Two common methods for imaging live cells and unstained tissues generate contrast by taking advantage of differences block containing the specimen has hardened, it is mounted on the arm of a microtome and slices are cut with a knive. Typical sections cut for electron microscopy 50-100 nm thick; sections cut for light microscopy are 0.5-50 ^m thick. The sections are collected either on microscope slides (light microscopy) or copper mesh grids (electron microscopy) and stained with an appropriate agent.
in the refractive index and thickness of cellular materials. These methods, called phase-contrast microscopy and differential interference contrast (DIC) microscopy (or Nomarski interference microscopy), produce images that differ in appearance and reveal different features of cell architecture. Figure 5-44 compares images of live, cultured cells obtained with these two methods and standard bright-field microscopy.
In phase-contrast images, the entire object and subcellular structures are highlighted by interference rings—concentric halos of dark and light bands. This artifact is inherent in the method, which generates contrast by interference between diffracted and undiffracted light by the specimen. Because the interference rings around an object obscure many details, this technique is suitable for observing only single cells or thin cell layers but not thick tissues. It is particularly useful for examining the location and movement of larger organelles in live cells.
DIC microscopy is based on interference between polarized light and is the method of choice for visualizing extremely small details and thick objects. Contrast is generated by differences in the index of refraction of the object and its surrounding medium. In DIC images, objects appear to cast a shadow to one side. The "shadow" primarily represents a difference in the refractive index of a specimen rather than its topography. DIC microscopy easily defines the outlines of large organelles, such as the nucleus and vacuole. In addition to having a "relief"-like appearance, a DIC image is a thin optical section, or slice, through the object. Thus details of the nucleus
▲ EXPERIMENTAL FIGURE 5-44 Live cells can be visualized by microscopy techniques that generate contrast by interference. These micrographs show live, cultured macrophage cells viewed by bright-field microscopy (left), phase-contrast microscopy (middle), and differential interference contrast (DIC) microscopy (right). In a phase-contrast image, cells in thick specimens (e.g., an intact Caenorhabditis elegans roundworm) can be observed in a series of such optical sections, and the three-dimensional structure of the object can be reconstructed by combining the individual DIC images.
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