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LIGHT MICROSCOPY

As in all experimental sciences, research in cell biology depends on the laboratory methods that can be used to study cell structure and function. Many important advances in understanding cells have directly followed the development of new methods that have opened novel avenues of investigation. An appreciation of the experimental tools available to the cell biologist is thus critical to understanding both the current status and future directions of this rapidly moving area of science. The two fundamental methods of studying the structure of cells microscopy and subcellular fractionation are described in the sections that follow. Other experimental approaches, including the methods of molecular biology, genomics, and proteomics, will be discussed in later chapters.

 

LIGHT MICROSCOPY

Because most cells are too small to be seen by the naked eye, the study of cells has depended heavily on the use of microscopes. Indeed, the very discovery of cells arose from the development of the microscope: Robert Hooke coined the term “cell” following his observations of a piece of cork with a simple light microscope in 1665 (Figure 1.24). Using a microscope that magnified objects up to about 300 times their actual size, Anton van Leeuwenhoek, in the 1670s, was able to observe a variety of different types of cells, including sperm, red blood cells, and bacteria. The proposal of the cell theory by Matthias Schleiden and Theodor Schwann in 1838 may be seen as the birth of contemporary cell biology. Microscopic studies of plant tissues by Schleiden and of animal tissues by Schwann led to the same conclusion: All organisms are composed of cells. Shortly thereafter, it was recognized that cells are not formed de novo but arise only from division of preexisting cells. Thus, the cell achieved its current recognition as the fundamental unit of all living organisms because of observations made with the light microscope.

Figure 1.24 The cellular structure of cork A reproduction of Robert Hooke’s drawing of a thin slice of cork examined with a light microscope. The “cells” that Hooke observed were actually only the cell walls remaining from cells that had long since died.

The light microscope remains a basic tool of cell biologists, with technical improvements allowing the visualization of ever-increasing details of cell structure. Contemporary light microscopes are able to magnify objects up to about a thousand times. Since most cells are between 1 and 100 μm in diameter, they can be observed by light microscopy, as can some of the larger subcellular organelles, such as nuclei, chloroplasts, and mitochondria (Figure 1.25). However, the light microscope is not powerful enough to reveal fine details of cell structure, for which resolution the ability of a microscope to distinguish objects separated by small distances is even more important than magnification. Images can be magnified as much as desired (for example, by projection onto a large screen), but such magnification does not increase the level of detail that can be observed.

Figure 1.25 Limits of microscopy The sizes of representative cells, organelles, and molecules are compared to the limits of resolution of light microscopy, super-resolution microscopy, and electron microscopy.

Figure 1.26 The light microscope Light is focused on the specimen by the condenser lens and then collected by the objective lens of the microscope. The numerical aperture is determined by the angle of the cone of light entering the objective lens (α) and by the refractive index of the medium (usually air or oil) between the lens and the specimen.



The diffraction of light limits the resolution of the light microscope to approximately 0.2 μm; two objects separated by less than this distance appear as a single image, rather than being distinguished from one another. This theoretical limitation of light microscopy is determined by two factors—the wave-length (λ) of visible light and the light-gathering power of the microscope lens (numerical aperture, NA)—according to the following equation:

Resolution = 0.61l / NA

The wavelength of visible light is 0.4 to 0.7 μm, so the value of λ is fixed at approximately 0.5 μm for the light microscope. The numerical aperture can be envisioned as the size of the cone of light that enters the microscope lens after passing through the specimen (Figure 1.26). It is given by the equation

NA = η sin α

where η is the refractive index of the medium through which light travels between the specimen and the lens. The value of η for air is 1.0, but it can be increased to a maximum of approximately 1.4 by using an oil-immersion lens to view the specimen through a drop of oil. The angle α corresponds to half the width of the cone of light collected by the lens. The maximum value of α is 90°, at which sin α = 1, so the highest possible value for the numerical aperture is 1.4.

The theoretical limit of resolution of the light microscope can therefore be calculated as follows:

Resolution = 0.61 x 0.5 / 1.4 × = 0.22 _m

This limitation of light microscopy is determined tained tissue Sec-Visuals Unlimited, Inc.) by the wavelength of visible light, and microscopes achieving this level of resolution had already been made by the end of the nineteenth century. However, as discussed below, new approaches have led to the development of novel methods (super-resolution microscopy) that have substantially increased the resolving power of fluorescence microscopy to reach beyond this limit.

Several different types of light microscopy are routinely used to study various aspects of cell structure. The simplest is bright-field microscopy, in which light passes directly through the cell and the ability to distinguish different parts of the cell depends on contrast resulting from the absorption of visible light by cell components. In many cases, cells are stained with dyes that react with proteins or nucleic acids in order to enhance the contrast between different parts of the cell. Prior to staining, specimens are usually treated with fixatives (such as alcohol, acetic acid, or formaldehyde) to stabilize and preserve their structures. The examination of fixed and stained tissues by bright-field microscopy is the standard approach for the analysis of tissue specimens in histology laboratories (Figure 1.27). Such staining procedures kill the cells, however, and therefore are not suitable for many experiments in which the observation of living cells is desired.

 

Figure 1.27 Bright-field micrograph of stained tissue Section of a benign kidney tumor.

Figure 1.28 Microscopic observation of living cells Photomicrographs of human cheek cells obtained with (A) phase-contrast, and (B) differential interference-contrast microscopy



Without staining, the direct passage of light does not provide sufficient contrast to distinguish many parts of the cell, limiting the usefulness of bright-field microscopy. However, optical variations of the light microscope can be used to enhance the contrast between light waves passing through regions of the cell with different densities. The two most common methods for visualizing living cells are phase-contrast microscopy and differential interference-contrast microscopy (Figure 1.28). Both kinds of microscopy use optical systems that convert variations in density or thickness between different parts of the cell to differences in contrast that can be seen in the final image. In bright-field microscopy, transparent structures (such as the nucleus) have little contrast because they absorb light poorly. However, light is slowed down as it passes through these structures so that its phase is altered compared with light that has passed through the surrounding cytoplasm. Phase-contrast and differential interference-contrast microscopy convert these differences in phase to differences in contrast, thereby yielding clear images of live, unstained cells.