projector lens image viewed directly
image viewed directly
captured by the objective lens and, because its wavelength is greater than 400 nm, will not be reflected by the dichroic mirror but will instead pass through. An emission filter, set to pass only blue light, cuts out any scattered UV light. The blue light now passes to the eye or camera in the usual way. Image b shows a field of cells cultured from rat brain (gift of Dr. Charles Krieger, Simon Fraser University) after staining with Hoechst. Only the nuclei are seen, as bright ovals.
Although some of the structures and chemicals found in cells can be selectively stained by specific fluorescent dyes, others are most conveniently revealed by using antibodies. In this technique an animal (usually a mouse, rabbit, or goat) is injected with a protein or other chemical of interest. The animal's immune system recognizes the chemical as foreign and generates antibodies that bind to (and therefore help neutralize) the chemical. Some blood is then taken from the animal and the antibodies purified. The antibodies can then be labeled by attaching a fluorescent dye. Images c and d show the same field of brain cells but with the excitation filter, dichroic mirror, and emission filter changed so as to reveal in c a protein called ELAV that is found only in nerve cells; then in d an intermediate filament protein (page 000) found only in glial cells. The antibody that binds to ELAV is labeled with a fluorescent dye that is excited by blue light and emits green light. The antibody that binds to the glial filaments is labeled with a dye that is excited by green light and emits red light. Because these wavelength characteristics are different, the location of the three chemicals—DNA, ELAV, and intermediate filament—can be revealed independently in the same specimen. See the CBASC website for an image of all three signals in color and superimposed.
The technique just described is called primary immunofluorescence and requires that the antibody to the chemical of interest be labeled with a dye. Only antibodies to chemicals that many laboratories study are so labeled. In order to reveal other chemicals, scientists use secondary immunofluorescence. In this approach, a commercial company injects an animal (e.g., a goat) with an antibody from another animal (e.g., a rabbit). The goat then makes "goat anti rabbit" antibody. This, called the secondary antibody, is purified and labeled with a dye. All the scientist has to do is make or buy a rabbit antibody that binds to the chemical of interest. No further modification of this specialized, primary antibody is necessary. Once the primary antibody has bound to the specimen and excess antibody rinsed off, the specimen is then exposed to the secondary antibody that binds selectively to the primary antibody. Viewing the stained preparation in a fluorescence microscope then reveals the location of the chemical of interest. The same dye-labeled secondary antibody can be used in other laboratories or at other times to reveal the location of many different chemicals because the specificity is determined by the unlabeled primary antibody.
The most commonly used type of electron microscope in biology is called the transmission electron microscope because electrons are transmitted through the specimen to the observer. The transmission electron microscope has essentially the same design as a light microscope, but the lenses, rather than being glass, are electromagnets that bend beams of electrons (Fig. 1.3b). An electron gun generates a beam of electrons by heating a thin, V-shaped piece of tungsten wire to 3000°C. A large voltage accelerates the beam down the microscope column, which is under vacuum because the electrons would be slowed and scattered if they collided with air molecules. The magnified image can be viewed on a fluorescent screen that emits light when struck by electrons. While the electron microscope offers great improvements in resolution, electron beams are potentially highly destructive, and biological material must be subjected to a complex processing schedule before it can be examined. The preparation of cells for electron microscopy is summarized in Figure 1.6.
A small piece of tissue (~1 mm3) is immersed in glutaraldehyde and osmium tetroxide. These chemicals bind all the component parts of the cells together; the tissue is said to be fixed. It is then washed thoroughly.
The tissue is dehydrated by soaking in acetone or ethanol.
The tissue is embedded in resin which is then baked hard.
Sections (thin slices less than 100 nm thick) are cut with a machine called an ultramicrotome.
The sections are placed on a small copper grid and stained with uranyl acetate and lead citrate. When viewed in the electron microscope, regions that have bound lots of uranium and lead will appear dark because they are a barrier to the electron beam.
Figure 1.6. Preparation of tissue for electron microscopy.
The transmission electron microscope produces a detailed image but one that is static, two-dimensional, and highly processed. Often, only a small region of what was once a dynamic, living, three-dimensional cell is revealed. Moreover, the picture revealed is essentially a snapshot taken at the particular instant that the cell was killed. Clearly, such images must be interpreted with great care. Electron microscopes are large and require a skilled operator. Nevertheless, they are the main source of information on the structure of the cell at the nanometer scale, called the ultrastructure.
Whereas the image in a transmission electron microscope is formed by electrons transmitted through the specimen, in the scanning electron microscope it is formed from electrons that are reflected back from the surface of a specimen as the electron beam scans rapidly back and forth over it (Fig. 1.3c). These reflected electrons are processed to generate a picture on a display monitor. The scanning electron microscope operates over a wide magnification range, from 10x to 100,000x. Its greatest advantage, however, is a large depth of focus that gives a three-dimensional image. The scanning electron microscope is particularly useful for providing topographical information on the surfaces of cells or tissues. Modern instruments have a resolution of about 1 nm.
IN DEPTH 1.2 Microscopy Rewarded_
Such has been the importance of microscopy to developments in biology that two scientists have been awarded the Nobel prize for their contributions to microscopy. Frits Zernike was awarded the Nobel prize for physics in 1953 for the development of phase-contrast microscopy and Ernst Ruska the same award in 1986 for the invention of the transmission electron microscope. Ruska's prize marks one of the longest gaps between a discovery (in the 1930s in the research labs of the Siemens Corporation in Berlin) and the award of a Nobel prize. Anton van Leeuwenhoek died almost two centuries before the Nobel prizes were introduced in 1901 and the prize is not awarded posthumously.
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