microscopy
The scanning electron microscope (SEM) is especially useful for detailed study of the topography of a specimen. The electron beam scans the surface of the sample, usually coated with a thin film of gold. The beam excites electrons on the surface, and these secondary electrons are detected by a device that translates the pattern of electrons into an electronic signal sent to a video screen. The result is an image of the specimen’s surface that appears three-dimensional. The transmission electron microscope (TEM) is used to study the internal structure of cells (see Figure 6.3). The TEM aims an electron beam through a very thin section of the specimen, much as a light microscope aims light through a sample on a slide. For the TEM, the specimen has been stained with atoms of heavy metals, which attach to certain cellular structures, thus enhancing the electron density of some parts of the cell more than others. The electrons passing through the specimen are scattered more in the denser regions, so fewer are transmitted. The image displays the pattern of transmitted electrons. Instead of using glass lenses, both the SEM and TEM use electromagnets as lenses to bend the paths of the electrons, ultimately focusing the image onto a monitor for viewing. Electron microscopes have revealed many subcellular structures that were impossible to resolve with the light microscope. But the light microscope offers advantages, especially in studying living cells. A disadvantage of electron microscopy is that the methods customarily used to prepare the specimen kill the cells and can introduce artifacts, structural features seen in micrographs that do not exist in the living cell. In the past several decades, light microscopy has been revitalized by major technical advances (see Figure 6.3). Labeling individual cellular molecules or structures with fluorescent markers has made it possible to see such structures with increasing detail. In addition, both confocal and deconvolution microscopy have produced sharper images of three-dimensional tissues and cells. Finally, a group of new techniques and labeling molecules developed in recent years, called super-resolution microscopy, has allowed researchers to “break” the resolution barrier and distinguish subcellular structures as small as 10–20 nm across. A recently developed new type of TEM called cryo-electron microscopy (cryo-EM) (see Figure 6.3) allows specimens to be preserved at extremely low temperatures. This avoids the use of preservatives, allowing visualization of structures in their cellular environment. This method is increasingly used to complement X-ray crystallography in revealing protein complexes and subcellular structures like ribosomes, described later. Cryo-EM has even been used to resolve some individual proteins. The Nobel Prize for Chemistry was awarded in 2017 to the developers of this valuable technique. Microscopes are the most important tools of cytology, the study of cell structure. Understanding the function of each structure, however, required the integration of cytology and biochemistry, the study of the chemical processes (metabolism) of cells.
The scanning electron microscope (SEM) is especially useful for detailed study of the topography of a specimen. The electron beam scans the surface of the sample, usually coated with a thin film of gold. The beam excites electrons on the surface, and these secondary electrons are detected by a device that translates the pattern of electrons into an electronic signal sent to a video screen. The result is an image of the specimen’s surface that appears three-dimensional. The transmission electron microscope (TEM) is used to study the internal structure of cells (see Figure 6.3). The TEM aims an electron beam through a very thin section of the specimen, much as a light microscope aims light through a sample on a slide. For the TEM, the specimen has been stained with atoms of heavy metals, which attach to certain cellular structures, thus enhancing the electron density of some parts of the cell more than others. The electrons passing through the specimen are scattered more in the denser regions, so fewer are transmitted. The image displays the pattern of transmitted electrons. Instead of using glass lenses, both the SEM and TEM use electromagnets as lenses to bend the paths of the electrons, ultimately focusing the image onto a monitor for viewing. Electron microscopes have revealed many subcellular structures that were impossible to resolve with the light microscope. But the light microscope offers advantages, especially in studying living cells. A disadvantage of electron microscopy is that the methods customarily used to prepare the specimen kill the cells and can introduce artifacts, structural features seen in micrographs that do not exist in the living cell. In the past several decades, light microscopy has been revitalized by major technical advances (see Figure 6.3). Labeling individual cellular molecules or structures with fluorescent markers has made it possible to see such structures with increasing detail. In addition, both confocal and deconvolution microscopy have produced sharper images of three-dimensional tissues and cells. Finally, a group of new techniques and labeling molecules developed in recent years, called super-resolution microscopy, has allowed researchers to “break” the resolution barrier and distinguish subcellular structures as small as 10–20 nm across. A recently developed new type of TEM called cryo-electron microscopy (cryo-EM) (see Figure 6.3) allows specimens to be preserved at extremely low temperatures. This avoids the use of preservatives, allowing visualization of structures in their cellular environment. This method is increasingly used to complement X-ray crystallography in revealing protein complexes and subcellular structures like ribosomes, described later. Cryo-EM has even been used to resolve some individual proteins. The Nobel Prize for Chemistry was awarded in 2017 to the developers of this valuable technique. Microscopes are the most important tools of cytology, the study of cell structure. Understanding the function of each structure, however, required the integration of cytology and biochemistry, the study of the chemical processes (metabolism) of cells.
