Steps

Rational

Light microscopy

TEM

SEM

Fixation

Preservation of structural detail according to level of resolution sought

Most common crosslinker: paraformaldehyde. Others fixatives: alcohols, ketones

Fixatives that penetrate rapidly into tissue: Osmium tetroxide (1%); glutaraldehyde (2.5%)

As for TEM

Dehydration

Avoids collapse of structure in vacuum

Not necessary

Essential. Done by sequential incubation in 50-100% ethanol/water mixes

As for TEM followed by critical point-drying and hexamethyldisilazane drying

Infiltration and Embedment

Provides support for sectioning

When done, paraffin is commonly used

More rigid media is essential because of thinner sections. Epoxy and acrylic resins are common

Not done

Sectioning

Avoids scattering, increases resolution

1-10 mm thick sections cut in rotary microtome

Ultrathin sections, typically 50 nm thick cut in ultramicrotome with diamond or fractured-glass knife

Not done as surfaces are imaged. Uniform, thin (e.g. 10 nm) gold/palladium coating is applied with sputter coating apparatus. Coating makes specimen electrically conductive, protects it from mass loss and prevents overheating and "charging" artifacts

Staining

Increases contrast

Histological stains Immunohistochemical and immunofluorescent techniques Others

Often begins with osmium fixation. Sections stained with heavy-atom containing compounds like uranyl acetate and lead citrate Immunohistochemical and immunogold techniques Others

Metal coating improves specific contrast

Information provided by: http://www.nmsu.edu