Histological Techniques for TEM

Question 1

Fixation

Answer 1

1st Step

Tissues are fixed for the same reasons one fixes for light microscopy.

The most common fixative is glutaraldehyde to preserve protein and carbohydrate, followed by postfixation in osmium tetroxide to preserve lipids.

Question 2

Dehydration

Answer 2

2nd Step

Water is removed from the tissue samples so that unpolymerized epoxy resin can infiltrate the tissue.

Increasing concentrations of ethyl alcohol are used until the tissue is in absolute alcohol.

Absolute alcohol is replaced with propylene oxide which is soluble in the plastic embedding medium.

Question 3

Embedding

Answer 3

3rd Step

An epoxy resin is used routinely as a TEM embedding medium (paraffin is used as a routine embedding medium for light microscopy).

Polymerization at 60oC or under a UV lamp causes hardening of the resin so that it can be sectioned.

Question 4

Sectioning

Answer 4

4th Step

Tissue sections are cut at about 60 nm thick by a glass or diamond knife in an apparatus called an ultramicrotome.

Question 5

Mounting

Answer 5

5th Step

Sections are picked up on a copper grid in the knife water trough. Spaces in the grid permit electrons to pass through.

Question 6

Staining

Answer 6

6th Step

Here chemical dyes are not used. Instead, salts of heavy metals such as uranyl acetate and lead citrate, are applied to sections supported on grids.

Question 7

Studying

Answer 7

7th Step

Grids are placed into the vacuum chamber of the electron microscope.

Electrons are absorbed by the heavy metals staining the specimen, while electrons pass through those regions of the specimen with little or no staining, and strike a fluorescent screen where an image appears and can be studied.

A permanent image can be recorded on a photographic film plate.