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 mitochondria (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.
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 high-voltage electron microscope, along two different axes, and about 160 different views recorded. the digital data allow individual thin slices of the complete three-dimensional 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.)
Looking at CeLLs anD MoLeCULes in the eLeCtron MiCrosCope
Figure 9–48 Combining cryoelectronmicroscope tomography and single-particle reconstruction. small, unfixed, rapidly frozen specimens can be examined while still frozen. in this example, the small nuclei of the amoeba Dictyostelium were gently isolated and then very rapidly frozen before a series of angled views were recorded with the aid of a tilting microscope stage. these digital views are combined by eM tomographs to produce a three-dimensional tomogram. two thin digital slices (10 nm) through this tomogram show (a) top views and (B) side views of individual nuclear pores (white arrows). (C) in the three-dimensional model, a surface rendering of the pores (blue) is seen embedded in the nuclear envelope (yellow). From a series of tomograms it was possible to extract data sets for nearly 300 separate nuclear pores, whose structures could then be averaged using the techniques of single-particle reconstruction. the surface-rendered view of one of these reconstructed pores is shown (D) from the nuclear face and (e) in (E) 50 nm cross section (compare with Figure 12–8). the pore complex is colored blue and the nuclear basket brown. (From M. Beck image, the SEM uses electrons that are scattered or emitted from the specimen’s et al., Science 306:1387–1390, 2004. With permission from aaas.) surface. The specimen to be examined is fixed, dried, and coated with a thin layer of heavy metal. Alternatively, it can be rapidly frozen, and then transferred to a cooled specimen stage for direct examination in the microscope. Often an entire plant part or small animal can be put into the microscope with very little preparation (Figure 9–49). The specimen is scanned with a very narrow beam of electrons. The quantity of electrons scattered or emitted as this primary beam bombards each successive point of the metallic surface is measured and used to control the intensity of a second beam, which moves in synchrony with the primary beam and forms an image on a computer screen. Eventually a highly enlarged image of the surface as a whole is built up (Figure 9–50).