Shaira J.

Austria August 17, 2017

BIO 120 A-4L Assignment

Specimen Preparation for Electron Microscopy

1. Fixation It is the killing the cells while remaining its life-like state. It also prevents the decay of
cells. Non-coagulants fixatives were applied to maintain the cross linkages of the specimens
molecules. Examples of which are glutaraldehyde and formalin.
2. Washing and Dehydration After fixation, the specimen would be washed, commonly with
water, to remove fixatives. The water will be removed using increasing concentration of ethanol
or acetone until water is gone.
3. Embedding It made sure that the sample specimen is hard by the addition of acrylic monomers
or epoxy resins. Hardness of the specimen is important for sectioning.
4. Sectioning Ultramicrotome is used to cut the specimen into ultra-thin cuts to enable electrons
to pass through them.
5. Staining Specimens are stained to exposed it to heavy metals that have specific affinities to
certain cellular components and that increase the electron -scattering capacity of those
components.
Special Electron Microscope Techniques
1. Negative Staining - Empty spaces of the specimens are stained by putting it to a droplet of
dense material like phosphotungsten. This stain appears to be dark.
2. Cryofixation It aims to preserve the life-like state of the cellular structure by rapid freezing in
dry ice, alcohol bath or liquid nitrogen or helium gases.
3. Shadowing It is done to create a three-dimensional effect on the specimen. It is placed in an
evacuated chamber and a heavy metal is evaporated at an angle above it to strike it only on one
side. The shadow that was formed would be coated with carbon film. The specimen would be
dissolved.
4. Freeze-fracturing The specimen would be frozen rapidly and would be cracked on the plane
through the tissue. After the separation, both surfaces would be shadowed with platinum film.
5. Freeze-etching - It is the sublimation of surface ice under vacuum to reveal details of the
fractured face of the specimen that were originally hidden. It is then shadowed and the platinum
replica studied.

(a) (c)

(b)

(a) negative staining (b) freeze-fracturing and etching

(c) cryofixation (d) shadowing
(d)
Figure 1. Specimen preparation
Figure 2. Special Electron Microscope Techniques