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nutsche filter dryer Original title: Critical point drying of scanning sample preparation method Share the latest scientific and technological information and release cutting-edge academic trends! Pay attention to the WeChat official account: mumuxilinj. More exciting content, news and information, and dry goods resources are waiting for you! Equipment and instruments, Shangmu Muxili! First, the principle The critical point drying method is a method for drying biological samples containing water, which uses the characteristic that the surface tension of liquid is eliminated in the critical state (the medium will be transformed into gas and liquid at a certain pressure and temperature), so that the liquid in the sample is gasified and finally completely dried. When the liquid is in a critical state, the phase interface is eliminated and the surface tension is equal to zero. Drying the sample under this condition can keep the structure of the sample, especially the surface structure, from deformation, which is an important method of biological sample preparation for scanning electron microscopy. Conditions of critical state: the liquid in a closed container is heated to a certain temperature (critical temperature) and critical pressure. Characteristics of critical state: the density of gas and liquid is equal, and the phase boundary disappears; the liquid becomes gas, and the surface tension of the liquid disappears to zero; no matter how much pressure is applied, the gas will not become liquid. II. CO2 critical point drying method Selection of critical drying fluid — — replacement fluid and intermediate fluid Replacement liquid: the liquid that plays the role of critical drying in the critical point dryer. Uch as liquid CO2. Intermediate liquid: The liquid used to replace the dehydrating agent and then replaced by the desiccant. Uch as acetone and isoamyl acetate. Water is a polar molecule with high surface tension. When dehydrating biological samples, the critical point of water (+ 374 ℃,cbd centrifugal extractor, 22 MPa) is inconvenient to operate and easy to damage the samples. The critical point of CO2 (+ 31 ℃, 7.4 MPa) is easy to reach and operate. However, CO2 is not easily mixed with water, so acetone/isoamyl acetate is usually used as an intermediate medium. The method has the advantages of avoiding the influence of surface tension and better preserving the fine structure of the sample. The operation is more convenient and quick. III. Sample preparation step 1. Draw materials & Fixed: Expand the full text 1) Use clean and sharp knives and scissors to cut tissues quickly, and avoid pulling,thin film distillation, sawing, pressing and other actions. 2) As soon as possible, put the tissue into 4 ℃ fixative (5% glutaraldehyde, 2.5% for samples without cell wall). If the sample floats on the fixative, it needs to be degassed (vacuumized) so that the sample is completely immersed in the fixative. 3) The length, width and height of the sample block shall not exceed 5mm. 4) The fixative was prepared with 0.1M phosphate buffer (PH = 7.2) to avoid cell atrophy or imbibition. The buffer concentration and PH can be adjusted according to the literature. Caution: 1) Fixation time 4-12h (12h with cell wall, 6h without cell wall). 2) The fixative can be dropped on the tissue or organ, and the sample can be obtained after half an hour, and then the sample can be soaked in the fixative to correct the size of the sample. The smaller the sample, the better. 3) When the sample is gathered, the pipette can be used to suck the fixative and resuspend the sample to ensure sufficient fixation. 2. Dehydration 1) Rinse and soak in 0.1M phosphate buffer solution (PH = 7.2) at room temperature for 3 times, nutsche filter dryer ,jacketed glass reactor, 20 min each time. 2) Absorb and discard the solution, add 30% ethanol, 4 ℃, 20 min. 3) Absorb and discard the solution, add 50% ethanol, 4 ℃, 20 min. 4) Absorb and discard the solution, add 70% ethanol, 4 ℃, 20 min. 5) Absorb and discard the solution, add 80% ethanol, 4 ℃, 20 min. 6) Absorb and discard the solution, add 95% ethanol, 4 ℃, 20 min. 7) Absorb and discard the solution, add 100% ethanol, 4 ℃, 20 min. 8) Absorb and discard the solution, replace it with 100% acetone (or isoamyl acetate), 4 ℃, twice, 20 min each time. If you need to stop the operation, please stay in step 4), and do not put the tissue in the high concentration alcohol solution for too long. Caution: 1) Try to prevent the sample from sticking to the wall or caking. Suck the fixing liquid back and forth by flicking the pipe wall or dropper to break up the sample. 2) Pay attention to fragile or very small samples, and replace the liquid quickly, otherwise an air film will be produced on the surface of the sample, affecting the effect of dehydration and drying. 3. Dry The sample is moved to the sample chamber to replace phosphoric acid acetone/isoamyl acetate with liquid CO2, and the CO2 is vaporized, exhausted and dried. Share the latest scientific and technological information and release cutting-edge academic trends! Pay attention to the WeChat official account: mumuxilinj. More exciting content, news and information, and dry goods resources are waiting for you! Equipment and instruments, Shangmu Muxili! 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