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.
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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.
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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.
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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.
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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.
