Biomedical Engineering Reference
In-Depth Information
compartments, including the endoplasmic reticulum, Golgi, and plasma
membrane.
D.
Electron Microscopy (EM)
Introduction
No matter how sophisticated the microscope or the software used to
resolve the images, light microscopes are limited by the physics of light:
objects less than 200 nm in size simply cannot be resolved,
although smaller images can be identified by fluorescent signals using
fluorescent microscopy. This limitation helped drive the development of
the electron microscope in the 1930s (38), which can magnify objects
>300,000 times, and can resolve objects of a nm or less in size! In-
stead of using light, electron microscopes use electrons as a source for
imaging. Ernst Ruska won the Nobel Prize in Physics in 1986 for the
development of the electron microscope, 4 years after chemist Aaron
Klug won the Nobel Prize for adapting the use of EM to help determine
the structure of nucleic acid-protein complexes in biologically relevant
organisms, especially viruses. One of Klug's earliest studies using EM
was published in 1964 (39).
Electrons are affected by any matter that they encounter, including
air. As a result, samples to be imaged in an electron microscope must
be kept in a vacuum. Consequently, live samples cannot be resolved!
In addition, a variety of methods must be employed to provide contrast
to biological membranes so that the object that is being resolved can
actually be seen.
Transmission electron microscope (TEM)
The TEM images electrons that pass through a sample, and can there-
fore visualize internal structures of cells and tissues. In TEM, the elec-
trons that pass through a sample are imaged; therefore, the darker areas
that are visualized from a phosphor image screen are the thicker and
denser areas, which allow fewer electrons to pass through.
For conventional TEM, sample specimens must be cut very thin and
should be dry (water is opaque to electrons). Because biological sam-
ples are primarily water, the water must be removed from the sample,
and the sample prepared so that they diffract electrons, which they do
not do naturally. Preparing samples for TEM that retain “native” structural
morphology is a challenge that many consider an art form. A number of
protocols have been developed for this purpose of the past decades, a
few of which are mentioned below.
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