Ultramicrotomy
Ultramicrotomy is a method for cutting specimens into extremely thin slices, called ultra-thin sections, that can be studied and documented at different magnifications in a
Ultramicrotomy process
Ultra-thin sections of specimens are cut using a specialized instrument called an "ultramicrotome". The ultramicrotome is fitted with either a diamond knife, for most biological ultra-thin sectioning, or a glass knife, often used for initial cuts. There are numerous other pieces of equipment involved in the ultramicrotomy process. Before selecting an area of the specimen block to be ultra-thin sectioned, the technician examines semithin or "thick" sections range from 0.5 to 2 μm. These thick sections are also known as survey sections and are viewed under a light microscope to determine whether the right area of the specimen is in a position for thin sectioning. "Ultra-thin" sections from 50 to 100 nm thick are able to be viewed in the TEM.
Tissue sections obtained by ultramicrotomy are compressed by the cutting force of the knife. In addition, interference microscopy of the cut surface of the blocks reveals that the sections are often not flat. With Epon or Vestopal as embedding medium the ridges and valleys usually do not exceed 0.5 μm in height, i.e., 5–10 times the thickness of ordinary sections (1).
A small sample is taken from the specimen to be investigated. Specimens may be from biological matter, like animal or plant tissue, or from inorganic material such as rock, metal, magnetic tape, plastic, film, etc., or other metal grid. Ideal section thickness for transmission electron microscopy with accelerating voltages between 50kV and 120kV is about 30–100 nm.
Advances
In 1952 Humberto Fernandez Morán introduced cryo ultramicrotomy, which is a similar technique but done at freezing temperatures between −20 and −150°C. Cryo ultramicrotomy can be used to cut ultra-thin frozen biological specimens. One of the advantages over the more "traditional" ultramicrotomy process is speed, since it should be possible to freeze and section a specimen in 1 to 2 hours.
References
- Helander HF (1969). "Surface topography of ultramicrotome sections". J Ultrastruct Res. 29: 373–382. PMID 4903906.