Diagnostic electron microscopy
Encyclopedia
The transmission electron microscope
(TEM) is used as an important diagnostic tool
to screen human tissues at high magnification (the ultrastructural level), often in conjunction with other methods, particularly light microscopy
and immunofluorescence techniques. The TEM was first used extensively for this purpose in the 1980s, especially for identifying the markers of cell differentiation
to identify tumours, and in renal disease
. Immunolabelling techniques are now generally used instead of the TEM for tumour diagnosis but the technique retains a critical role in the diagnosis of renal disease and a range of other conditions.
Specifically, TEM should be used for diagnostic purposes when it: (1) provides useful (complementary) information in the context of a carefully considered differential diagnosis
; (2) provides an ‘improved’ diagnosis that results in better treatment strategies and; (3) is time & cost effective
in respect to alternative techniques. For diagnostic purposes solid tissues are prepared for TEM in the same way as other biological tissues, they are fixed in glutaraldehyde and osmium tetroxide
then dehydrated and embedded in epoxy resin
. The epoxy resin block is trimmed and the target tissue is selected using a light microscope
by viewing semithin sections stained with toluidine blue. The block is then retrimmed and the specific area for observation is ultrathin sectioned, preferably using a diamond knife. The ultrathin sections are collected on 3mm copper (mesh) grids and stained with uranyl acetate
and lead citrate to make the contents of the tissue electron dense (and thus visible in the electron microscope
).
Transmission electron microscopy
Transmission electron microscopy is a microscopy technique whereby a beam of electrons is transmitted through an ultra thin specimen, interacting with the specimen as it passes through...
(TEM) is used as an important diagnostic tool
Diagnosis
Diagnosis is the identification of the nature and cause of anything. Diagnosis is used in many different disciplines with variations in the use of logics, analytics, and experience to determine the cause and effect relationships...
to screen human tissues at high magnification (the ultrastructural level), often in conjunction with other methods, particularly light microscopy
Microscopy
Microscopy is the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye...
and immunofluorescence techniques. The TEM was first used extensively for this purpose in the 1980s, especially for identifying the markers of cell differentiation
Cellular differentiation
In developmental biology, cellular differentiation is the process by which a less specialized cell becomes a more specialized cell type. Differentiation occurs numerous times during the development of a multicellular organism as the organism changes from a simple zygote to a complex system of...
to identify tumours, and in renal disease
Kidney
The kidneys, organs with several functions, serve essential regulatory roles in most animals, including vertebrates and some invertebrates. They are essential in the urinary system and also serve homeostatic functions such as the regulation of electrolytes, maintenance of acid–base balance, and...
. Immunolabelling techniques are now generally used instead of the TEM for tumour diagnosis but the technique retains a critical role in the diagnosis of renal disease and a range of other conditions.
Specifically, TEM should be used for diagnostic purposes when it: (1) provides useful (complementary) information in the context of a carefully considered differential diagnosis
Differential diagnosis
A differential diagnosis is a systematic diagnostic method used to identify the presence of an entity where multiple alternatives are possible , and may also refer to any of the included candidate alternatives A differential diagnosis (sometimes abbreviated DDx, ddx, DD, D/Dx, or ΔΔ) is a...
; (2) provides an ‘improved’ diagnosis that results in better treatment strategies and; (3) is time & cost effective
Cost-effectiveness
Cost-effectiveness analysis is a form of economic analysis that compares the relative costs and outcomes of two or more courses of action. Cost-effectiveness analysis is distinct from cost-benefit analysis, which assigns a monetary value to the measure of effect...
in respect to alternative techniques. For diagnostic purposes solid tissues are prepared for TEM in the same way as other biological tissues, they are fixed in glutaraldehyde and osmium tetroxide
Osmium(VIII) oxide
Osmium tetroxide is the chemical compound with the formula OsO4. The compound is noteworthy for its many uses, despite the rarity of osmium...
then dehydrated and embedded in epoxy resin
Epoxy
Epoxy, also known as polyepoxide, is a thermosetting polymer formed from reaction of an epoxide "resin" with polyamine "hardener". Epoxy has a wide range of applications, including fiber-reinforced plastic materials and general purpose adhesives....
. The epoxy resin block is trimmed and the target tissue is selected using a light microscope
Optical microscope
The optical microscope, often referred to as the "light microscope", is a type of microscope which uses visible light and a system of lenses to magnify images of small samples. Optical microscopes are the oldest design of microscope and were possibly designed in their present compound form in the...
by viewing semithin sections stained with toluidine blue. The block is then retrimmed and the specific area for observation is ultrathin sectioned, preferably using a diamond knife. The ultrathin sections are collected on 3mm copper (mesh) grids and stained with uranyl acetate
Uranyl acetate
Uranyl acetate is the acetate salt of uranium and is a yellow crystalline solid made up of yellow rhombic crystals and has a slight acetic odor. Uranyl acetate is slightly radioactive, the precise radioactivity depends on the isotopes of uranium present...
and lead citrate to make the contents of the tissue electron dense (and thus visible in the electron microscope
Electron microscope
An electron microscope is a type of microscope that uses a beam of electrons to illuminate the specimen and produce a magnified image. Electron microscopes have a greater resolving power than a light-powered optical microscope, because electrons have wavelengths about 100,000 times shorter than...
).