Electronmicroscopy of biological and sensitive samples
Samples for electronmicroscopy should not containwater, because they are exposed to high vacuum in a high-resolution electronmicroscope and thewaterwould be rapidly released fromwet specimens. This would not only lead to their degradation, but also complicate the work of accelerated electrons, which would be colliding with molecules of water. Therefore it is necessary to modify the biological material that contains a high percentage of water, so that it does not store any water before observation under the microscope. There are several approaches how to prepare hydrated samples:
- Drying substrate in air (the simplest way, but the sample is often destructed due to the effect of surface tension).
- Chemical fixation (goal is tomaintain cell ultrastructurewithminimal changes fromthe native state).
- Dehydration of the sample (ethanol or acetone series), and drying using substances with low surface tension (e.g. HMDS) or using the critical point (CPD).
- Physical (cryo-)methods,where the native state of the sample is preserved using amethod of rapid freezing and very helpful is the subsequent sublimation of water vapor; at present, it is the most advanced method of the preparation of sensitive and biological samples with water content.
- Metal coating of sample surface (e.g. Au, Pt,W) which increases the signal.
