Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

LOOKING AT CELLS AND MOLECULES IN THE ELECTRON MICROSCOPE
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spindle pole body
0.5 µm
Figure 9–45 Localizing proteins in
electron microscopy. Immunogold
electron microscopy is used here to find
the specific location of four different protein
components within the spindle pole body
of yeast. At the top is a thin section of a
yeast mitotic spindle showing the spindle
microtubules that cross the nucleus and
connect at each end to spindle pole
bodies embedded in the nuclear envelope.
A diagram of the components of a single
spindle pole body is shown below. On
separate sections, antibodies against
four different proteins of the spindle pole
body are used, together with colloidal
gold particles (black dots), to reveal where
within the complex structure each protein is
located. (Courtesy of John Kilmartin.)
Spc72 Cnm67 Spc29 Spc110
electron microscopy—can be used in the electron microscope. The usual procedure
is to incubate a thin section first with a specific primary antibody, and then
with a secondary antibody to which a colloidal gold particle has been attached.
The gold particle is electron-dense and can be seen as a black dot in the electron
microscope (Figure 9–45). Different antibodies can be conjugated to different
sized gold particles so multiple proteins can be localized in a single sample.
A complication for immunogold labeling is that the antibodies and colloidal
MBoC6 m9.46/9.45
gold particles do not penetrate into the resin used for embedding; therefore, they
detect antigens only at the surface of the section. This means that the method’s
sensitivity is low, since antigen molecules in the deeper parts of the section are
not detected. Furthermore, we may get a false impression regarding which structures
contain the antigen and which do not. One solution is to label the specimen
before embedding it in plastic, when cells and tissues are still fully accessible to
labeling reagents. Extremely small gold particles, about 1 nm in diameter, work
best for this procedure. Such small gold particles are usually not easily visible in
the final sections, so additional silver or gold is nucleated around the tiny 1 nm
gold particles in a chemical process very much like photographic development.
Different Views of a Single Object Can Be Combined to Give a
Three-Dimensional Reconstruction
Thin sections often fail to convey the three-dimensional arrangement of cellular
components viewed in a TEM, and the image can be very misleading: a linear
structure such as a microtubule may appear in section as a pointlike object, for
example, and a section through protruding parts of a single irregularly shaped
solid body may give the appearance of two or more separate objects (Figure 9–46).
The third dimension can be reconstructed from serial sections, but this is a lengthy
and tedious process. Even thin sections, however, have a significant depth compared
with the resolution of the electron microscope, so the TEM image can also
be misleading in an opposite way, through the superimposition of objects that lie
at different depths.