Because of the limited resolution of the light microscope, analysis of the details of cell structure has required the use of more powerful microscopic techniques namely electron microscopy, which was developed in the 1930s and first applied to biological specimens by Albert Claude, Keith Porter, and George Palade in the 1940s and 1950s. The electron microscope can achieve a much greater resolution than that obtained with the light microscope because the wavelength of electrons is shorter than that of light. The wavelength of electrons in an electron microscope can be as short as 0.004 nm about 100,000 times shorter than the wavelength of visible light. Theoretically, this wavelength could yield a resolution of 0.002 nm, but such a resolution cannot be obtained in practice, because resolution is determined not only by wave-length, but also by the properties of the microscope lens and the specimen being examined. Consequently, for biological samples the practical limit of resolution of the electron microscope is 1–2 nm. Although this resolution is much less than that predicted simply from the wavelength of electrons, it represents more than a hundredfold improvement over the resolving power of the light microscope (see Figure 1.25).
Two types of electron microscopy transmission and scanning are widely used to study cells. In principle, transmission electron microscopy is similar to the observation of stained cells with the bright-field light microscope. Specimens are fixed and stained with salts of heavy metals, which provide contrast by scattering electrons. A beam of electrons is then passed through the specimen and focused to form an image on a fluorescent screen. Electrons that encounter a heavy metal ion as they pass through the sample are deflected and do not contribute to the final image, so stained areas of the specimen appear dark.
Positive staining Transmission electron micrograph of a white blood cell positively stained by heavy metal salts.
Specimens to be examined by transmission electron microscopy can be prepared by either positive or negative staining. In positive staining, tissue specimens are cut into thin sections and stained with heavy metal salts (such as osmium tetroxide, uranyl acetate, and lead citrate) that react with lipids, proteins, and nucleic acids. These heavy metal ions bind to a variety of cell structures, which consequently appear dark in the final image (Figure 1.37). Alternative positive-staining procedures can also be used to identify specific macromolecules within cells. For example, antibodies labeled with electron-dense heavy metals (such as gold particles) are frequently used to determine the subcellular location of specific proteins in the electron microscope. This method is similar to the use of antibodies labeled with fluorescent dyes in fluorescence microscopy. Three-dimensional views of structures with resolutions of 2–10 nm can also be obtained using the technique of electron tomography, which generates three-dimensional images by computer analysis of multiple two-dimensional images obtained over a range of viewing directions.
Negative staining Transmission electron micrograph of negatively stained actin filaments.
Negative staining is useful for the visualization of intact biological structures such as bacteria, isolated subcellular organelles, and macromolecules (Figure 1.38). In this method, the biological specimen is deposited on a supporting film, and a heavy metal stain is allowed to dry around its surface. The unstained specimen is then surrounded by a film of electron-dense stain, producing an image in which the specimen appears light against a stained dark background. The second type of electron microscopy, scanning electron microscopy, is used to provide a three-dimensional image of cells (Figure 1.39). In scanning electron microscopy the electron beam does not pass through the specimen. Instead, the surface of the cell is coated with a heavy metal, and a beam of electrons is used to scan across the specimen. Electrons that are scattered or emitted from the sample surface are collected to generate a three-dimensional image as the electron beam moves across the cell.
Scanning electron microscopy Scanning electron micrograph of a macrophage.