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Understanding the structural organization of cells is essential for learning how they function. In this chapter, we briefly describe some of the principal microscopy methods used to study cells. Optical microscopy will be our starting point because cell biology began with the light microscope, and it is still an indispensible tool. The development of methods for the specific labeling and imaging of individual cellular constituents and the reconstruction of their threedimensional architecture has meant that, far from falling into disuse, optical microscopy continues to increase in importance. One advantage of optical microscopy is that light is relatively nondestructive. By tagging specific cell components with fluorescent probes, such as intrinsically fluorescent proteins, we can watch their movement, dynamics, and interactions in living cells. Although conventional optical microscopy is limited in resolution by the wavelength of visible light, new methods cleverly bypass this limitation and allow the position of even single molecules to be mapped. By using a beam of electrons instead of visible light, electron microscopy can image the interior of cells, and their macromolecular components, at almost atomic resolution, and in three dimensions.
This chapter is intended as a companion, rather than an introduction, to the chapters that follow; readers may wish to refer back to it as they encounter applications of microscopy to basic biological problems in the later pages of the book.
Looking at CeLLs in the Light MiCrosCope
A typical animal cell is 10–20 μm in diameter, which is about onefifth the size of the smallest object that we can normally see with the naked eye. Only after good light microscopes became available in the early part of the nineteenth century did Schleiden and Schwann propose that all plant and animal tissues were aggregates of individual cells. Their proposal in 1838, known as the cell doctrine, marks the formal birth of cell biology.
Animal cells are not only tiny, but they are also colorless and translucent. The discovery of their main internal features, therefore, depended on the development, in the late nineteenth century, of a variety of stains that provided sufficient contrast to make those features visible. Similarly, the far more powerful electron microscope introduced in the early 1940s required the development of new techniques for preserving and staining cells before the full complexities of their internal fine structure could begin to emerge. To this day, microscopy often relies as much on techniques for preparing the specimen as on the performance of the microscope itself. In the following discussions, we therefore consider both instruments and specimen preparation, beginning with the light microscope.
The images in Figure 9–1 illustrate a stepwise progression from a thumb to a cluster of atoms. Each successive image represents a tenfold increase in magnification. The naked eye can see features in the first two panels, the light microscope allows us to see details corresponding to about the fourth or fifth panel, and the electron microscope takes us to about the seventh or eighth panel. Figure 9–2 shows the sizes of various cellular and subcellular structures and the ranges of size that different types of microscopes can visualize.
Looking at CeLLs in the Light MiCrosCope
Looking at CeLLs anD MoLeCULes in the eLeCtron MiCrosCope 20 mm 2 mm 0.2 mm 20 µm 2 µm 0.2 µm 20 nm 2 nm 0.2 nm the Light Microscope Can resolve Details 0.2 μm apart