Biomedical Engineering Reference
In-Depth Information
Negative staining
While the addition of heavy metals can be used to provide contrast to
intracellular organelles, these positive staining methods do not always
provide the best contrast for surface structures, such as the plasma
membrane. For this purpose negative staining procedures are often em-
ployed. These involve the embedding of cells or other particles, such as
viruses, in metals that provide contrast. Because the biologic sample is
less dense than its surroundings, this method is referred to as “negative”
staining.
Freeze-fracture and deep etching
This method has been developed to allow for a very high resolution
3-dimensional image to be obtained of a sample, both of surface and
internal structures. It incorporates a method known as rotary shadowing.
In freeze-fracture, a “replica” is made of a sample, and this rather than
the sample, is examined by TEM. The sample is frozen and fractured
while under vacuum. The sample is “etched” by allowing it to dehydrate
under vacuum (some techniques expose samples to extensive freeze
drying, which leads to “deep etching”), and then a replica is made using a
thin coating of a heavy metal such as platinum (the shadowing), which
is then coated with carbon. Using this method, the thickness of the
accumulated metal varies with the surface topography, and very high
resolution images, with a 3-dimensional effect, is revealed.
Variations on TEM
A wide variety of methods has been and continues to be developed to
improve the capabilities of TEM. For example, new mixed procedures,
such as correlative video light electron microscopy, combine fluorescent
microscopy and TEM for analyzing dynamic structures within cells. Cells
expressing a fluorescent reporter such as GFP can be monitored by flu-
orescence, and then analyzed by TEM after fixation. Three-dimensional
reconstructive methods are then used to overlay the two images.
Scanning electron microscope (SEM)
SEM was developed in 1942 but was not commercialized until the
1960s. The SEM (NOT to be confused with a scanning tunneling electron
microscope; Table 4) is capable of imaging electrons emitted from a
sample, and therefore is used to study surface structures. It uses lenses
to focus a magnetized electron beam at a surface, and the interactions of
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