Electron Microscopy
The resolution of a microscope is limited by the
wavelength of light passing through the sample.
For visible microscopes using 400 nm light (blue
light), the limit of resolution is one half the
wavelength, or 200nm. This is some two to three
orders of magnitude larger than many cellular
structures. Electrons, like photons, have wavelike
properties, but, unlike light, electrons can be
accelerated to wavelengths well below 1nm. This
has allowed the development of the Electron
Microscope (EM), with resolutions down to
below 1nm. Although electron wavelengths would
theoretically allow resolution down to below
0.01nm, in practice, mechanical limitations on the
construction of the apparatus have prevented this
limit from being approached.
Two types of electron microscopes are in wide
use: the transmission electron microscope (TEM)
and the scanning electron microscope (SEM). The
TEM operates on the same principles as a light
microscope. A beam of electrons, accelerated
from a tungsten filament, is focused on a sample,
and the transmitted electrons are focused into an
image. "Electron dense" areas of the sample
(often made dense by staining techniques) scatter
electrons, leading to dark areas in the image. The
image itself, being made up of electrons, is
invisible to the eye, so it is visualized by
projection onto a fluorescent screen, which emits
light when it is struck by electrons. For permanent
records, photographic film, which is exposed by
electrons, is used in place of the screen.
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Comparison of the imaging paths of the
transmission and scanning electron
microscopes.
Of the organelles made clearly visible by the
transmission electron micrograph of the
hepatic cell above (rat), only the nucleus could
have been resolved with a light microscope.
Organelles: (I) Nucleus, (II) Endoplasmic
reticulum, (III) Mitochondria, (IV) Golgi
apparatus, (V) Bile canaliculus, (VI) Plasma
membrane, (VII) Desmosome, (VIII)
Secretory granule.
The SEM is used to visualize surface details of the
sample. The image develops by means of the
scattering of electrons by the surface of the
sample when the beam hits it. A narrowly focused
beam (10nm diameter) is "scanned" across the
sample surface, and the secondary electrons which
are reflected from the surface are amplified and
used to determine characteristics of the sample
surface at the probe position.
The preparation of samples for transmission
electron microscopy parallels tissue preparation
for visible microscopy. Tissue samples must be
fixed, processed, embedded, sectioned and
mounted before viewing. The sections produced
must be thinner and stronger than those for light
microscopy, and the level of detail observable
mandates that the tissue structure be exceptionally
well preserved. Specialized fixing and processing
techniques have been refined to meet these
requirements. Furthermore, staining techniques
have been developed to produce electron dense
zones instead of colored or fluorescent areas.
(Techniques of sample preparation for scanning
electron microscopy are not covered here. For a
good overview of the process, the reader is
referred to: Robinson, G & Gray T. (1990)
Electron Microscopy 3: Specialized Techniques,
in Bancroft, J. & Stevens A. (ed.) Theory and
Practice of Histological Techniques, 3rd edn.
London. Churchill.)
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Microscopy