rapid freezing

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.