Step 1: Primary fixation with aldehydes (proteins)
Proteins are crosslinked by glutaraldehyde and formaldehyde to stabilise the ultrastructure before further processing.
Step 2: Secondary fixation with osmium tetroxide (lipids)
Blipid membranes are fixed to prevent their extraction by solvents during dehydration. The black osmium precipitate which is formed during this process increases sample conductivity and minimizes image distortions resulting from charging.
Step 3: Dehydration series with solvent (ethanol or acetone)
A fixed specimen is dehydrated by incubation in a series of ethanol or acetone solutions. Solvent concentration is increased gradually so that water is removed gently, without causing specimen shrinkage.
Step 4: Drying
Allowing acetone or ethanol to simply evaporate from sample surface would create artefacts as these solvents have relatively high surface tension and would create micro-ripping of the surface upon leaving. To prevent this, dehydration solvents are replaced either with Hexamethyldisilazane (HMDS) or liquid CO2. HMDS can be used in cell preparations and after a short (3 minute) incubation it is removed and excess is left to evaporate. Liquid CO2 on the other hand is applied to tissues in a critical point drier where it is brought to a critical temperature and pressure point at which it vaporises.
Step 5: Mounting on a stub
The specimen is mounted on a metal stub using a sticky carbon disc which increases conductivity. Silver-containing glue can additionally be applied for even more conductivity.
Step 6: Sputter coating with conductve material
To prevent charge buildup on specimen surface, it is coated with a conductive material, most commonly gold. The metal is applied in a controlled manner in a sputter coater. It is critical that the coating is thick enough to prevent charging (typically around 10 nm) but not thick enough to obscure specimen surface details.