The transmission electron microscope is an imaging system that theoretically permits very high resolution (0.1 nm) (Figure 1–8). In practice, however, the resolution obtained by most good instruments is around 3 nm. This high resolution allows magnifications of up to 400,000 times to be viewed with detail. Unfortunately, this level of magnification applies only to isolated molecules or particles. Very thin tissue sections can be observed with detail at magnifications of up to about 120,000 times.
Figure 1–8Photograph of the JEM-1230 transmission electron microscope. (Courtesy of JEOL USA, Inc., Peabody, MA.)
The transmission electron microscope functions on the principle that a beam of electrons can be deflected by electromagnetic fields in a manner similar to light deflection in glass lenses. In the electron microscope, electrons are released by heating a very thin metallic (usually tungsten) filament (the cathode) in a vacuum. The electrons released are then submitted to a voltage difference of 60–120 kV between the cathode and the anode, which is a metallic plate with a hole in its center (Figure 1–9). Electrons are thus attracted to the anode and accelerated to high speeds. They pass through the central opening in the anode, forming a constant stream (or beam) of electrons that penetrates the tube of the microscope. The beam passes inside electric coils and is deflected in a way roughly analogous to what occurs in optical lenses, because electrons change their path when submitted to electromagnetic fields. For this reason, the electric coils of electron microscopes are called electromagnetic lenses.
Schematic view of a transmission electron microscope with its lenses and the pathway of the electrons. CCD, charged coupled device.
The configuration of the electron microscope is very similar to that of the optical microscope, although the optics of the electron microscope are usually placed upside down (Figure 1–9). The first lens is a condenser that focuses the beam of electrons on the section. Some electrons interact with atoms of the section and continue their course, whereas others simply cross the specimen without interacting. Most electrons reach the objective lens, which forms a magnified image that is then projected through other magnifying lenses. Because the human eye is not sensitive to electrons, the image is finally projected on a fluorescent screen or is registered by photographic plates or a charged coupled device camera. Because most of the image in the transmission electron microscope is produced by the balance between the electrons that hit the fluorescent screen (or the photographic plate) and the electrons that are retained in the tube of the microscope, the resulting image is always in black and white. Dark areas of an electron micrograph are usually called electron dense, whereas light areas are called electron lucent.
To provide a good interaction between the specimen and the electrons, electron microscopy requires very thin sections (40–90 nm); therefore, embedding is performed with a resin that becomes very hard. The blocks thus obtained are so hard that glass or diamond knives are usually necessary to section them. The extremely thin sections are collected on small metal grids and transferred to the interior of the microscope to be analyzed.
Freezing techniques allow the examination of tissues by electron microscopy without the need for fixation and embedding. There are fewer artifacts than with conventional tissue preparation, although the technique is usually arduous. Frozen tissues may be sectioned and submitted to cytochemistry or immunocytochemistry or may be fractured (cryofracture, freeze fracture) to reveal details of the internal structure of the membranes.
Scanning Electron Microscopy
Scanning electron microscopy permits pseudo-three-dimensional views of the surfaces of cells, tissues, and organs. This electron microscope produces a very narrow electron beam that is moved sequentially (scanned) from point to point across the specimen. Unlike the electrons in the transmission electron microscope, those in the scanning electron microscope do not pass through the specimen (Figure 1–10). The electron beam interacts with a very thin metal coating previously applied to the specimen and produces reflected or emitted electrons. These electrons are captured by a detector that transmits them to amplifiers and other devices so that in the end the signal is projected into a cathode ray tube (a monitor), resulting in a black-and-white image. The resulting photographs are easily understood, since they present a view that appears to be illuminated from above, just as our ordinary macroscopic world is filled with highlights and shadows caused by illumination from above. The scanning electron microscope shows only surface views. The inside of organs can be analyzed by freezing the organs and fracturing them to expose their internal surfaces. Examples of scanning electron microscopy can be seen in Figures 12–3 and 12–4.
Schematic view of a scanning electron microscope.