A complication for immunogold labeling is that the antibodies and colloidal 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 threedimensional 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.
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.)
Because of the large depth of field of electron microscopes, all the parts of the threedimensional 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 threedimensional 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, threedimensional 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 threedimensional 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.