Sample preparation method of the hottest scanning

2022-10-16
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The sample preparation method of scanning electron microscope

the sample preparation of scanning electron microscope is simpler than that of transmission electron microscope, and there is no need to embed and slice. The preparation of scanning electron microscope samples must meet the following requirements: ① maintain intact tissue and cell morphology; ③ Fully expose the parts to be observed; ③ Good conductivity and high secondary electron yield; ④ Keep it fully dry

some biological materials with low water content and not easy to deform can be observed directly under low accelerating voltage without fixing and drying, such as animal hair, insects, plant seeds, pollen, etc., but the image quality is poor, and the observation and taking of photos must be as fast as possible. For most biological materials, chemical or physical methods should be used to fix, dehydrate and dry them first, and then spray carbon and metal to improve the conductivity and secondary electron yield of the materials

preparation of samples by chemical methods

the 2014 City financial key work promotion conference was held in Tianjin auditorium. The procedures for preparing samples by chemical methods are usually: cleaning, chemical fixation, drying, metal spraying

1. Cleaning: some biomaterials are often covered by foreign bodies such as blood, cell debris, food residues in the digestive tract, bacteria, lymph and mucus, which cover the parts to be observed. Therefore, it is necessary to clean the attachments with normal saline or isotonic buffer before fixation. These foreign matters can also be removed by washing with 5% sodium carbonate or enzyme digestion

2. Fixation: aldehydes (mainly glutaraldehyde and paraformaldehyde) and osmium tetroxide are usually used for double fixation, and osmium tetroxide can also be used for single fixation. Osmium tetroxide fixation can not only preserve the tissue and cell structure well, but also increase the conductivity and secondary electron yield of the material, and improve the quality of scanning electron microscopy images. This is very important for high-resolution scanning electron microscopy. 3. It is often important to keep equipment and computers clean. In order to enhance this effect, osmium tetroxide tannic acid or osmium tetroxide zhucha dichala and other materials can be repeatedly treated to combine them with more heavy metal osmium, which is conductive dyeing

3. Drying: critical point drying method is usually used after fixation. The principle is: properly select the temperature and pressure to make the liquid reach the critical state (the interface between liquid and gas disappears), so as to avoid the sample deformation caused by the surface tension of water in the drying process. The critical temperature and pressure of water should not be too high (37.4 ℃, 218 PA) when drying aqueous biomaterials directly at the critical point. Usually, ethanol or acetone is used to dehydrate the material, and then an intermediate medium, such as amyl acetate, is used to replace the dehydrating agent, and then the intermediate medium is replaced with liquid or solid carbon dioxide, freon 13 and nitrous oxide in the dryer, which still has a high impact strength at (4) 0 ℃ for critical drying

4. Spray metal: stick the dry sample to the metal sample table with a conductive adhesive or other adhesives, and then spray a layer of 50 ~ 300 angstrom thick metal film in the vacuum evaporator to improve the conductivity and secondary electron yield of the sample, improve the image quality, and prevent the sample from heating and radiation damage. If the ion sputtering coater is used to spray metal, a uniform thin metal coating with fine particles can be obtained, and the quality of scanning electronic image can be improved

preparation of samples by freezing method

low temperature scanning electron microscopy is a rapidly developing and widely used method in the 1980s. It includes freeze fixation, freeze drying, freeze cutting of biological samples and scanning electron microscopy of frozen water samples

1. Freeze fixation: put biological materials into cryogenic refrigerant, such as liquid helium, liquid nitrogen, liquid Freon and propane. Rapid freezing can make the structure and chemical composition of biological tissue cells close to the living state. Biological samples frozen and fixed can be transferred to a scanning electron microscope with a low-temperature sample stage under low temperature conditions for direct observation without further treatment or only in frozen sample 1 The surface of the dispensing experimental device is sprayed with a thin layer of metal. This method is not only fast and simple, but also can eliminate the illusion of shrinkage caused by drying method. It is especially suitable for the study of biomaterials with high water content

2. Freeze drying: after the biological sample is frozen and fixed, the water in it freezes into ice, and the surface tension disappears; Then put the frozen sample in a vacuum to gradually sublime the ice into steam. The dried sample obtained in this way avoids the morphological changes caused by surface tension to a certain extent

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