急速凍結

rapid freezing

SEMで生物、食品、塗料等の含水試料を観察するために、試料を急速に凍結して本来の状態に近い形態に保持する方法。
冷凍庫やドライアイスで緩慢に凍結させると水分の流動や体積膨張、氷晶形成により試料が変形する恐れがある。急速に凍結すると、それらを小さく抑えることができ試料が持っている本来の微細形態が保持される。凍結の速度は、大気圧下では10⁴°C/sec以上が必要であり、この条件を満たす浸漬法と金属圧着法が広く用いられている。
浸漬法とは沸騰が起きにくい冷媒に試料を投入する方法である。冷媒としては融点と沸点の差が大きい液体エタンや液体プロパンがよく用いられるが、液体窒素中に固体窒素が分散したスラッシュ窒素も用いられる。試料本来の微細形態が保持される深度は表面から5～10 µmほどと言われている。普通の液体窒素に試料を投入すると、試料周囲に窒素ガス層が形成され、液体窒素が試料に接触し難いため冷却効率が落ち試料が変形する場合がある。しかし、含水率が低く変形しづらい試料や試料変形の度合いと解像度とのバランスで低倍率での観察には、液体窒素が用いられることがある。
金属圧着法は液体窒素や液体ヘリウムで冷却された金属に試料を圧着する方法である。試料本来の微細形態が保持される深度は表面から20 µmほどと言われている。金属は熱伝導率の良い純銅がよく用いられる。浸漬法や高圧凍結法と比較し、広い面の凍結を行うことができる。
凍結した試料は、クライオSEM法を用いて観察するか、凍結置換および凍結乾燥を行った後に常温のSEMで観察する。

Rapid freezing is one of physical fixation methods to rapidly freeze water-containing specimens such as living organisms, foods and paints, to keep them close to their original form for scanning electron microscope (SEM) observation.
If a specimen is slowly frozen in a refrigerator or with dry ice, the specimen may deform due to water flow from the specimen, volume expansion of the specimen or ice-crystal formation in the specimen. If the specimen is rapidly frozen, the phenomena are greatly suppressed and the original fine structure of the specimen is preserved. The freezing rate is needed to be 10⁴°C/sec or better under atmospheric pressure. To achieve this fast freezing rate, the immersion method and the metal mirror freezing (slam freezing) method are widely used.
In the immersion method, the specimen is placed in a coolant which does not cause boiling. As a coolant, liquid ethane or liquid propane which has a large difference between the melting point and boiling point, is widely used, but slush nitrogen, which is solid nitrogen dispersed in liquid nitrogen, is also used. The freezing depth to which the original fine structure of the specimen can be preserved is said to be about 5-10 µm below the specimen surface. When the specimen is placed in ordinary liquid nitrogen, a layer of nitrogen gas is formed around the specimen, making it difficult for the liquid nitrogen to contact with the specimen, thus reducing the cooling efficiency and possibly deforming the sample. However, liquid nitrogen can be used as a coolant for specimens with low water content, which are hard to deform, or for specimens with low-magnification observations to balance the degree of specimen deformation with image resolution.
In the metal mirror freezing method, the specimen is punched against a metal block cooled by liquid nitrogen or liquid helium. The freezing depth to which the original fine structure of the specimen can be preserved is said to be about 20 µm below the specimen surface. A metal suitable for this method is pure copper which has a high thermal conductivity. Compared with the immersion method and the high pressure freezing method, metal mirror freezing enables freezing over a large area.
The frozen specimen is observed with a cryo-SEM while keeping its frozen state, or observed at room temperature with an ordinary SEM after freeze substation and freeze drying.