CN113640323A - Preparation method of transmission electron microscope sample with small number of cells - Google Patents
Preparation method of transmission electron microscope sample with small number of cells Download PDFInfo
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- CN113640323A CN113640323A CN202111042231.5A CN202111042231A CN113640323A CN 113640323 A CN113640323 A CN 113640323A CN 202111042231 A CN202111042231 A CN 202111042231A CN 113640323 A CN113640323 A CN 113640323A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/02—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material
- G01N23/04—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by transmitting the radiation through the material and forming images of the material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2223/00—Investigating materials by wave or particle radiation
- G01N2223/03—Investigating materials by wave or particle radiation by transmission
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2223/00—Investigating materials by wave or particle radiation
- G01N2223/10—Different kinds of radiation or particles
- G01N2223/102—Different kinds of radiation or particles beta or electrons
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2223/00—Investigating materials by wave or particle radiation
- G01N2223/60—Specific applications or type of materials
- G01N2223/612—Specific applications or type of materials biological material
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Abstract
The invention provides a preparation method of a transmission electron microscope sample with a small number of cells, which relates to the technical field of transmission electron microscopes and comprises the following steps: adding cells into an EP tube, adding the collected cells into precooled glutaraldehyde stationary liquid, centrifuging the EP tube, taking out supernatant, adding 1% agarose gel, picking up cell blocks to wrap the cell blocks in the agarose gel, cooling to a fixed state, cutting, adding the glutaraldehyde stationary liquid again, washing by using sodium dimethylkunmi buffer solution, and gradually dehydrating by using ethanol; soaking the cells; gradually heating and polymerizing the cell sample to form an embedding block; polishing the cell dense block area in the embedding block and cutting the cell dense block area into small resin blocks; and cutting the sample into semi-thin slices, dyeing and positioning, polishing only the residual target area, and cutting the ultrathin slices by using a diamond cutter with the target area as the center to obtain the transmission electron microscope sample. The preparation method reduces the centrifugation times, thereby avoiding the change of the cell morphology and being capable of truly reflecting the real morphology of the substances in the cells.
Description
Technical Field
The invention relates to the technical field of transmission electron microscopes, in particular to a preparation method of a transmission electron microscope sample with a small number of cells.
Background
Transmission Electron Microscopy (TEM) is a very important tool and means for observing microstructures in the IC industry. Many key dimensions of online detection need to be compared with the measured value of a transmission electron microscope, and the method is also an important mode for solving many problems of online detection.
The conventional transmission electron microscope embedding method has high requirements on the number of cell samples, and basically adopts a method of centrifuging and taking precipitates to perform sample treatment and embedding. Too many centrifugation times can change the appearance of the tangible substance of the sample, and the operation steps are complex and time-consuming. When the number of cell samples is not enough, cells are lost to different degrees due to repeated centrifugation and subsequent operation, so that the cells are difficult to embed, and after embedding, ultrathin sections and electron microscope observation are carried out in the later stage, so that inconvenience is caused.
Disclosure of Invention
The invention aims to solve the defects that cells are required to be too much and are easy to lose in the prior art.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of a transmission electron microscope sample with a small number of cells comprises the following steps:
s1: preparing cells, adding the cells to an EP tube,
s2: fixing a specimen: adding the cells collected in the S1 into precooled glutaraldehyde stationary liquid, centrifuging by using an EP tube, taking out supernatant, adding 1% agarose gel, picking up cell blocks at the bottom of the EP tube to wrap the cell blocks in the agarose gel, cooling to a fixed state, cutting and adding the glutaraldehyde stationary liquid again, washing for multiple times by using sodium dimethylkunmu buffer solution, and then washing by using pure water;
s3: dehydrating with ethanol step by step;
s4: respectively soaking cells by using a mixed solution of propylene oxide, propylene oxide and a pure Epon-812 solution and Epon-812 epoxy resin;
s5: gradually heating and polymerizing the cell sample in the S4 to form an embedding block;
s6: observing the embedded block in S5, polishing the cell dense block area and cutting into small resin blocks;
s7: and slicing the small resin blocks to form semi-thin slices, dyeing and positioning the cell area, selecting a target area according to the image prompt of the semi-thin slices, trimming and polishing the semi-thin slices, cutting the ultrathin slices with the thickness of 70-90nm by using a diamond cutter with the target area at the center after only the target area is left, and obtaining the cell section of the target area, namely the transmission electron microscope sample.
Preferably, the S1 specifically includes the following steps: cells cultured in a culture dish or a culture flask were washed with PBS, and the washed cells were added to a 1.5ml EP tube, and after centrifugation, the cells were aggregated at the bottom of the tube.
Preferably, the cells in S1 in S2 are added into 1ml of pre-cooled glutaraldehyde fixing solution, the EP tube is stabilized during the addition, and after the addition is completed, the cells are fixed for 2h or overnight at 4 ℃.
Preferably, the agarose gel in S2 is prepared in advance, and is heated before adding, then naturally cooled to 40-50 ℃, the adding amount of the agarose gel is 100 ul, and after naturally cooled to the agarose gel fixed state, the agarose gel is cut into 1: 1: 1, small square.
Preferably, the washing with sodium dimethylkunmunoate buffer solution used in S2 is performed 6 times for 15min, then fixed with 1% osmic acid for 2h, and then washed with pure water for 6 times for 5min, at 4 ℃.
Preferably, the ethanol is dehydrated step by step once by using 30%, 50%, 70%, 80%, 90% ethanol, and then dehydrated twice by using absolute ethanol.
Preferably, the S4 specifically includes the following steps:
b1: displacement with propylene oxide twice;
b2: propylene oxide and pure Epon-812 were used as 2: 1, soaking the cells by the mixed solution;
b3: propylene oxide and pure Epon-812 were used as 1: 2, soaking the cells by the mixed solution according to the proportion;
b4: and (3) soaking the cells by using Epon-812 epoxy resin at the soaking temperature of 37 ℃ for 7h to obtain a cell sample.
Preferably, the step-by-step heating polymerization sequence in S5 is: firstly, putting the mixture into a 37 ℃ oven for 24 hours; then putting the mixture into a 45 ℃ oven for 24 hours; then putting the mixture into an oven at 60 ℃ for 24 hours; finally, the mixture is put into a 37 ℃ oven.
Preferably, in S7, the cell region is doubly stained with uranium acetate-lead citrate, and after the staining is completed, the cell region is rinsed with pure water and dried by irradiation of infrared light.
Preferably, the uranium acetate is uranyl acetate, 25% saturated uranium acetate is used, the uranium acetate is prepared by 50-70% ethanol, and the formula of the lead citrate is 1.33 g of lead nitrate, 1.76 g of sodium citrate and 30 ml of distilled water; shaking for half an hour, adding 5ml of 1N sodium hydroxide, and adding distilled water to prepare 50 ml.
The preparation method of the transmission electron microscope sample with a small number of cells can avoid the influence of multiple times of centrifugation on the change of the cell appearance, can truly reflect the real appearance of substances inside the cells, has no too many requirements on the number of the cell samples, and can be applied to the transmission electron microscope observation of various cell samples or samples which are similar to cells with small volume and easy to loosen in the actual operation.
Drawings
FIG. 1 is a schematic view of an Epon-812 epoxy resin embedded block of a transmission electron microscope cell sample obtained by the method of the present invention;
FIG. 2 is a schematic representation of an embedded block of epoxy resin of an Epon-812 sample of a transmission electron microscope cell obtained by a conventional method.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments of the present disclosure.
A preparation method of a transmission electron microscope sample with a small number of cells comprises the following steps:
s1: cell preparation:
specifically, cells cultured in a culture dish or a culture flask are rinsed with PBS, in one embodiment, the pH of PBS is 7.2, the number of rinsing is 3, and each rinsing time is 0.5 min; the washed cells were then added to a 1.5ml EP tube, and in one embodiment, the cells were added to a 1.5ml EP tube using trypsinization, which in one embodiment uses 2.5% pancreatin concentration, and the digestion time was determined according to the cell status. After centrifugation, the cells accumulated at the bottom of the tube, and in one embodiment, the centrifugation speed was 1500r/min and the centrifugation speed was 5 min. In other embodiments, the cells may be added to a 1.5ml EP tube using a doctor blade or other method depending on the experimental requirements.
S2: fixing a specimen:
a1: initial fixation of the specimen: and (3) adding the cells collected in the S1 into 1ml of precooled glutaraldehyde fixing solution, adding into an EP tube with the volume of 1.5ml stabilized in the process so as not to cause the cells to be dispersed and not to form lumps, and fixing for 2h or overnight at 4 ℃ after the addition is finished.
A2: and (3) agarose gel wrapping: the agarose gel is prepared in advance at 1%, added into a microwave oven for heating, and then naturally cooled to 40-50 ℃, and in one embodiment, the agarose gel used is agarose with the concentration of 1% and without EB.
Centrifuging the EP tube in A1, discarding the supernatant, adding 100 ul of agarose gel, then picking up the cell mass at the bottom of the EP tube to wrap in the agarose gel sugar, the picking up process needs to be gentle in action, the cell mass is prevented from being damaged, naturally cooling to the agarose gel to a fixed state, and cutting to be 1: 1: 1, and cut as small as possible without destroying the gel-coated cells, and then 1ml of 4% glutaraldehyde fixing solution was added again and left to stand at 4 ℃.
A3: post-fixing of the specimen: washing with 0.1mol/L sodium dimethylkunmu buffer solution for 6 times, each time for 15min, then fixing with 1% osmic acid for 2h, and washing with pure water for 6 times, each time for 5min, at 4 deg.C.
S3: ethanol dehydration:
dehydrating with 30%, 50%, 70%, 80%, and 90% ethanol for 15min, and dehydrating with anhydrous ethanol for 30 min.
In another embodiment, dehydration with 70% ethanol is carried out over night.
S4: soaking:
b1: the reaction mixture was replaced twice with propylene oxide, each time for 30min, at a temperature of 28 ℃.
B2: propylene oxide and pure Epon-812 were used as 2: 1, soaking the cells by the mixed solution at the soaking temperature of 30 ℃ for 4 h.
B3: propylene oxide and pure Epon-812 were used as 1: 2, soaking the cells in the mixed solution at the soaking temperature of 35 ℃ for 5 hours.
B4: using Epon-812 epoxy resin to soak cells, wherein the soaking temperature is 37 ℃, and the soaking time is 7h to obtain a cell sample, in one embodiment, the soaking operation is performed on a machine, and the cell sample needs to be shaken back and forth in the soaking process, so that the Epon-812 epoxy resin gradually and completely replaces a dehydrating agent in the sample, and all gaps inside and outside the cells are filled with pure Epon-812 epoxy resin. The Epon-812 epoxy resin formula is as follows: DDSA (dodecenylsuccinic anhydride, curing agent), MNA (methyl endomethyltetrahydrophthalic anhydride, curing agent), DMP-30 (accelerator).
S5: embedding:
specifically, the cell sample obtained in S4 is put into a mold filled with a pure Epon-812 epoxy resin embedding agent, and then heated for polymerization, and the heated polymerization is carried out, and in one embodiment, the cell sample is firstly put into a 37 ℃ oven for 24 hours; then putting the mixture into a 45 ℃ oven for 24 hours; then putting the mixture into an oven at 60 ℃ for 24 hours; finally, the mixture is placed into a 37 ℃ oven to form an embedded block. The pure Epon-812 epoxy resin embedding medium is liquid before polymerization, can permeate into cells, and is heated to polymerize into solid for subsequent ultrathin section.
S6: block repairing:
the embedding block in S5 is first observed to select a cell dense block region, in one embodiment, the black region on the embedding block is the cell dense block region, and the cell dense block is ground and cut into a resin small block in a trapezoidal or rectangular shape. Wherein the dense block of cells turns black as a result of osmate oxidation.
S7: slicing and staining:
specifically, in one embodiment, the method includes the following steps:
slicing: cutting the cut resin small blocks into semi-thin slices with the thickness of 1-2 um by using a semi-thin slicer;
dyeing: dyeing and positioning a cell area, carrying out double dyeing by using uranium acetate-lead citrate, washing by using pure water, and irradiating and drying by using an infrared lamp; in one embodiment, the uranium acetate is uranyl acetate, the uranium staining solution is 2-5% saturated uranium acetate, and the uranium staining solution is prepared by 50-70% ethanol; the formula of the lead citrate staining solution is as follows: 1.33 g of lead nitrate, 1.76 g of sodium citrate and 30 ml of distilled water; shaking for half an hour, adding 5ml of 1N sodium hydroxide, and adding distilled water to prepare 50 ml.
Polishing: and selecting a target area according to the semi-thin slice image prompt, repairing and polishing the embedding block again to leave the target area, and cutting an ultrathin slice with the thickness of 70-90nm by using a diamond cutter with the target area as the center to obtain a cell section of the target area, namely the transmission electron microscope sample.
Referring to fig. 1 and 2, fig. 1 is a schematic diagram of an epoxy resin embedded block of a cell sample Epon-812 obtained by a transmission electron microscope using the method of the present invention, which requires a significantly smaller number of cells than fig. 2, and the cell aggregates are easy to aggregate, not easy to disperse, and easy to observe under an electron microscope.
The preparation method of the transmission electron microscope sample with a small number of cells can avoid the phenomenon that the appearance of the cells is changed due to the influence of repeated centrifugation, can truly reflect the real appearance of substances in the cells, does not have too many requirements on the number of the cell samples, and can be applied to the transmission electron microscope observation of various cell samples or samples which are similar to cells and are isometric too small and easy to loosen in actual operation.
The above description is only an example of the present invention, and is not intended to limit the present invention, and it is obvious to those skilled in the art that various modifications and variations can be made in the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Claims (10)
1. A preparation method of a transmission electron microscope sample with a small number of cells is characterized by comprising the following steps: comprises the following steps:
s1: preparing cells, adding the cells to an EP tube,
s2: fixing a specimen: adding the cells collected in the S1 into precooled glutaraldehyde stationary liquid, centrifuging by using an EP tube, taking out supernatant, adding 1% agarose gel, picking up cell blocks at the bottom of the EP tube to wrap the cell blocks in the agarose gel, cooling to a fixed state, cutting and adding the glutaraldehyde stationary liquid again, washing for multiple times by using sodium dimethylkunmu buffer solution, and then washing by using pure water;
s3: dehydrating with ethanol step by step;
s4: respectively soaking cells by using a mixed solution of propylene oxide, propylene oxide and a pure Epon-812 solution and Epon-812 epoxy resin;
s5: gradually heating and polymerizing the cell sample in the S4 to form an embedding block;
s6: observing the embedded block in S5, polishing the cell dense block area and cutting into small resin blocks;
s7: and slicing the small resin blocks to form semi-thin slices, dyeing and positioning the cell area, selecting a target area according to the image prompt of the semi-thin slices, trimming and polishing the semi-thin slices, cutting the ultrathin slices with the thickness of 70-90nm by using a diamond cutter with the target area at the center after only the target area is left, and obtaining the cell section of the target area, namely the transmission electron microscope sample.
2. The method for preparing a transmission electron microscope sample with a small number of cells according to claim 1, wherein the method comprises the following steps: the S1 specifically includes the following steps: cells cultured in a culture dish or a culture flask were washed with PBS, and the washed cells were added to a 1.5ml EP tube, and after centrifugation, the cells were aggregated at the bottom of the tube.
3. The method for preparing a transmission electron microscope sample with a small number of cells according to claim 1, wherein the method comprises the following steps: in the S2, the cells in the S1 are added into 1ml of pre-cooled glutaraldehyde fixing solution, an EP tube is stabilized during the addition, and after the addition is completed, the cells are fixed for 2 hours or overnight at the temperature of 4 ℃.
4. The method for preparing a transmission electron microscope sample with a small number of cells according to claim 1, wherein the method comprises the following steps: and preparing the agarose gel in the S2 in advance, heating the agarose gel before adding the agarose gel, naturally cooling the agarose gel to 40-50 ℃, wherein the adding amount of the agarose gel is 100 ul, naturally cooling the agarose gel until the agarose gel is in a fixed state, and cutting the agarose gel to form a shape of 1: 1: 1, small square.
5. The method for preparing a transmission electron microscope sample with a small number of cells according to claim 1, wherein the method comprises the following steps: the washing with sodium dimethylkunmunoate buffer solution used in S2 was performed 6 times for 15min, followed by fixation with 1% osmic acid for 2h, and further washing with pure water for 6 times for 5min each, at 4 ℃.
6. The method for preparing a transmission electron microscope sample with a small number of cells according to claim 1, wherein the method comprises the following steps: the ethanol is dehydrated step by using 30 percent, 50 percent, 70 percent, 80 percent and 90 percent of ethanol for dehydration once respectively, and then dehydrated twice by using absolute ethanol.
7. The method for preparing a transmission electron microscope sample with a small number of cells according to claim 1, wherein the method comprises the following steps: the S4 specifically includes the following steps:
b1: displacement with propylene oxide twice;
b2: propylene oxide and pure Epon-812 were used as 2: 1, soaking the cells by the mixed solution;
b3: propylene oxide and pure Epon-812 were used as 1: 2, soaking the cells by the mixed solution according to the proportion;
b4: and (3) soaking the cells by using Epon-812 epoxy resin at the soaking temperature of 37 ℃ for 7h to obtain a cell sample.
8. The method for preparing a transmission electron microscope sample with a small number of cells according to claim 1, wherein the method comprises the following steps: the polymerization sequence of the stepwise heating in the S5 is as follows: firstly, putting the mixture into a 37 ℃ oven for 24 hours; then putting the mixture into a 45 ℃ oven for 24 hours; then putting the mixture into an oven at 60 ℃ for 24 hours; finally, the mixture is put into a 37 ℃ oven.
9. The method for preparing a transmission electron microscope sample with a small number of cells according to claim 1, wherein the method comprises the following steps: in the S7, the cell area is doubly stained by uranium acetate-lead citrate, and after the staining is finished, the cell area is rinsed by pure water and is irradiated and dried by an infrared lamp.
10. The method for preparing a transmission electron microscope sample with a small number of cells according to claim 9, wherein the method comprises the following steps: the uranium acetate is uranyl acetate, 25% saturated uranium acetate is used, the uranium acetate is prepared by 50-70% ethanol, and the formula of the lead citrate is 1.33 g of lead nitrate, 1.76 g of sodium citrate and 30 ml of distilled water; shaking for half an hour, adding 5ml of 1N sodium hydroxide, and adding distilled water to prepare 50 ml.
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