Cryo-Electron Microscopy
Plunge freezing
Rapidly plunging a specimen into a liquid cryogen is certainly the simplest means of achieving cryofixation. There is an intimate contact between the surface of the sample and the cryogen giving good thermal conductivity. Plunge freezing vitrifies samples of up to a 500 nm thick.
The temperature of liquid nitrogen at ambient pressures is very low (-196°C), and while it is cheap and readily available, its thermal conductivity is very poor making it unsuitable as a cryogen for freezing biological samples. It has a very low heat capacity, meaning only a small amount of energy needs to be transferred to liquid nitrogen to drastically raise its temperature. The boiling point of liquid nitrogen is about -196°C so as soon as something warmer comes in contact, it starts to boil. An insulating layer of nitrogen gas forms between the liquid and the specimen, preventing it from freezing quickly. This is called the Leidenfrost effect.
Odd as it sounds, under reduced pressure liquid nitrogen will undergo a phase change and become solid. So, when liquid nitrogen is put under low vacuum it will become a semi-solid slush, and its temperature is reduced to -210°C, well below the boiling point. Cooling rates can be improved using this slush as when a specimen is plunged into it, it envelops the specimen closely and extracts heat from it more efficiently without the Leidenfrost effect. This is often used for freezing of samples for low resolution cryo-SEM studies.
The most efficient cryogens are ethane and propane. Their boiling points are much higher, -89°C and -42°C respectively, so no insulating gas layer is formed between the liquid and the specimen, resulting in a very fast freezing rate. However, to liquify these it is necessary to cool them with liquid nitrogen. Ethane and propane are highly flammable and are even more so when condensed, so it is always important to take the right safety precautions when using them. Check with your local facility on safe handling procedures.
This method is only suitable for relatively thin samples as the freezing rate drops off with distance from the freezing liquid, leading to increased ice crystal growth and tissue damage with increasing tissue depth. It can be used for slightly larger samples if the region of interest lies at the surface. For cryo-SEM cooling rates often aren’t so critical especially if the study does not require high resolution.
Specific instruments have been designed to allow reproducible results with the plunge freezing process. These also provide a controlled environment for the sample prior to freezing, especially in relation to temperature and humidity, to ensure the specimen is in optimal condition before freezing. However, for cryo-SEM applications it is possible often to plunge the sample by hand.
After freezing, the specimen is then stored in liquid nitrogen before being observed in a cryo-EM or processed further.
