Ultramicrotomy is a specialized technique to provide thin specimens for transmission electron microscopy observation, which is the most commonly used technique to study the ultrastructure of cells and tissues in biology, and is also a key technique for other electron microscopy techniques in biology, such as electron microscopy radioautography, electron microscopy enzyme cytochemistry, and immunoelectron microscopy. Almost all of the current knowledge about the ultrastructure of various cells and tissues of living organisms is provided by this technique. Using this technique to observe and study virus specimens, it is possible to see both the internal microstructure of the virus and the relationship between the virus and the host cell, but the study of the ultramicro-morphological changes induced by the host cell, as well as the mechanisms of virus adsorption, entry into the host cell, and proliferation and replication within the host cell, require that ultrathin slices of the host animal's tissues or cultured cells be made before they can be observed with transmission electron microscopy The thickness of the sections is 10-100 nm. Sections with a thickness of 10-100 nm are called ultrathin sections. The technique of making such sections is called ultrathin section technique, which includes a series of extremely complicated processes such as material extraction, fixation, rinsing, dehydration, immersion, embedding and polymerization, sectioning, and staining, etc. Compared with paraffin sections for light microscopy, the operation requirements are even more stringent. Among them, sampling, fixation and staining are important aspects of this technique. Source: Virology Testing
Operation method
basic program Move 1. sampling requirements for general animal tissue sampling are as follows: (1) the specimen material should be fresh, the speed of sampling should be fast: due to biological tissues after leaving the body, the cells will immediately release a variety of hydrolytic enzymes and cause cell autolysis, so that its microstructure changes and produce false impressions, so try to keep the material as fresh as possible, after dissection, surgery or biopsy of the material is quickly put into the fixative, in order to try to maintain the structure of the cells in the living state. (2) Small sampling, sampling site to be accurate: due to the fixative used for electron microscopy specimen preparation of its penetration rate is extremely slow, while the area of ultra-thin section is very small, the general sampling is not more than 0. 5-1mm3, so the sampling must pay attention to the sampling site. If the sampling site is inappropriate, the expected results can not be obtained. (3) The fixative and equipment used must be pre-cooled to minimize the activity of hydrolytic enzymes in isolated cells and to reduce cell autolysis. Generally, the fixative is kept in a refrigerator at 4°C, and the scissors or scalpel blades and slides used for the removal of the material can be placed on ice and pre-cooled. (4) The knives and scissors for cutting tissues must be very sharp, and mechanical damage caused by pulling, tugging, sawing and pressing should be avoided during operation. If the specimen is an isolated culture of a virus, attention must be paid to its quantity and concentration. This culture can be taken after the plastic centrifuge tube centrifugation to take its precipitate, the amount of centrifugal precipitation must be the size of a grain of rice, such as the amount is too small, it will not be able to carry out the subsequent operation. Specific methods of sampling: according to the number of specimens taken to prepare clean vials (bottles must be rigorously washed), labeled or numbered on each vial, the vials are injected with 4 ℃ fixative 1-2 ml. In addition, clean carrier sheet and surgical blades and a small amount of ice are prepared, and the carrier sheet is placed on the ice. Immediately after the tissue is removed from the body, place it on the carrier sheet and put a few drops of 4℃ fixative on it, then use a sharp blade to cut the tissue into small pieces of 0.5~1 mm3 and immediately put it into the fixative and place it in the 4℃ refrigerator for fixation. 2. Fixation (1) Fixation methods: there are two kinds of fixation methods: physical and chemical. The former is the use of freezing, microwave irradiation, critical point drying and other means to preserve the cell structure; the latter is to use certain chemical reagents to fix the structure of the cell, these chemical reagents are called fixative, which can chemically combine with proteins to form cross-links to stabilize intracellular proteins, and can make the fat, sugar to maintain the life of the state and position of the fine morphology and structure of the preservation. Tissues are usually fixed mostly by chemical methods. Ideal fixative should have the following conditions: ① can quickly and uniformly penetrate into the cell, immediately kill the cell to minimize the "post-mortem changes; ② can stabilize the various structural components to ensure that in the subsequent processing will not be dissolved or lost; ③ can preserve the enzyme activity of the cell and antigenicity for cytochemistry or immunocytochemistry of the determination of the mother) does not affect the cell contraction or expansion to maintain a variety of structures in the state of life; ④ no artificial artifacts or deformation to ensure the authenticity of the electron microscope image. In order to achieve the above requirements, it is also necessary to pay attention to the pH value, osmotic pressure and electrolyte concentration of the fixation solution. 3. Staining Specific staining method: Since uranium and lead have different electron staining effects, the current sections are generally double staining method, i.e., first stained with axial staining, and then stained with lead staining solution. Specific operation steps: place a wax plate or melted paraffin-impregnated filter paper in a clean Petri dish, drop the uranyl acetate dye on the wax plate or impregnated filter paper, put the carrier net with the slices on the dye (the slices are in contact with the dye) and dye for 15~30 min, rinse the slices in double-distilled water, dry them on filter paper, and then put the carrier net on lead citrate dye (in order to prevent lead carbonate precipitation, put a few solid sodium hydroxide in the Petri dish). (in order to prevent the formation of lead carbonate precipitation, a few grains of solid sodium hydroxide can be put into the Petri dish), dye for 5~10 min, rinse the sections with double-distilled water, absorb the dryness, and put them into a clean Petri dish for spare time, and then examine them by electron microscopy. Caveat The correctness or otherwise of the sampling will be closely related to the preparation of the specimen can meet the observation requirements, is a key link in the ultra-thin sectioning technology.The purpose of fixation: ① to maintain the structure of the cells in the living state; ② in the fixed after rinsing, dehydration and embedding, to maintain the cellular components will not be lost and dissolved, and make the organization of the appropriate hardening, so that the organization of the various substances within the cells of the chemical reaction to change the minimum; ③ for the specimen for the subsequent treatment process (including staining and subjected to electron-beam bombardment) in order to prepare.Several commonly used fixatives: 1) Osmium acid (Osmium tetroxide, OsO4Osmium tetroxide (OsO 4) is a light yellow lumpy or needle-like crystal with a melting point of 40-41°C and a molecular weight of 254. 2. It is soluble in water, alcohol, ether and chloroform. Its vapor is strongly irritating and toxic to the mucous membranes of the eyes, nose and throat, so it should be operated in a fume hood for protection. Osmium acid is a strong oxidizing agent, has great affinity for nitrogen, can form cross-links with proteins, stabilize various structural components of proteins without precipitation. It has a good protective effect on lipids, can be combined with unsaturated fatty acid chains to form complexes, is the only fixative that can fix lipids. In addition, the high density of metal starvation and the combination of fixed tissue components, by the electron beam irradiation can scatter a large number of electrons, so that the image contrast increases, playing the role of "electron staining". Osmium fixation can avoid the contraction or expansion of the tissue block, so that the tissue block soft and hard moderate, conducive to the production of ultra-thin sections. The main disadvantage of osmium acid is that the molecules are large, and the penetration rate of the tissue is slow (0.1~0.3mm/h), which makes it easy to produce uneven fixation, so the volume of the tissue block should not be more than 1mm.3Therefore, the volume of tissue block should not exceed 1mm 3. Osmium acid is not effective in fixation of carbohydrates, causing more loss of enzyme activity and destroying antigenicity, so it is not suitable for enzyme cytochemistry and immunocytochemistry studies. The concentration of osmiic acid fixative is 1%, and the fixation time is 1~4h, such as fixing monolayer culture cells for 15~30 min.2) Glutaraldehyde (C2) Glutaraldehyde (C5)H8C 5 H 82): molecular weight 100. 12. Boiling point 73~75°C, absorption spectrum 280nm. Commercially available glutaraldehyde is a 25% or 50% aqueous solution with a pH of 4.0~5.0. Most of the refined glutaraldehyde is used for electron microscopy, the glutaraldehyde stored for a long time can lose aldehyde group due to polymerization at high temperature, oxygen, neutral or alkaline pH, and the fixation effectiveness is obviously reduced, so the original solution of glutaraldehyde should be kept in a low-temperature place. The prepared glutaraldehyde should be stored at 4℃. Freshly prepared glutaraldehyde fixative should be used as much as possible. Glutaraldehyde has strong permeability to tissues, fast fixation speed (0.4mm/h) and active affinity to intracellular structures, especially to some volatile intracellular structures, such as microtubules, mitotic spindle filaments, and cellular matrix has a better fixation effect, and it reacts with proteins and amino acids by cross-linking with tissues and proteins in solution, which stabilizes the cellular components. It preserves glycogen, immobilizes nuclear proteins, preserves enzyme activity and is suitable for cytochemical studies. Another advantage of glutaraldehyde is that glutaraldehyde-fixed tissue blocks can be stored in the fixative for a longer period of time (weeks or even 1~2 months) without any ultrastructural changes, which is especially suitable for preservation of materials taken from remote clinical, laboratory or field sites. However, glutaraldehyde is also not an ideal fixative, it can not preserve fat, no electronic staining effect, can not increase image contrast, and the osmotic pressure of the buffer requirements are high. At present, most of the fixation method uses glutaraldehyde and osmium acid, glutaraldehyde for pre-fixation, osmium acid for post-fixation, complement each other's strengths and weaknesses, the fixation effect is better. The tissues fixed by glutaraldehyde must be rinsed repeatedly with buffer, otherwise the residual glutaraldehyde will affect the osmium penetration of osmium fixation. The concentration of glutaraldehyde fixative was 1%~5%, and the fixation time was 0.5~2 h. At present, the commonly used ultrathin fixative for electron microscopy is osmium.Currently, the commonly used stains for ultrathin sections in electron microscopy are uranium and lead salts. Uranium can be combined with most of the cellular components, especially easy to combine with nucleic acids, and the staining is more detailed, true, and not easy to appear precipitation particles, but uranium is radioactive, the use of special attention. Lead has affinity for various structures of cells and tissues, easy to combine with proteins, especially for glycogen that can not be stained by osmium tetroxide has a staining effect. However, lead staining is more troublesome, and lead is easy to combine with airborne CO2Combination of lead and air CO2 to form insoluble lead carbonate precipitation, contamination of the section. Therefore, when staining, we should try to avoid staining with airborne CO2contact with airborne CO2. Generally, some NaOH is placed in a covered staining dish to minimize the contact of lead with airborne CO2. For more product details, please visit Aladdin Scientific website.