To prepare samples for SEM analysis, the following steps can be followed:
1. Primary fixation with aldehydes: This step involves fixing the proteins in the sample using aldehydes. Aldehydes help to preserve the structure of proteins and prevent degradation.
2. Secondary fixation with osmium tetroxide: After primary fixation, the sample is subjected to secondary fixation with osmium tetroxide. This step helps to fix the lipids in the sample and provides contrast for imaging.
3. Dehydration series with solvent: The sample is then dehydrated using a series of solvents such as ethanol or acetone. Dehydration removes water from the sample and prepares it for drying.
4. Drying: Once the sample is dehydrated, it needs to be dried. This can be done using various methods such as critical point drying, freeze drying, or simply air drying. The goal is to remove all traces of solvent from the sample.
5. Mounting on a stub: The dried sample is then mounted on a stub, which is a small metal cylinder or disk. The stub provides a stable platform for the sample during imaging.
6. Sputter coating with conductive material: To prevent charging and improve conductivity, the sample is coated with a thin layer of conductive material such as gold or carbon using a sputter coater. This coating ensures that the electron beam can interact properly with the sample during SEM analysis.
It is important to note that the specific sample preparation techniques may vary depending on the nature of the sample and the specific requirements of the SEM analysis. Therefore, it is essential to consult the instrument manufacturer's guidelines and protocols for sample preparation.
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