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

558 Chapter 9: Visualizing Cells
Because of the large depth of field of electron microscopes, all the parts of the
three-dimensional specimen are in focus, and the resulting image is a projection
(a superimposition of layers) of the structure along the viewing direction. The lost
information in the third dimension can be recovered if we have views of the same
specimen from many different directions. The computational methods for this
technique are widely used in medical CT scans. In a CT scan, the imaging equipment
is moved around the patient to generate the different views. In electron-microscope
(EM) tomography, the specimen holder is tilted in the microscope,
which achieves the same result. In this way, we can arrive at a three-dimensional
reconstruction, in a chosen standard orientation, by combining different views
of a single object. Each individual view will be very noisy but by combining them
in three dimensions and taking an average, the noise can be largely eliminated.
Starting with thick plastic sections of embedded material, three-dimensional
reconstructions, or tomograms, are used extensively to describe the detailed anatomy
of specific regions of the cell, such as the Golgi apparatus (Figure 9–47) or
the cytoskeleton. Increasingly, microscopists are also applying EM tomography
to unstained frozen, hydrated sections, and even to rapidly frozen whole cells
or organelles (Figure 9–48). Electron microscopy now provides a robust bridge
between the scale of the single molecule and that of the whole cell.
Images of Surfaces Can Be Obtained by Scanning Electron
Microscopy
A scanning electron microscope (SEM) directly produces an image of the
three-dimensional structure of the surface of a specimen. The SEM is usually
smaller, simpler, and cheaper than a transmission electron microscope. Whereas
the TEM uses the electrons that have passed through the specimen to form an
Figure 9–46 A three-dimensional
reconstruction from serial sections.
Single thin sections in the electron
microscope sometimes give misleading
impressions. In this example, most sections
through a cell containing a branched
mitochondrion seem to contain two or
three separate MBoC6 mitochondria m9.47/9.46 (compare
Figure 9–44). Sections 4 and 7, moreover,
might be interpreted as showing a
mitochondrion in the process of dividing.
The true three-dimensional shape can
be reconstructed from a complete set of
serial sections.
1
2
3
4
5
6
7
8
9
(A)
(B)
(C)
250 nm
Figure 9–47 Electron-microscope (EM)
tomography. Samples that have been
rapidly frozen, and then freeze-substituted
and embedded in plastic, preserve their
structure in a condition that is very close
to their original living state (Movie 9.2).
This example shows the three-dimensional
structure of the Golgi apparatus from
a rat kidney cell. Several thick sections
(250 nm) of the cell were tilted in a highvoltage
electron microscope, along two
different axes, and about 160 different
views recorded. The digital data allow
individual thin slices of the complete threedimensional
data set, or tomogram, to
be viewed; for example, the serial slices,
each only 4 nm thick, are shown in (A) and
(B). Very little changes from one slice to
the next, but using the full data set, and
manually color-coding the membranes (B),
one can obtain a full three-dimensional
reconstruction, at a resolution of about
7 nm, of the complete Golgi complex and
its associated vesicles (C). (From M.S.
Ladinsky et al., J.Cell Biol. 144:1135–1149,
1999. With permission from the authors.)