4.2 Light Microscopy: Exploring Cell Structure and Visualizing Proteins Within Cells

The cellular basis of life was first appreciated using primitive light microscopes. Since then, progress in cell biology has paralleled, and has often been driven by, technological advances in light microscopy (Figure 4-8). Here we discuss each of these major developments and how they advanced the study of cellular processes. First we describe basic uses of a light microscope to observe unstained cells and structures. Next we describe the development of fluorescence microscopy and its use to localize specific proteins in fixed cells. By using molecular genetic techniques to fuse a protein of interest with a naturally fluorescent protein and express the resulting chimeric protein in cells, it is possible to follow the movement of specific proteins in live cells—an ability that has revealed how dynamic the organization of live cells is. In parallel with these advances in specimen preparation, optical advances were being made to enhance and sharpen the images provided by fluorescence microscopy to reveal cellular structure with unprecedented clarity. Many specialized technologies have emerged from these advances, and we describe some of the more important ones.

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FIGURE 4-8 Development of the light microscope. (a) Early microscopes, like the ones used by Robert Hooke in the 1660s, used lenses or a mirror to illuminate the specimen. (b) Optics in general, and light microscopes in particular, developed enormously during the nineteenth century. By the middle of the twentieth century, highly sophisticated microscopes limited only by the resolution of light were common. (c) In the second half of the twentieth century, fluorescence microscopy and digital imaging, together with confocal techniques, were developed to yield the versatile microscopes of today.
[Part (a) SSPL/Getty Images; parts (b) and (c) courtesy of A. Bretscher.]

Many of the techniques we describe allow one to examine live cells in a microscope. These advances not only permit video microscopy, but also allow one to examine the responses of live cells or their components to specific stimuli or their interactions with other cells. As we discuss in this section, they have provided scientists with the ability to probe the functioning of individual components in live cells.