CN114166878A - Cryoelectron microscope sample preparation method for eukaryotic cell sample - Google Patents
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Abstract
The invention discloses a sample preparation method for a eukaryotic cell sample by a cryoelectron microscope, and belongs to the field of sample preparation by the cryoelectron microscope. The method comprises the following steps: taking a grid of a cryoelectron microscope as a substrate, loading a nano alumina film on the substrate, and then treating the nano alumina film in liquid ethane at the temperature of-175 ℃ to-160 ℃ for 3-5 min; and adding a sample to be tested on the frozen net to form an ultrathin liquid film, and putting the ultrathin liquid film in liquid nitrogen for freezing and storing for later use. The invention directly loads the nano alumina film on the cryoelectron microscope as the supporting film, and the operation is simple and easy to realize; the properties of strong conductivity, high mechanical property, good hydrophilicity and the like of the nano alumina film are utilized, and the nano alumina film is loaded on a grid of a cryoelectron microscope, and the nano alumina film has abundant mismatch bonds and oxygen deficiency bonds on the surface, has abundant holes, is easy to adsorb cells, promotes the uniform and single-layer distribution of the cells, and is favorable for improving the observation effect of cell sample freezing sample preparation.
Description
Technical Field
The invention relates to the field of sample preparation of a cryoelectron microscope, in particular to a cryoelectron microscope sample preparation method for a eukaryotic cell sample.
Background
The cryoelectron microscope is an ultralow temperature freezing sample preparation and transmission technology for a scanning electron microscope, and can realize direct observation of liquid, semi-liquid and samples sensitive to electron beams. Since the 70's of the last century to date, cryo-electron microscopy has been able to be well-established for the observation of different samples. Although the cryoelectron microscopy technology has been developed for many years and has made obvious breakthroughs in data processing and camera hardware succession, in frozen sample preparation, different testers are still required to search sample preparation conditions according to different sample states, so that the repeatability of the samples is still low.
With the continuous maturity and perfection of the later image processing technology, the problem of the early sample preparation is increasingly highlighted, and the key to the preparation of the frozen sample is how to prepare the sample with proper thickness and uniform sample distribution, and the sample can keep good molecular properties. At present, a layer of porous carbon film is generally covered on a sample supporting net prepared by a cryoelectron microscope sample, and a supporting film is additionally covered on the sample supporting net, if the supporting film is not covered, most samples in holes have gas-liquid interfaces, and biomacromolecules existing in the gas-liquid interfaces are often denatured to further influence the structure, so that the resolution ratio is low. Researchers have tried to use materials such as silicon nitride or gold as the support film, but they have not succeeded in wide use for the carrier net in the end because of problems such as poor conductivity and mechanical strength. Therefore, the search for an ideal supporting film for the grid of the cryoelectron microscope is one of the key factors for solving the problem of low repetition rate of the conventional freezing sample preparation. In addition, clinical samples such as cell tissues have problems of cell aggregation, cell containing a large number of organelles, activation state of cell tissues and the like, and are one of the important reasons for poor observation effect of the cryoelectron microscope. Therefore, it is necessary to find a new sample preparation method for a cryoelectron microscope in order to overcome the defects of the current sample preparation method for a cryoelectron microscope.
Disclosure of Invention
The invention aims to provide a cryo-electron microscope sample preparation method for a eukaryotic cell sample, which aims to solve the problems in the prior art, and utilizes the properties of strong conductivity, high mechanical property, good hydrophilicity and the like of a nano alumina film to load the nano alumina film on a cryo-electron microscope carrier, and simultaneously, the nano alumina film has rich mismatch bonds and oxygen deficiency bonds on the surface, has rich holes, is easy to adsorb cells, promotes the uniform and single-layer distribution of the cells, and is favorable for improving the observation effect of cell sample freezing sample preparation.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a cryoelectron microscope sample preparation method for a eukaryotic cell sample, which comprises the following steps:
taking a grid of a cryoelectron microscope as a substrate, loading a nano alumina film on the substrate, and then treating the nano alumina film in liquid ethane at the temperature of-175 ℃ to-160 ℃ for 3-5 min;
and adding a sample to be tested on the frozen net to form an ultrathin liquid film, and putting the ultrathin liquid film in liquid nitrogen for freezing and storing for later use.
Preferably, the grid is a graphene grid.
Preferably, the loading of the nano alumina thin film on the substrate comprises the following steps:
(1) dissolving polyvinylidene fluoride and polyvinylpyrrolidone in N, N-dimethylacetamide, and performing ultrasonic treatment for 30-40min to form a membrane coating solution;
(2) placing the carrying net in the film coating liquid for processing, and then adding 1.5% of nano Al by mass2O3And 0.05 percent of dispersant, and carrying out ultrasonic reaction for 15-20h, and then soaking the carrier net in deionized water to form a film for later use.
Preferably, the mass ratio of the polyvinylidene fluoride to the polyvinylpyrrolidone to the N, N-dimethylacetamide is (15-20): (86-90): 1.
Preferably, the dispersant is sodium pyrophosphate, sodium polymetaphosphate or sodium tartrate.
Preferably, in the step (1), the ultrasonic treatment temperature is 63 ℃;
preferably, in the step (2), the ultrasonic reaction temperature is 55 ℃, and a defoaming treatment is further included after the ultrasonic reaction and before the soaking in deionized water.
Preferably, the sample to be tested is a cell sample.
Preferably, the cell sample is obtained by culturing the collected cells to a confluency of 85% -95%, and after subculturing, taking the cells in the logarithmic growth phase, and then terminating the reaction with a Tris-HCl buffer solution having a pH of 7.0.
Preferably, the thickness of the nano alumina film is 5-10 nm.
The invention discloses the following technical effects:
the invention discloses a method for preparing a sample for a cryoelectron microscope of a eukaryotic cell sample, which changes a supporting film-carbon film covered on a conventional cryoelectron microscope carrying net into a nano alumina film, wherein the preparation condition of the nano alumina film is simple and easy to control, and a single-layer film completely covering the carrying net is formed on the carrying net, so that the background noise of the sample in the imaging of the cryoelectron microscope can be greatly reduced. By the verification of candida glabrata cells, the fact that the nano alumina film covers the cryoelectron microscope carrier net can achieve good resolution.
Compared with an ultrathin carbon film serving as a grid-carrying support film of a cryoelectron microscope, the nano-alumina film adopted by the invention has stronger conductivity and mechanical property, and can be used for manufacturing frozen samples at ultralow temperature, thereby obviously reducing the drift caused by electrons during photographing exposure. Because the surface of the nano alumina film formed by the modified alumina has rich mismatched bonds and under-oxygen bonds, and the thin layer contains rich holes, the porous film with high surface activity can be obtained, because the film is directly formed on the net, the film is completely covered according to the structure of the net, and after a sample to be detected is added, the surface activity of the nano alumina film can increase the adsorption effect on cells, so that the cells are uniformly distributed, and because of the single-layer porous structure of the nano alumina film, the cells are fixed on the surface, and simultaneously, uniform single-layer cells are formed, and a multi-layer structure with few agglomerated cells is formed, so that the background noise can be remarkably reduced. In addition, in order to observe the cell state of a certain specific stage of cells, cells in a proper stage need to be selected and observed after the cells are stopped, so that the physiological condition of the specific stage of cells can be accurately analyzed, and technical support is provided for clinical data analysis.
Drawings
FIG. 1 is a representation of a cryoelectron microscopic cell using the nano-alumina thin film prepared in example 2 of the present invention as a support film, a: observing the morphology of the cells by a cryoelectron microscope; b: a partial enlarged view in box.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
This example is prepared prior to cryoelectron microscopy using Candida glabrata cells (purchased from ATCC cell bank) as an example.
Candida glabrata is cultured by a conventional method by using a conventional culture medium. By the logarithmic phase, cells were counted at 105-106And/ml, washing with PBS, adding culture solution, and resuspending for later use.
Example 2
Preparation of nano-alumina film
(1) Dissolving polyvinylidene fluoride and polyvinylpyrrolidone in N, N-dimethylacetamide (the mass ratio of polyvinylidene fluoride to polyvinylpyrrolidone to N, N-dimethylacetamide is 15:86: 1), and carrying out ultrasonic treatment at 63 ℃ for 30min to form a membrane coating solution;
(2) placing the carrier net in a film coating liquid for processing, and then adding 1.5 mass percent of nano Al2O3And 0.05 percent of sodium pyrophosphate, performing ultrasonic reaction for 15 hours at the temperature of 55 ℃, standing and defoaming, and then soaking the carrier net in deionized water to form a film for later use.
Example 3
Preparation of nano-alumina film
(1) Dissolving polyvinylidene fluoride and polyvinylpyrrolidone in N, N-dimethylacetamide (the mass ratio of polyvinylidene fluoride to polyvinylpyrrolidone to N, N-dimethylacetamide is 18:88: 1), and carrying out ultrasonic treatment at 63 ℃ for 35min to form a membrane coating solution;
(2) placing the carrier net in a film coating liquid for processing, and then adding 1.5 mass percent of nano Al2O3And 0.05 percent of sodium pyrophosphate, performing ultrasonic reaction for 17 hours at the temperature of 55 ℃, standing and defoaming, and then soaking the carrier net in deionized water to form a film for later use.
Example 4
Preparation of nano-alumina film
(1) Dissolving polyvinylidene fluoride and polyvinylpyrrolidone in N, N-dimethylacetamide (the mass ratio of polyvinylidene fluoride to polyvinylpyrrolidone to N, N-dimethylacetamide is 20:90: 1), and carrying out ultrasonic treatment at 63 ℃ for 40min to form a membrane coating solution;
(2) placing the carrier net in a film coating liquid for processing, and then adding 1.5 mass percent of nano Al2O3And 0.05 percent of sodium pyrophosphate, performing ultrasonic reaction for 20 hours at the temperature of 55 ℃, standing and defoaming, and then soaking the carrier net in deionized water to form a film for later use.
Test example 1
Respectively taking out the cryoelectron microscope carrier net covered with the nano alumina film prepared in the embodiment 2-4 from deionized water, standing for 20s, and then treating the cryoelectron microscope carrier net in liquid ethane at the temperature of-175-160 ℃ for 3-5 min; then, 4. mu.L of Candida glabrata cells cultured in example 1 were dropped onto the nano alumina film, liquid adsorption was performed from one side by a Leica EMGP plunger, liquid nitrogen was added, sampling was performed, freezing was performed in liquid nitrogen, and then characterization was performed by a cryoelectron microscope.
Comparative example 1
The cryoelectron microscope was covered with a carbon film as a support film, and the procedure was otherwise the same as in example 2 of test example 1.
Comparative example 2
The Candida glabrata cells were added with the nano alumina film and then were frozen in liquid nitrogen, and then the reaction was terminated by adding a Tris-HCl buffer solution of pH7.0, and the other steps were the same as those of the related art protocol of example 2 in Experimental example 1.
Comparative example 3
The procedure of experimental example 1 was the same as that of the related art in example 2, except that reduced graphene oxide was used as a support film to cover the cryoelectron microscope carrier mesh as control 1.
The results show that: the observation of candida glabrata cell samples with the nano alumina thin film as the supporting film in experimental example 1 clearly can obtain better integrity of cell structures compared with comparative examples 1-3, and has no drift phenomenon, and can already resolve to near atomic resolution (wherein the characterization chart of example 2 is shown in fig. 1). In contrast, in comparative example 1, cell aggregation and low resolution were observed; comparative example 2 shows that there is a small amount of stacking aggregation phenomenon, which affects the resolution of the specific cell structure, but the reason for this may be that the cell reaction is terminated first, and the mismatched bond and the oxygen deficient bond on the nano alumina membrane preferentially adsorb the cells on the cell surface, while if the termination reaction is performed after the nano alumina membrane is added, due to the activity of the cells themselves, part of the cells may preferentially enter the cavities of the nano alumina membrane, causing stacking aggregation of part of the cells, which further affects the resolution; comparative example 3 is advantageous for uniform distribution of cells, but it is difficult to form a monolayer structure by itself when preparing redox graphene, and reduced redox graphene has a large number of oxygen-containing groups on the surface, but the oxygen-containing groups can adsorb a part of cells, but more enter the cytoplasmic region, and uniform distribution of cells is extremely limited. Therefore, through comparison, the cell cryoelectron microscope sample preparation method is more favorable for observing the fine internal structure of the cell, and is favorable for assisting in clinically and visually observing whether a certain specific cell is diseased or not.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
Claims (10)
1. The cryoelectron microscope sample preparation method for the eukaryotic cell sample is characterized by comprising the following steps of:
taking a grid of a cryoelectron microscope as a substrate, loading a nano alumina film on the substrate, and then treating the nano alumina film in liquid ethane at the temperature of-175 ℃ to-160 ℃ for 3-5 min;
and adding a sample to be tested on the frozen net to form an ultrathin liquid film, and putting the ultrathin liquid film in liquid nitrogen for freezing and storing for later use.
2. The cryoelectron microscopy sample preparation method for eukaryotic cell samples according to claim 1, wherein the grid is graphene.
3. The cryoelectron microscopy sampling method for eukaryotic cell samples according to claim 1, wherein the loading of the nano-alumina thin film on the substrate comprises the following steps:
(1) dissolving polyvinylidene fluoride and polyvinylpyrrolidone in N, N-dimethylacetamide, and performing ultrasonic treatment for 30-40min to form a membrane coating solution;
(2) placing the carrying net in the film coating liquid for processing, and then adding 1.5% of nano Al by mass2O3And 0.05 percent of dispersant, and carrying out ultrasonic reaction for 15-20h, and then soaking the carrier net in deionized water to form a film for later use.
4. The cryoelectron microscopy sample preparation method for eukaryotic cell samples as claimed in claim 3, wherein the mass ratio of the polyvinylidene fluoride, the polyvinylpyrrolidone and the N, N-dimethylacetamide is (15-20): (86-90): 1.
5. The cryoelectron microscopy sample preparation method for eukaryotic cell samples according to claim 3, wherein the dispersing agent is sodium pyrophosphate, sodium polymetaphosphate or sodium tartrate.
6. The cryoelectron microscopy sample preparation method for eukaryotic cell samples according to claim 3, wherein in step (1) the sonication temperature is 63 ℃.
7. The cryoelectron microscopy specimen preparation method for eukaryotic cell samples as claimed in claim 3, wherein in step (2), the ultrasonic reaction temperature is 55 ℃, and the method further comprises a defoaming treatment after the ultrasonic reaction and before the soaking in deionized water.
8. The cryoelectron microscopy sample preparation method for eukaryotic cell samples according to claim 1, wherein the sample to be tested is a cell sample.
9. The cryoelectron microscopy sample preparation method for eukaryotic cell samples according to claim 8, wherein the cell samples are obtained by culturing the collected cells to 85% -95% confluency, subculturing, taking the cells in logarithmic growth phase, and terminating the reaction with Tris-HCl buffer solution with pH 7.0.
10. The cryoelectron microscopy sample preparation method for eukaryotic cell samples according to claim 1, wherein the thickness of the nano-alumina film is 5-10 nm.
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| CN202210131271.5A CN114166878B (en) | 2022-02-14 | 2022-02-14 | Cryoelectron microscope sample preparation method for eukaryotic cell sample |
| ZA2023/00067A ZA202300067B (en) | 2022-02-14 | 2023-01-03 | Sample preparation method of cryo-electron microscope for eukaryotic cell samples |
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| CN202210131271.5A CN114166878B (en) | 2022-02-14 | 2022-02-14 | Cryoelectron microscope sample preparation method for eukaryotic cell sample |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111908455A (en) * | 2019-10-30 | 2020-11-10 | 清华大学 | Reduced graphene oxide film and preparation method thereof |
| CN113960078A (en) * | 2020-07-20 | 2022-01-21 | 清华大学 | Application of multifunctional graphene grid in three-dimensional reconstruction of cryoelectron microscope |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111908455A (en) * | 2019-10-30 | 2020-11-10 | 清华大学 | Reduced graphene oxide film and preparation method thereof |
| CN113960078A (en) * | 2020-07-20 | 2022-01-21 | 清华大学 | Application of multifunctional graphene grid in three-dimensional reconstruction of cryoelectron microscope |
Non-Patent Citations (3)
| Title |
|---|
| K.MIWA ET AL.: ""Fabrication of microchannel-TEM grid for in situ liquid observation of interfacial chemical reaction"", 《2018 INTERNATIONAL CONFERENCE ON ELECTRONICS PACKAGING AND IMAPS ALL ASIA CONFERENCE (ICEP-IAAC)》 * |
| YANGNING ZHANG ET AL.: "A "Tips and Tricks" Practical Guide to the Synthesis of Metal Halide Perovskite Nanocrystals", 《AMERICAN CHEMICAL SOCIETY》 * |
| 张立人: "超亲水氧化铝复合网的制备及其在油水分离中的研究", 《中国优秀硕士学位论文全文数据库》 * |
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| CN114166878B (en) | 2022-04-26 |
| ZA202300067B (en) | 2023-03-29 |
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