Przygotowanie próbek do mikroskopii elektronowej

Biological samples must be prepared for electron microscopy to protect the sample from damage by the powerful electron beam and the low pressure created by the vacuum.

Sample preparation starts with chemical fixation and complete dehydration. Chemical fixatives such as glutaraldehyde or formaldehyde cross-link the proteins, while osmium tetroxide is often used to fix the membrane lipids.

Next, the sample is dehydrated gradually with increasing concentrations of ethanol or acetone to avoid sample shrinkage.

For TEM, samples are embedded in a hard epoxy resin. An ultramicrotome then slices the sample into ultrathin cross-sections that electrons can pass through.

These sections are stained with heavy metals, such as uranyl acetate. Molecules that bind the metals appear darker and provide contrast to the image.

For SEM, samples are further dried using liquid carbon dioxide in a process called critical point drying. Samples are then sputter-coated with a thin layer of conductive metal, such as gold, preventing thermal damage to the sample and providing better image quality.