Critical drying technique

　　Sem samples are observed in a high vacuum. Whether it is water or dehydrated solution, it will produce violent vaporization in high vacuum, which will not only affect the vacuum degree, pollute the sample, but also destroy the fine structure of the sample. Therefore, the sample must be dried before being observed with electron microscopy。

　　Three drying methods are mainly introduced: air drying method, critical point drying method, freeze drying ；

　　Advantages and disadvantages of drying methods

　　Air drying advantages: easy to operate, save time

　　Disadvantages: Due to the effect of surface tension when the dehydrating agent volatilizes, the tissue block will shrink and deform

　　The advantages of critical point drying are: the fine structure of the sample can be preserved better; The operation is convenient, fast 2 ~ 3 hours can be completed, commonly used

　　Disadvantages: Need special equipment - critical point dryer。

　　Advantages of direct freeze-drying of water-containing samples: no dehydration, no sample shrinkage, the earlier used method

　　Disadvantages: It takes a long time, consumes more liquid nitrogen, is easy to produce ice crystal damage, and is not widely used。

　　Advantages of freeze-drying after dehydration: ice crystal damage can be reduced, and the drying time is short

　　Disadvantages: The organic solvent has the extraction effect on the sample composition, resulting in the loss of some inclusions。

　　First, air drying method

　　Basic description: The dehydrated sample is exposed to the air to gradually evaporate the dehydrating agent and dry, or it can be dried directly (without dehydration) in the dryer。

　　common method

　　Method 1: Single or double fixation → dehydration → Sample placed in 100% dehydrating agent → natural drying in air；

　　Method 2: Fixation → (tissue conduction treatment) → immersion in buffer and ether 1:1 for 5min→ air drying；

　　Method 3: Single or double fixation → ethanol dehydration → immersion in HMDS for 15min twice each time → ventilation and kitchen drying for 1h

　　Second, critical point drying method(CPD: critical point dryer)

　　1.Basic meaning: The critical point drying method is to use the material in a critical state, its surface tension is equal to zero characteristics, so that the liquid of the sample is completely vaporized, and discharged in a gas way, to achieve the purpose of complete drying。

　　2.Critical point: The general term for the temperature and pressure at which the gaseous and liquid phases of a substance reach the same density and become a uniform fluid. The temperature at this time is called the critical point temperature, and the pressure is called the critical point pressure。

　　3.Intermediate: A liquid used to replace a dehydrating agent and then replaced by a desiccant。

　　4.Commonly used desiccant

　　co2，F-13，F-23，F-116，N2O

　　5.Common intermediate：

　　Intermediate: A liquid used to replace a dehydrating agent and then replaced by a desiccant。

　　Reagents: acetic acid (isoamyl), acetone

　　6.Drying process: fixed dehydration → transfer to intermediate solution → transfer to sample chamber → replacement of isoamyl acetate with liquid CO2 →CO2 vaporization exhaust → drying

　　7.Reasons for critical point drying failure (incomplete)：

　　(1) Incomplete dehydration

　　(2) Isoamyl acetate reflux

　　(3) Excessive isoamyl acetate was found on the surface

　　(4) Too many samples

　　(5) The temperature of the sample chamber is high, and it is difficult to inject CO2

　　(6) There is a leak in the sample chamber

　　Three. Freeze drying

　　1. Basic description: The process of placing the frozen sample in a high vacuum and removing the water or dehydrating agent in the sample by sublimation。

　　2.The routine steps of direct freeze-drying of aqueous samples are: sampling → fixation → freeze protection → rapid freezing → sublimation (10-2 torR) drying → platform plating

　　3.Sample freeze-drying after dehydration Basic description: The sample is dehydrated with ethanol or acetone and transitioned to certain volatile organic solvents, which are then frozen together with these solvents and sublimed in a vacuum to achieve drying

　　supplementary materials：

　　（1）For small biological samples such as microorganisms, if you want to operate easily, you can choose natural drying method or dry drying method; If you want to obtain better results, it is recommended to use the critical point drying method or vacuum drying method。

　　（2）In general, plant tissues with less water content and thicker cell walls and waxy layers are more likely to use natural drying, while young, more water-containing tissues need to choose other methods. Critical point drying and tert-butanol vacuum drying are suitable for almost all types of plant samples, but due to the complexity of the operation, they are generally not used when simpler drying methods are available。

　　（3）Critical point drying is the preferred drying method for animal samples. Considering the simplicity of operation, natural drying method can also be used for animal samples with low water content and relatively hard observation site, but critical point drying or tert-butanol vacuum drying method is recommended for samples with high water content and relatively soft。