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Plunge Freezing
| The use of unstained specimens in the electron microscope was an elusive goal for a long time. At the begining, the biggest hurdle in the pursuit of high resolution electron microscopy of biological specimens was that of radiation damage. This comes from the fact that the building blocks of organic molecules tend to have a low atomic number, and thus are more easily damaged by the electron beam than heavy weight atoms. The use of negative stains was an adaptation to circumvent the problem, but in these cases, the image arises from the stain surrounding the specimen, and not from the specimen itself.
The solution to the problem came in the form of cryogenic specimens. At low temperatures, the radiation damage is reduced significantly. Furthermore, if the temperature is lowered sufficiently fast, the water molecules do not have enough time to rearrange into crystalline arrays, and remain in a disordered state similar to that of a glass. This has the advantage that as the density of the water remains similar to that present in the liquid form, the structure of the molecules embedded in this ice is preserved. Fast freezing of the sample cannot be achieved at normal pressures with liquid nitrogen. To do this, one has to use a cryogen with a much higher hear capacity. The usual cryogen used for freezing samples is ethane. Propane can also be used to this end, but ethane has the advantage of being lighter than air, and therefore it is not as dangerous to use as propane. At the NYSBCÂ we have two dedicated plungers for the preparation of frozen-hydrated specimens. One is a home-made, manual plunger, operated by the gravitational force of the planet (which is quite reliable), and a Gatan CP3. The Gatan instrument is very easy to operate. The staff will be happy to show any affiliate how to prepare frozen specimens.
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