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We know that the technique used in TEM sample preparation involves multiple steps, one of the most important of them is fixation.

Fixation can be of two types:

  1. Cryofixation, that suggests that the specimen are subjected to freezing temperature to perserve the cell's living material before we observe them.

  2. Chemical fixation involves the use of certain chemicals, such as formalin, glutaraldehyde, etc. for almost same purpose.

The question is when it is mandatory to use one of them only? Such as, cryofixation or chemical one? At some instances, have the researchers ever need to use both of the techniques at the same time?

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Cryofixation usually preserves the organelles inside the cells better, but is more work and usually results in TEM images that are lower contrast. Chemical fixation is easier and gives good contrast, but is more likely to destroy details in delicate samples. Chemical fixation will usually be used unless it is found or expected to not work well for a particular sample. It is possible to mix the methods, for example by including glutaraldehyde in freeze substitution solutions. Here is an article that describes everything you asked about in more detail, if you can access it: A comparison of cryo- versus chemical fixation in the soil green algae Jaagiella

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