Figure 9–51 Scanning electron microscopy. (a) a scanning electron micrograph of the stereocilia projecting from a hair cell in the inner ear of a bullfrog. For comparison, the same structure is shown by (B) differential-interferencecontrast light microscopy (Movie 9.3) and (C) thin-section transmission electron microscopy. (Courtesy of richard Jacobs and James hudspeth.) being plunged into a coolant. A special sample holder keeps this hydrated specimen at –160°C in the vacuum of the microscope, where it can be viewed directly without fixation, staining, or drying. Unlike negative staining, in which what we see is the envelope of stain exclusion around the particle, hydrated cryoelectron microscopy produces an image from the macromolecular structure itself. However, the contrast in this image is very low, and to extract the maximum amount of structural information, special imageprocessing techniques must be used, as we describe next.
Multiple images Can Be Combined to increase resolution
As we saw earlier (p. 532), noise is important in light microscopy at low light levels, but it is a particularly severe problem for electron microscopy of unstained macromolecules. A protein molecule can tolerate a dose of only a few tens of electrons per square nanometer without damage, and this dose is orders of magnitude below what is needed to define an image at atomic resolution.
The solution is to obtain images of many identical molecules—perhaps tens of thousands of individual images—and combine them to produce an averaged image, revealing structural details that are hidden by the noise in the original images. This procedure is called single-particle reconstruction. Before combining all the individual images, however, they must be aligned with each other. Sometimes it is possible to induce proteins and complexes to form crystalline arrays, in which each molecule is held in the same orientation in a regular lattice. In this case, the alignment problem is easily solved, and several protein structures have been determined at atomic resolution by this type of electron crystallography. In principle, however, crystalline arrays are not absolutely required. With the help of a computer, the digital images of randomly distributed and unaligned molecules can be processed and combined to yield highresolution reconstructions (see Movie 13.1). Although structures that have some intrinsic symmetry make the task of alignment easier and more accurate, this technique has also been used for objects like ribosomes, with no symmetry. Figure 9–54 shows the structure of
Figure 9–52 The nuclear pore. rapidly frozen nuclear envelopes were imaged in a high-resolution seM, equipped with a field emission gun as the electron source. these views of each side of a nuclear pore represent the limit of resolution of the seM (compare with Figure 12–8). (Courtesy of Martin goldberg and terry allen.)
We know in detail about many cell processes, such as Dna replication and transcription and rna translation, but will we ever be able to visualize such rapid molecular processes in action in cells?
Will we ever be able to image intracellular structures at the resolution of the electron microscope in living cells?
how can we improve crystallization and single-particle cryoelectron microscopy techniques to obtain high-resolution structures of all important membrane channels and transporters? What new concepts might these structures reveal?
Figure 9–53 Negatively stained actin filaments. in this transmission electron micrograph, each filament is about 8 nm in diameter and is seen, on close inspection, to be composed of a helical chain of globular actin molecules. (Courtesy of roger Craig.) the protein capsid inside a human immunodeficiency virus (HIV) that has been determined at high resolution by the combination of many particles, multiple views, and molecular modeling.