Ultrarapid freezing, when used in conjunction with appropriate accessory processing methods has long been recognized to provide reservation of ultrastructure, localization of soluble inorganic and organic species, and preservation of antigenicity that is superior to that obtainable through conventional chemical fixation techniques and the use of cryoprotectants. Furthermore, it is the only procedure currently available for capturing events that occur on timescales less than a few seconds. Successful ultrarapid freezing requires that heat be withdrawn so rapidly from an unfixed un-cryoprotected sample that no ice crystal damage is visible at the electron microscopic level of analysis. This is not a trivial undertaking, and until recently, the improvements gained through ultrarapid freezing were often paid for in terms of decreasing productivity. However, over the last several years, improvements in methodology and the introduction of refined equipment for the ultrarapid freezing of biological samples has significantly improved both the ease of application of technique and the yield of well preserved samples. The EMS-002 Cryo Workstation allows for a reliable virtually routine freezing of biological samples.
The EMS-002 achieves ultra-rapid cooling by quenching the sample in liquid propane or other suitable cryogen, cooled to near liquid nitrogen temperature. The EMS-002 accomplishes this by using a stainless steel vacuum insulated Dewar and copper condensation chamber. Liquid nitrogen is poured into the Dewar to a level determined by the user. The initial cooling of the cryogen condensation chamber takes about 15 to 20 minutes.
Gaseous propane is then admitted to the condensation chamber slowly enough to allow it to condense and fill the chamber with liquid propane. The propane is led to cool to near liquid nitrogen temperature supervised by the digital temperature controller while the sample is being prepared for freezing. The device requires only about 10 minutes to cool to operating temperature after the first filling of propane, and less than five minutes after subsequent fillings.
The operator can increase or decrease the rate of cooling by adjusting the level of liquid nitrogen in the Dewar. Heaters built into the chamber wall, powered by the temperature controller, evenly warms the propane above its melting point. The controllers’ temperature is set for the kind of cryogen being used, for instance, 185°C for commercial grade propane.
Chemically pure propane (map. 190°C) can also be used, but it is significantly more expensive than commercial grade propane and confers no particular advantage. Ethane and a non-flammable cryogen, SUVA® 124 produced by Dupont can also be used as a cryogen in this instrument. When using flammable cryogens like Propane & Ethane we recommend using the EMS-002 in a fume hood.
The sample can be prepared using any favorite size EM Grid if the system is being used for Cryo-TEM or Panama hat-shaped specimen carriers for Freeze- Fracture or Freeze Substitution. Once the sample is applied to the EM grid for instance, the excess must be carefully blotted away with a small piece of filter paper. Remember, the goal is to form a thin meniscus of specimen across the holes in the grid. Once achieved, insert the specimen holder in the“Drop Anvil” of the EMS-002.
The technique provides higher rates of cooling than can be achieved by plunging the sample into the cryogen by hand because of the high velocity and accuracy the sample enters the cryogen. The optimal plunging speed is approximately 100m per/sec it provides more efficient heat exchange. The freezing is fast enough to capture rapid events and labile structures that are not seen in chemically fixed material. The technique has been applied to many types of specimens: aqueous suspensions and emulsions, tissue culture cells, suspensions of a cells, and tissues. Specimens frozen with the EMS-002 may be freeze fractured, freeze substituted, frozen thin sectioned, or freeze dried. The samples may also be used for Cryo-SEM or Cryo-TEM.
|Cat #: 37000
|Description: EMS 002 Plunge Freezer
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|Cat #: 37015
|Description: Propane Transfer System
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|Cat #: 37457
|Description: Environmental Chamber
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