is nickel, copper, or silver.

[0017] In some embodiments, the metal is nickel, and the nickel deposition solution is composed of nickel sulfate, trisodium citrate, lactic acid, dimethylaminoborane, and ammonia.

[0018] In some embodiments, the concentration of nickel sulfate is 30-50 g / L, the concentration of trisodium citrate is 10-30 g / L, the concentration of lactic acid is 5-15 g / L, the concentration of dimethylaminoborane is 0.5-2.0 g / L, and the pH is adjusted to 6-12 with ammonia.

[0019] In some embodiments, the method further includes a washing step following the metal deposition of the cells.

[0020] In some embodiments, one or more of water, ethanol, and methanol solvents are used for cleaning.

[0021] This application also provides a cell sample prepared by the rapid cell sample preparation method described in any embodiment of this application, wherein the cell sample is the cell, and further, the cell surface has deposited metal.

[0022] In some embodiments, the metal is nickel.

[0023] The embodiments of this application achieve the following beneficial effects: This application presents a method for rapidly preparing cell scanning electron microscopy (SEM) samples via metal deposition on the cell surface. The method fixes cell morphology through metal deposition on the cell surface, thus preparing SEM samples. Preparing SEM samples via cell metallization offers several advantages: firstly, the deposited metal enhances the mechanical strength of the cell surface micro / nano structures, making the cell surface morphology less susceptible to damage during dehydration and drying, eliminating the need for gradient dehydration and critical carbon dioxide drying processes; secondly, the deposited metal imparts conductivity to the cell samples, allowing direct use for SEM imaging without the need for gold sputtering; furthermore, the selection of nickel as the deposited metal imbues the cells with magnetism, facilitating the rapid collection of suspended cell samples. In summary, the cell SEM sample preparation method provided in this application offers advantages such as short preparation time, minimal disruption of cell surface structures, and independence from any specialized equipment. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 Scanning electron microscope image of HCT-8 cells after nickel metallization; Figure 2 Scanning electron microscope image of MCF-7 cells after copper metallization, which adhere to the cell wall. Figure 3 Scanning electron microscope image of MCF-7 cells that have adhered to the wall without metallization. Detailed Implementation

[0026] The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit the scope of the present application. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0027] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0028] The first aspect of this application provides a method for rapidly preparing cell samples, comprising the following steps: fixing the cell sample with a fixative; incubating it in a catalyst solution; depositing metal onto the cells in a metal deposition solution; and obtaining the cell sample.

[0029] In some embodiments, the cells are animal cells.

[0030] In some embodiments, the cell is a mammalian cell.

[0031] In some embodiments, the fixative is any one or more of formaldehyde, paraformaldehyde, and glutaraldehyde.

[0032] In some embodiments, the fixative is a mixed solution of paraformaldehyde and glutaraldehyde.

[0033] In some embodiments, the concentration of paraformaldehyde ranges from 1% to 5%, for example, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, and 5%; the concentration of glutaraldehyde ranges from 0.1% to 0.5%, for example, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, and 0.5%.

[0034] In some embodiments, the fixed time is 10 to 20 minutes, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 minutes.

[0035] In some embodiments, the catalyst is a palladium-containing compound.

[0036] In some embodiments, the catalyst is ammonium tetrachloropalladium, more specifically 2 to 15 mM ammonium tetrachloropalladium, such as 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, or 15 mM ammonium tetrachloropalladium.

[0037] In some embodiments, the incubation time is not less than 5 minutes.

[0038] In some embodiments, the incubator is washed 1 to 5 times with deionized water or PBS buffer.

[0039] In some embodiments, the metal is nickel, copper, or silver.

[0040] In some embodiments, the metal is nickel. One method for depositing metallic nickel on the cell surface involves preparing a nickel deposition solution, and then subjecting the cells (after adsorbing the catalyst) and the nickel deposition solution to electroless metal deposition at room temperature for 1-10 minutes.

[0041] In some embodiments, the nickel deposition solution consists of nickel sulfate, trisodium citrate, lactic acid, dimethylaminoborane, and ammonia. The concentration of nickel sulfate is 30-50 g / L, the concentration of trisodium citrate is 10-30 g / L, the concentration of lactic acid is 5-15 g / L, the concentration of dimethylaminoborane is 0.5-2.0 g / L, and the pH is adjusted to 6-12 with ammonia.

[0042] In some embodiments, the metal is copper. One method for depositing metallic copper on the cell surface involves preparing a copper deposition solution, and then subjecting the cells (after adsorbing the catalyst) and the copper deposition solution to electroless metal deposition at room temperature for 1-10 minutes.

[0043] In some embodiments, the copper deposition solution is composed of sodium hydroxide, copper sulfate, potassium sodium tartrate, and formaldehyde, wherein the concentration of sodium hydroxide is 2-20 g / L, the concentration of copper sulfate is 2-20 g / L, the concentration of potassium sodium tartrate is 5-30 g / L, and the concentration of formaldehyde is 0.5-5.0 mL / L.

[0044] In some embodiments, the method further includes a washing step following the metal deposition of the cells.

[0045] In some embodiments, one or more of water, ethanol, and methanol solvents are used for cleaning.

[0046] The second aspect of this application provides a cell sample prepared by the method provided in any embodiment of the first aspect of this application, wherein the cell sample is the cell, and further, the cell surface has deposited metal.

[0047] In some embodiments, the metal is nickel.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0049] Unless otherwise specified, the quantitative analysis experiments in the following examples are all repeated three times, and the results are averaged.

[0050] Example 1: Preparation of nickel-metallized cell samples 1.1 Experimental Materials HCT-8 cells (human colorectal cancer cell line, purchased from the National Experimental Cell Resource Sharing Platform) were cultured in RPMI 1640 cell culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. When the cell density reached approximately 90%, trypsin digestion and cell collection were performed.

[0051] 1.2 Preparation steps After digesting the cultured HCT-8 cells, they were resuspended and dispersed as follows: The HCT-8 cells were placed in a sterile working area of ​​a laminar flow hood, and the original culture medium was discarded. An appropriate amount of biological buffer PBS was added to gently wash the cells for 10-20 seconds, and then the washing solution was discarded. An appropriate amount of trypsin digestion solution was added (1 mL of trypsin (Trypsin-EDTA (0.25%); catalog number 25200056; ThermoFisher Scientific Inc.) was added to a 25 mL culture flask). The culture flask was gently shaken to spread the trypsin evenly across the cell layer at the bottom, and the flask was placed in a 37°C incubator for digestion. When the cells showed significant shrinkage and increased spacing, but were not completely detached, 2-3 mL of RPMI 1640 cell culture medium was added to stop the trypsin digestion. The adherent cells were repeatedly pipetted to disperse them into a single-cell suspension. The cell suspension was mixed and transferred to a centrifuge tube, and centrifuged at 1000 rpm for 3 minutes. After centrifugation, the supernatant was discarded, and the cells were gently resuspended and dispersed by agitation with fixative.

[0052] HCT-8 cells were fixed at room temperature for 15 minutes in a fixative mixture of 3% paraformaldehyde and 0.1% glutaraldehyde.

[0053] The pretreated cells were placed in a 5 mM ammonium tetrachloropalladium solution and incubated in the dark for 15 minutes, then washed 5 times with deionized water.

[0054] A nickel deposition solution was prepared containing 40 g / L nickel sulfate, 20 g / L trisodium citrate, 10 g / L lactic acid, and 1 g / L dimethylaminoborane, with the pH adjusted to 7.5 using ammonia. HCT-8 cells with adsorbed catalyst were added to the nickel deposition solution for metallization at room temperature for 5 minutes. The nickel-metallized HCT-8 cells were characterized by SEM, and the results are shown below. Figure 1 As shown. From Figure 1 It can be seen that the dispersed suspended cells have an approximately spherical appearance, and the nanostructures on the cell surface can be clearly observed. This indicates that the cell structure was not destroyed during the preparation of the cell scanning electron microscope sample. This is because metallic nickel is rapidly and uniformly deposited on the cell surface, which increases the mechanical strength of the cell structure, especially the micro and nano structures, so that the cells can still maintain their original morphology during the direct drying process (without ethanol gradient dehydration and carbon dioxide critical drying).

[0055] Example 2: Preparation of copper-metallized cell samples 2.1 Experimental Materials MCF-7 cells (human breast cancer cell line, purchased from the National Experimental Cell Resource Sharing Platform) were cultured in DMEM cell culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C and 5% CO2. When the cell density reached approximately 90%, trypsin digestion and cell collection were performed.

[0056] 2.2 Preparation steps The process of digesting cultured MCF-7 cells to induce adhesion is as follows: MCF-7 cells are placed in a sterile working area of ​​a laminar flow hood, and the original culture medium is discarded. Then, an appropriate amount of biological buffer PBS is added to gently wash the cells for 10-20 seconds, and the washing solution is discarded. An appropriate amount of trypsin digestion solution (1 mL trypsin-EDTA (0.25%); catalog number 25200056; ThermoFisher Scientific Inc.) is added to a 25 mL culture flask. The flask is gently shaken to evenly distribute the trypsin across the bottom cell layer, and then placed in a 37°C incubator for digestion. When the cells show significant shrinkage and increased spacing, but are not completely detached, 2-3 mL of complete culture medium is added to stop the trypsin digestion. The adherent cells are repeatedly pipetted to disperse them into a single-cell suspension. The cell suspension is mixed and transferred to a centrifuge tube. The cells are centrifuged at 1000 mL / min. Centrifuge at rpm for 3 minutes; after centrifugation, discard the supernatant, add an appropriate amount of fresh culture medium, gently resuspend and disperse the cells, and evenly inoculate the cell suspension into a culture dish (place a glass slide cut into 1 cm × 1 cm at the bottom of the culture dish), and then place it in an incubator for 20 minutes to allow the cells to adhere to the surface of the glass slide.

[0057] MCF-7 cells were fixed at room temperature for 15 minutes in a fixative mixture of 3% paraformaldehyde and 0.1% glutaraldehyde.

[0058] The pretreated cells were placed in a 5 mM ammonium tetrachloropalladium solution and incubated in the dark for 15 minutes, then washed 5 times with deionized water.

[0059] A copper deposition solution was prepared containing 6 g / L sodium hydroxide, 6.5 g / L copper sulfate pentahydrate, 14.5 g / L potassium sodium tartrate tetrahydrate, and 5 mL / L formaldehyde. MCF-7 cells with catalyst adsorption were added to the copper deposition solution and metallized at room temperature for 5 minutes. The copper-metallized MCF-7 cells were characterized by SEM, and the results are shown below. Figure 2 As shown. From Figure 2 It can be seen that the cells adhering to the surface of the slide have an approximately spherical shape at their center, and extend outwards from the periphery through filamentous pseudopodia and lamellar pseudopodia. The micro- and nano-structures on the cell surface can be clearly observed, indicating that the cell structure was not destroyed during the preparation of the cell scanning electron microscope sample. This is because metallic copper is rapidly and uniformly deposited on the cell surface, which increases the mechanical strength of the cell structure, especially the micro- and nano-structures, so that the cells can still maintain their original morphology during the direct drying process (without ethanol gradient dehydration and carbon dioxide critical drying).

[0060] Preparation of comparative unmetallized cell samples Experimental materials The same experimental materials as in Example 2.1 were used.

[0061] Preparation steps The procedure was similar to that in Example 2.2, except that the MCF-7 cells after catalyst adsorption were not subjected to metal deposition but were directly dried. The dried cell samples were then sputter-coated with gold. The unmetallized MCF-7 cells were characterized by SEM, and the characterization results are as follows: Figure 3 As shown. From Figure 3 It can be seen that the surface morphology of cells adhering to the surface of the slide is severely damaged during the direct drying process (without ethanol gradient dehydration and carbon dioxide critical drying) without metal deposition, and the micro- and nano-structures on the cell surface cannot be clearly observed.

[0062] The above results indicate that the method for rapidly preparing cell scanning electron microscopy samples by metal deposition on the cell surface involved in this application has the advantages of short sample preparation time and fewer preparation steps. Moreover, the cell samples prepared by the method of rapidly preparing cell scanning electron microscopy samples by metal deposition on the cell surface involved in this application have better cell structure integrity and clearer structure compared with conventionally treated, especially unmetallized cell samples, and have good application prospects.

[0063] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0064] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A method for rapidly preparing cell samples, characterized in that, Includes the following steps: Cell samples were fixed with a fixative. Incubate in a catalyst solution; Cellular metal deposition was carried out in a metal deposition solution; The cell sample was obtained.

2. The method according to claim 1, characterized in that, The metal is deposited on the cell surface.

3. The method according to claim 1, characterized in that, The cells are animal cells, preferably mammalian cells.

4. The method according to claim 1, characterized in that, The fixative is any one or more of formaldehyde, paraformaldehyde, and glutaraldehyde; Preferably, the fixative is a mixed solution of paraformaldehyde and glutaraldehyde; More preferably, the fixative is a mixed solution of 1-5% paraformaldehyde and 0.1-0.5% glutaraldehyde; Choose any time, with a fixed duration of 10-20 minutes.

5. The method according to any one of claims 1-4, characterized in that, The catalyst is a palladium-containing compound, preferably ammonium tetrachloropalladium, more preferably 2-15 mM ammonium tetrachloropalladium.

6. The method according to any one of claims 1-5, characterized in that, The incubation time shall not be less than 5 minutes; Optionally, wash 1 to 5 times with deionized water or PBS buffer after incubation.

7. The method according to any one of claims 1-6, characterized in that, The metal is nickel, copper, or silver.

8. The method according to claim 7, characterized in that, The metal is nickel, and the nickel deposition solution is composed of nickel sulfate, trisodium citrate, lactic acid, dimethylaminoborane, and ammonia. Preferably, the concentration of nickel sulfate is 30-50 g / L, the concentration of trisodium citrate is 10-30 g / L, the concentration of lactic acid is 5-15 g / L, the concentration of dimethylaminoborane is 0.5-2.0 g / L, and the pH is adjusted to 6-12 with ammonia.

9. The method according to any one of claims 1-8, characterized in that, It also includes a washing step following the metal deposition in the cells; Optionally, one or more of water, ethanol, and methanol solvents may be used for cleaning.

10. A cell sample prepared by the method of any one of claims 1-9, characterized in that, The cell sample is the cell, preferably the cell surface has deposited metal; Preferably, the metal is nickel.

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