CN110907252A - Biological sample preparation device and sample preparation method thereof - Google Patents
Biological sample preparation device and sample preparation method thereof Download PDFInfo
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- CN110907252A CN110907252A CN201911296614.8A CN201911296614A CN110907252A CN 110907252 A CN110907252 A CN 110907252A CN 201911296614 A CN201911296614 A CN 201911296614A CN 110907252 A CN110907252 A CN 110907252A
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- 239000012472 biological sample Substances 0.000 title claims abstract description 65
- 238000002360 preparation method Methods 0.000 title claims abstract description 38
- 238000005464 sample preparation method Methods 0.000 title abstract description 7
- 239000000523 sample Substances 0.000 claims abstract description 215
- 239000007788 liquid Substances 0.000 claims abstract description 52
- 239000011521 glass Substances 0.000 claims abstract description 32
- 238000003825 pressing Methods 0.000 claims abstract description 29
- 238000000034 method Methods 0.000 claims abstract description 8
- 230000004083 survival effect Effects 0.000 claims description 29
- 238000003384 imaging method Methods 0.000 claims description 16
- 229910001069 Ti alloy Inorganic materials 0.000 claims description 3
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 claims description 3
- 239000010935 stainless steel Substances 0.000 claims description 3
- 229910001220 stainless steel Inorganic materials 0.000 claims description 3
- 229910052719 titanium Inorganic materials 0.000 claims description 3
- 239000010936 titanium Substances 0.000 claims description 3
- 241000244206 Nematoda Species 0.000 description 22
- 239000002609 medium Substances 0.000 description 22
- 239000000499 gel Substances 0.000 description 7
- 230000003287 optical effect Effects 0.000 description 6
- 230000007613 environmental effect Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 239000011543 agarose gel Substances 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 230000003444 anaesthetic effect Effects 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000006059 cover glass Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 241000252212 Danio rerio Species 0.000 description 2
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
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- 238000013461 design Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
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- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 239000004812 Fluorinated ethylene propylene Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004061 bleaching Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- HQQADJVZYDDRJT-UHFFFAOYSA-N ethene;prop-1-ene Chemical group C=C.CC=C HQQADJVZYDDRJT-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000003193 general anesthetic agent Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
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- 238000007254 oxidation reaction Methods 0.000 description 1
- 229920009441 perflouroethylene propylene Polymers 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
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- 230000000087 stabilizing effect Effects 0.000 description 1
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- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- 238000012546 transfer Methods 0.000 description 1
- 238000004506 ultrasonic cleaning Methods 0.000 description 1
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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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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Sampling And Sample Adjustment (AREA)
- Microscoopes, Condenser (AREA)
Abstract
The invention provides a biological sample preparation device, which comprises a sample groove, a glass slide, a transparent film and a sample pressing ring, wherein the sample groove can be placed in a liquid environment; the bottom of the sample tank is designed to be hollow and transparent, and the transparent film isolates the biological sample from the liquid environment. The invention also provides a method for preparing samples by using the biological sample preparation device. The biological sample preparation device and the sample preparation method thereof provided by the invention can isolate a biological sample from a liquid environment, can be widely applied to an upright microscope, an inverted microscope or an upright and inverted combined microscope, and can image the sample for a long time.
Description
Technical Field
The invention relates to the technical field of sample preparation devices, in particular to a biological sample preparation device and a sample preparation method thereof.
Background
The upright microscope is widely applied due to the advantages of short optical path, easy finding of key areas, better imaging effect and the like, and the upright microscope with high numerical aperture usually needs to use a liquid medium for imaging. For example, the optical sheet type positive microscope is widely used in real-time high-spatial-temporal-resolution imaging research of small-sized model organisms due to its fast and low-bleaching characteristics, but most of the optical sheet type positive microscopes use a plurality of water immersion objectives for imaging, and a sample needs to be immersed in a liquid environment for imaging. Referring to fig. 1, a biological sample remains viable in a liquid environment when viewed with a high numerical aperture upright microscope whose objective lens is immersed in the liquid environment to image the biological sample.
However, since many biological samples cannot be imaged under liquid conditions, the application range of the upright microscope is greatly limited. In the case of nematodes, which are about 1mm long and are characterized by semitransparent body, and the nematode can not acquire images due to violent body vibration in a liquid environment, a biological sample is usually fixed in gel to isolate liquid and then imaged by an upright microscope at present, but the dissolution temperature of the gel is high and reaches the stress temperature of many samples such as nematodes, so that the existing high-resolution upright microscope cannot be applied to many samples. At present, an inverted microscope, such as a laser confocal microscope, is used for imaging nematodes, and the laser confocal microscope cannot meet the research requirements in terms of time resolution and spatial resolution.
If the nematode can be isolated from the liquid environment and the refractive index from the liquid to the nematode is not changed, the nematode can be collected more quickly and deeply at the subcellular level by using an upright microscope, and the application range of the upright microscope such as a light sheet type upright microscope is expanded.
Disclosure of Invention
The invention aims to provide a biological sample preparation device and a biological sample preparation method, which can isolate a biological sample from a liquid environment.
In order to solve the technical problems, the invention provides a biological sample preparation device, which comprises a sample groove capable of being placed in a liquid environment, a glass slide arranged in the sample groove and used for bearing a biological sample, a transparent film which covers the sample groove and has the same refractive index as the liquid environment, and a sample pressing ring which is fastened on the sample groove and fixes the transparent film on the sample groove; the bottom of the sample tank is designed to be hollow and transparent, and the transparent film isolates the biological sample from the liquid environment.
Further, a layer of light-permeable sample survival medium is arranged between the glass slide and the biological sample, the thickness of the sample survival medium is equal to the distance from the upper surface of the glass slide to the top end of the sample groove, and the transparent film covering the sample groove can fix the biological sample on the sample survival medium.
Further, the sample pressing ring is fixed in the sample groove, a slide glass in the sample groove is fixed in the sample groove, and the biological sample on the slide glass is immersed in the liquid environment.
Further, the thickness of the film is 0.025 mm-0.5 mm.
Further, the sample groove is circular, square or other shapes, and the shape and the size of the slide are matched with those of the sample groove so as to ensure that the sample groove can hold the slide.
Further, the sample groove is an arm sample groove or a non-arm sample groove.
Furthermore, the sample tank is made of pure titanium, titanium alloy or stainless steel.
The invention also provides a method for preparing samples by using the biological sample preparation device, which comprises the following steps:
placing the slide in a sample tank;
placing the sample survival medium over the slide such that a sample survival medium plane is formed within the sample trench;
placing the sample on a sample survival medium plane;
covering a transparent film on the sample groove, and pressing the sample on a sample survival medium plane in the sample groove;
fastening the sample pressing ring on the sample groove to fix the transparent film on the sample groove;
and placing the sample groove on an upright microscope for observation and imaging.
The invention provides a biological sample preparation device and a sample preparation method thereof, wherein a biological sample is sealed in a sample groove by using a film with the refractive index same as that of environmental liquid, so that the biological sample is isolated from the external liquid environment, the observation imaging of the biological sample by an optical sheet type normal microscope is not influenced, the biological sample is not polluted and deformed, the biological sample can be stored for a long time and repeatedly used for many times, and meanwhile, the phase difference caused by the change of the refractive index is avoided. In addition, the biological sample preparation device provided by the invention has the advantages that the biological samples are placed on the glass slide in the sample groove, a large number of biological samples can be placed at the same time, high-throughput screening can be rapidly carried out, and the extraction efficiency is improved. In addition, the sample tank material selection and the geometric dimension are designed aiming at microscopic observation, so that the sample tank can be suitable for observation of most optical microscopes, the application range of a sample preparation device is expanded, the application range of an observed and imaged biological sample is expanded, and the study of the nematode on the subcellular level is promoted if the nematode is observed by an upright microscope such as a slide and the like.
Drawings
FIG. 1 is a schematic diagram of a prior art configuration for imaging a biological sample immersed in a liquid environment with an objective lens of an upright microscope;
FIG. 2 is a schematic structural diagram of a biological sample preparation device according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of a sample cell with an arm used in a biosample apparatus according to an embodiment of the present invention;
fig. 4 is a schematic structural diagram of a conventional biological sample preparation device in a liquid environment according to another embodiment of the present invention.
Detailed Description
Referring to fig. 2, the biological sample preparation device according to the embodiment of the present invention includes a sample tank 1 capable of being placed in a liquid environment, a slide 2 placed in the sample tank 1 for carrying a biological sample, a transparent film 3 covering the sample tank 1 and having the same refractive index as the liquid environment, and a sample pressing ring 4 fastened to the sample tank 1 for fixing the transparent film 3 on the sample tank 1. Wherein, 1 bottom in sample groove is the fretwork design, can bear the cover glass, and consequently light can follow 1 bottom fretwork part in sample groove and see through to just put microscope's observation, the part of not fretwork in 1 bottom in sample groove is used for bearing slide 2. The thickness of the transparent film 3 is 0.025 mm-0.5 mm, and the size of the inner diameter of the sample pressing ring 4 is slightly smaller than the sum of the outer diameter of the sample groove 1 and 2 times of the film thickness. And, the sample cell 1 lateral wall is trapezoidal design, like this after pressing appearance ring 4 to detain on sample cell 1, can play anti-skidding fixed action along with pressing the process frictional force increase of appearance ring 4 down to can guarantee to press appearance ring 4 tightly to press film 3 on sample cell 1. Thus, when the sample tank 1 is placed in a liquid environment and observed by an upright microscope, the transparent film 3 can isolate the biological sample in the sample tank 1 from the external liquid environment and can prevent environmental liquid from permeating into the sample tank 1. In order not to change the refractive index of the objective lens to the sample, the refractive index of the film 3 should be the same as that of the environmental liquid, for example, a fluorinated ethylene propylene film or other film with the same refractive index as the environmental liquid can be selected according to the environmental liquid. Of course, the sample preparation device can isolate the sample from the liquid environment, so that the upright microscope can be used for observation imaging, and the inverted microscope and the microscope combining the upright and inverted microscope can also be used for observation imaging.
The size and shape of the sample groove 1 can be selected according to specific situations so as to meet the requirements of different devices, and the shape of the sample groove 1 can be specifically selected from a circle, a square or other shapes. The shape and the size of the slide glass 2 are matched with the shape and the size of the sample groove 1, so that the sample groove 1 can be used for bearing the slide glass 2. The sample tank 1 is made of pure titanium, titanium alloy or stainless steel, and the sample tank 1 made of the materials is not easy to deform, has good chemical stability and high and low temperature tolerance, and can be recycled. The assembled sample tank 1 can be disassembled and cleaned as a whole, and can also be placed in an ultraviolet irradiation or strong acid environment for cleaning. After cleaning and airing, the sample tank 1 can be stably stored at room temperature, and is not easy to generate oxidation, rusting and other phenomena.
Wherein, the sample cell 1 is a sample cell without an arm, referring to fig. 3, the sample cell 1 can also be made into a sample cell with an arm, the sample arm 6 is fixedly connected with the sample cell 1 through a bolt, and the tail end of the sample arm 6 can be fixed on a sample operation platform through a screw, so that the biological sample in the sample cell 1 can be observed and imaged only by fixing the sample arm 6 in the operation process. Because the size of the sample groove 1 can be designed into various specifications according to the size of the glass slide 2, and the sample groove 1 is made of materials which are not easy to deform and have good chemical stability and high and low temperature tolerance, and is designed to be convenient for microscope observation, the sample prepared by the sample preparation device provided by the invention can be used for observation of most optical microscopes, and can be directly used by users without related experience.
In order to ensure the activity of the biological sample during the test, it is usually possible to place a layer of sample survival medium 5 on the slide 2 and then place the biological sample on the sample survival medium 5, which facilitates the long-term observation of the biological sample. The thickness of the sample survival medium 5 should be equal to the distance from the upper surface of the slide 2 to the top end of the sample groove 1, after the transparent film 3 is covered on the sample groove 1 and fastened on the sample groove 1 by the sample pressing ring 4, the sample groove 1 is immersed in a liquid environment, the biological sample is isolated from the liquid environment in the sample groove 1, and the film 3 provides downward pressure to the biological sample under the action of the sample pressing ring 4, so that the biological sample on the sample survival medium 5 can be fixed, and the biological sample is ensured to be in a stable state. As a specific embodiment of the present invention, if the biological sample is a nematode, a zebrafish or a drosophila, etc., the sample survival medium 5 is a solid agarose gel capable of ensuring the activity of the nematode, the zebrafish or the drosophila. Of course, if other biological samples are selected, the sample survival medium 5 is selected from other media that ensure the viability of the biological sample.
The invention provides a sample preparation method of a biological sample preparation device, which comprises the following steps:
placing the glass slide 2 in the sample groove 1;
placing the sample survival medium 5 over the slide 2 such that a sample survival medium plane is formed within the sample trench 1;
placing the sample on a sample survival medium plane;
covering the sample groove 1 with a transparent film 3, and pressing the sample on the sample survival medium plane in the sample groove 1;
fastening the sample pressing ring 4 on the sample groove 1 to fix the transparent film 3 on the sample groove 1;
thus, the sample cell 1 is placed in a liquid environment and observed and imaged on an upright microscope.
The following description specifically describes the use method of the biological sample preparation device provided by the embodiment of the present invention by taking the preparation of nematode samples as an example.
1. Required material
A sample tank 1; a slide 2; an equal water refractive index film 3; a nematode sample; a sample pressing ring 4; sample survival medium 5, agarose gel; sterilizing deionized water; anesthetic agents, and the like.
2. Preparation of the experiment
1) Adjusting the temperature to 20 ℃, and starting the equipment for precooling;
2) ultrasonically cleaning the glass slide 2;
3) agarose gel was prepared at a ratio of 3% (W/V):
3. in the sample preparation process, as the body of the nematode has violent swing or can not survive in the liquid environment, a device for isolating the sample from the liquid environment is needed when the nematode sample is prepared.
1) Embedding the slide 2 in the sample tank 1;
2) dripping liquid agarose gel 5 above the glass slide 2 in the sample groove 1;
3) covering the gel with a cover glass, flattening, and extruding out excessive gel;
4) after molding, the cover glass is taken out, a gel plane is formed above the sample groove 1, and redundant solid gel outside the sample groove is removed;
5) sucking and dripping 1-3 mu L of nematode anesthetic on a gel plane by using a liquid transfer device, and then picking nematodes in different development stages into the anesthetic;
6) before the anesthetic is dried, the nematodes are placed in a required direction, and then the nematode is covered with a transparent film 3 with equal water refractive index;
7) the sample pressing ring 4 is buckled on the film and pressed downwards, the film 3 is arranged between the sample pressing ring and the sample groove, the friction force ensures that the sample pressing ring is not easy to fall off, meanwhile, the film 3 presses the nematodes on the sample survival medium 5 in the sample groove 1 to ensure the survival of the sample, and meanwhile, the pressure on the nematodes further limits the swinging of the nematodes;
8) the model is loaded, can be immersed in a liquid environment and isolated from the liquid, and the refractive index from the objective lens to the sample is not changed, and then observation imaging is carried out by using an upright microscope.
The device in the specific assembling process comprises the following steps from top to bottom in sequence: a sample pressing ring 4, an equal water refractive index film 3, a biological sample, a sample survival medium 5, a glass slide 2 and a sample groove 1. The whole device isolates the liquid environment, and the transparent film 3 on the surface of the sample provides downward pressure, thereby providing double guarantee for stabilizing the sample. It is worth mentioning that, because the slide 2 and the sample survival medium 5 at the bottom of the sample are both transparent, the sample can be imaged from the bottom by using an inverted microscope at the same time, and the sample preparation device provided by the invention can realize multifunctional application.
Of course, as another embodiment, the present invention provides a biological sample preparation device, which can also be used for conventional sample preparation in a liquid environment. Referring to fig. 4, for biological samples whose bodies do not swing violently in a liquid environment and which cannot be used for image acquisition, the biological samples can be attached to the glass slide 2, then the glass slide 2 attached with the biological samples is placed at the bottom of the sample groove 1, the sample pressing ring 4 with the outer diameter slightly smaller than the inner diameter of the sample groove 1 is used for fixing the sample pressing ring in the sample groove 1, and the glass slide 2 at the bottom of the sample groove 1 is fixed. Finally, the sample tank 1 is placed in a liquid environment, and an upright microscope is used for observation and imaging. The sample tank 1 placed in the liquid environment is sequentially from bottom to top: sample tank 1-slide 2-sample-press ring 4.
The following provides a detailed description of a method for using a biological sample preparation device according to another embodiment of the present invention, taking cell sample preparation as an example. The cells need to keep alive in the liquid environment, so that a device for isolating the sample from the liquid environment is not needed during cell sampling, and the cells can be directly immersed in the liquid environment for observation.
1. Required material
The present sample preparation device; a sample tank 1; a slide 2; a cell culture medium; a sample pressing ring 4; cells and cell culture conditions;
2. preparation of the experiment
1) The equipment is started and preheated to 37 ℃, and the carbon dioxide of the living cell workstation is set to be 5%;
2) ultrasonic cleaning glass slide 2
3. Sample preparation:
1) spreading the cells on a glass slide 2, and placing the glass slide in a 5% carbon dioxide incubator at 37 ℃ overnight; performing plasmid transfection operation on the cells the next day;
2) 16 hours after transfection, slide 2 was removed and placed in sample well 1 of the sample preparation device.
3) Placing a sample pressing ring 4 with the outer diameter similar to the inner diameter of the sample groove 1 on the glass slide 2 in the sample groove 1 and pressing the glass slide 2 tightly, and keeping the stability of the sample pressing ring 4 by depending on the friction force between the sample pressing ring 4 and the inner wall of the sample groove 1;
4) the sample tank 1 is filled with culture medium, so that the activity of the cell sample can be maintained;
5) the bottom of the glass slide 2 is dried and placed on an inverted microscope device for imaging.
4. Storage and use
After the sample preparation device is used, the sample preparation device is wiped clean by alcohol, and the sample preparation device is dried and stored at room temperature. And can be recycled indefinitely.
The biological sample preparation device provided by the invention expands the application range of an upright microscope such as a light sheet microscope, and also develops the use of a non-liquid environment imaging sample under the upright microscope. Meanwhile, the device is simple and convenient to operate and can be recycled.
Finally, it should be noted that the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to examples, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (8)
1. A biological sample preparation device is characterized in that: the sample holder comprises a sample groove (1) which can be placed in a liquid environment, a glass slide (2) which is arranged in the sample groove (1) and used for bearing a biological sample, a transparent film (3) which covers the sample groove (1) and has the same refractive index as the liquid environment, and a sample pressing ring (4) which is fastened on the sample groove (1) and used for fixing the transparent film (3) on the sample groove (1); the bottom of the sample tank (1) is designed to be hollow and transparent, and the transparent film (3) isolates a biological sample from the liquid environment.
2. The biosample apparatus of claim 1, wherein: a layer of light-transmitting sample survival medium (5) is arranged between the glass slide (2) and the biological sample, the thickness of the sample survival medium (5) is equal to the distance from the upper surface of the glass slide (2) to the top end of the sample groove (1), and the biological sample on the sample survival medium (5) can be fixed by the transparent film (3) covered on the sample groove (1).
3. The biosample apparatus of claim 1, wherein: the sample pressing ring (4) is fixed in the sample groove (1), a glass slide (2) in the sample groove (1) is fixed in the sample groove (1), and a biological sample on the glass slide (2) is immersed in the liquid environment.
4. The biosample apparatus of claim 1, wherein: the thickness of the film is 0.025 mm-0.5 mm.
5. The biosample apparatus of claim 1, wherein: the sample groove (1) is round, square or other shapes, and the shape and size of the glass slide (2) are matched with those of the sample groove (1) so as to ensure that the sample groove (1) can hold the glass slide (2).
6. The biosample apparatus of claim 1, wherein: the sample groove (1) is a sample groove with an arm or a sample groove without an arm.
7. The biosample apparatus of claim 1, wherein: the sample tank (1) is made of pure titanium, titanium alloy or stainless steel.
8. The method for preparing a biological sample preparation device according to any one of claims 1 to 7, comprising the steps of:
placing the glass slide (2) in the sample groove (1);
arranging a sample survival medium (5) above the glass slide (2) so that a sample survival medium plane is formed in the sample groove (1);
placing the sample on a sample survival medium plane;
covering a transparent film (3) on the sample groove (1), and pressing the sample on the sample survival medium plane in the sample groove (1);
fastening the sample pressing ring (4) on the sample groove (1) to fix the transparent film (3) on the sample groove (1);
and (3) placing the sample groove (1) on an upright microscope for observation and imaging.
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