Many light-microscope techniques are available for observing cells. Cells that have been fixed and stained can be studied in a conventional light microscope, whereas antibodies coupled to fluorescent dyes can be used to locate specific molecules in cells in a fluorescence microscope. Living cells can be seen with phase-contrast, differential-interference-contrast, dark-field, or bright-field microscopes. All forms of light microscopy are facilitated by digital image-processing techniques, which enhance sensitivity and refine the image. Confocal microscopy and image deconvolution both provide thin optical sections and can be used to reconstruct three-dimensional images.
Techniques are now available for detecting, measuring, and following almost any desired molecule in a living cell. Fluorescent indicator dyes can be introduced to measure the concentrations of specific ions in individual cells or in different parts of a cell. Virtually any protein of interest can be genetically engineered as a fluorescent fusion protein, and then imaged in living cells by fluorescence microscopy. The dynamic behavior and interactions of many molecules can be followed in living cells by variations on the use of fluorescent protein tags, in some cases at the level of single molecules. Various superresolution techniques can circumvent the diffraction limit and resolve molecules separated by distances as small as 20 nm.
Looking at CeLLs anD MoLeCULes in the eLeCtron MiCrosCope
Light microscopy is limited in the fineness of detail that it can reveal. Microscopes using other types of radiation—in particular, electron microscopes—can resolve much smaller structures than is possible with visible light. This higher resolution comes at a cost: specimen preparation for electron microscopy is complex and it is harder to be sure that what we see in the image corresponds precisely to the original living structure. It is possible, however, to use very rapid freezing to preserve structures faithfully for electron microscopy. Digital image analysis can be used to reconstruct threedimensional objects by combining information either from many individual particles or from multiple tilted views of a single object. Together, these approaches extend the resolution and scope of electron microscopy to the point at which we can faithfully image the structures of individual macromolecules and the complexes they form.
the electron Microscope resolves the Fine structure of the Cell
The formal relationship between the diffraction limit to resolution and the wavelength of the illuminating radiation (see Figure 9–6) holds true for any form of radiation, whether it is a beam of light or a beam of electrons. With electrons, however, the limit of resolution is very small. The wavelength of an electron decreases as its velocity increases. In an electron microscope with an accelerating voltage of 100,000 V, the wavelength of an electron is 0.004 nm. In theory, the resolution of such a microscope should be about 0.002 nm, which is 100,000 times that of the light microscope. Because the aberrations of an electron lens are considerably harder to correct than those of a glass lens, however, the practical resolving power of modern electron microscopes is, even with careful image processing to correct for lens aberrations, about 0.05 nm (0.5 Å) (Figure 9–40). This is because only the very center of the electron lenses can be used, and the effective numerical aperture is tiny. Furthermore, problems of specimen preparation, contrast, and radiation damage have generally limited the normal effective resolution for biological objects to 1 nm (10 Å). This is nonetheless about 200 times better than the resolution of the light microscope. Moreover, the performance of electron microscopes is improved by electron illumination sources called field emission guns. These very bright and coherent sources substantially improve the resolution achieved.
In overall design, the transmission electron microscope (TEM) is similar to a light microscope, although it is much larger and “upside down” (Figure 9–41).
0.14 nm