ALPF Medical Research

Microscopic Techniques The Instruments

Even today, one of the most important tools for studying microorganisms is the light microscope, which, like van Leeuwenhoek's instrument, uses visible light for observing objects. These instruments can magnify images approximately 1,000x, making it relatively easy to observe the size, shape, and motility of prokaryotic cells. The electron microscope, introduced in 1931, can magnify images in excess of 100,000x. This microscope revealed many fine details of cell structure. A major advancement came in 1981 with the introduction of the first scanning probe microscope. This, in turn, led to even more sophisticated technologies that allow scientists to view individual atoms. The types of microscopes are summarized in table 3.1.

Principles of Light Microscopy: The Bright-Field Microscope

In light microscopy, light typically passes through a specimen and then through a series of magnifying lenses. The most common type of light microscope, and the easiest to use, is the bright-field microscope, which evenly illuminates the field of view.

Magnification

The modern light microscope has two magnifying lenses—an objective lens and an ocular lens—and is called a compound

Table 3.1 A Summary of Microscopic Instruments and Their Characteristics
Instrument	Mechanism	Uses/Comment
Light Microscopes	Visible light passes through a series of lenses to produce a magnified image.	Relatively easy to use. Considerably less expensive than confocal and electron microscopes.
Bright-field	Illuminates the field of view evenly.	Most common type of microscope.
Phase-contrast	Amplifies differences in refractive index to create contrast.	Makes unstained cells more readily visible.
Interference	Two light beams pass through the specimen and then recombine.	Causes the specimen to appear as a three-dimensional image.
Dark-field	Light is directed toward the specimen at an angle.	Makes unstained cells more readily visible; organisms stand out as bright objects against a dark background.
Fluorescence	Projects ultraviolet light, causing fluorescent molecules in the specimen to emit longer wavelength light.	Used to observe cells that have been stained or tagged with a fluorescent dye.
Confocal	Mirrors scan a laser beam across successive regions and planes of a specimen. From that data, a computer constructs an image.	Used to construct a three-dimensional image structure; provides detailed sectional views of intact cells.
Electron Microscopes	Use electron beams in place of visible light to produce the magnified image.	Can clearly magnify images 100,000x.
Transmission	Transmits a beam of electrons through a specimen.	Elaborate specimen preparation, which may introduce artifacts, is required.
Scanning	A beam of electrons scans back and forth over the surface of a specimen.	Used for observing surface details; produces a three-dimensional effect.
Scanning Probe Microscopes	Make it possible to view images on an atomic scale.	Produce a map showing the bumps and valleys of the atoms on that surface.
Scanning tunneling	A sharp metallic probe causes electrons to tunnel between the probe and a conductive surface.	First scanning probe microscope developed.
Atomic force	A tip bends in response to even the slightest force between it and the sample.	Can operate in air and in liquids.

microscope (figure 3.1). These lenses in combination visually enlarge an object by a factor equal to the product of each lens' magnification. For example, an object is magnified 1,000-fold when it is viewed through a 10x ocular lens and an objective lens with a power of100x. Most compound microscopes have a selection of objective lenses that are of different powers—typically 4x, 10x, 40x, and 100x. This makes a choice of different magnifications possible with the same instrument.

The condenser lens does not affect the magnification but, positioned between the light source and the specimen, is used to focus the illumination on the specimen.

Eyepiece. A magnifying lens, usually about 10X,

Specimen stage.

Condenser. Focuses the light.

Iris diaphragm. Controls the amount of light that enters the objective lens.

Resolution

The usefulness of a microscope depends not so much on its degree of magnification, but on its ability to clearly separate, or resolve, two objects that are very close together. The resolving power is defined as the minimum distance existing between two objects when those objects can still be observed as separate entities. The resolving power therefore determines how much detail actually can be seen (figure 3.2).

Resolving power of a microscope depends on the quality and type of lens, magnification, and how the specimen under observation has been prepared. At higher magnifications it can also be limited by the wavelength of the light used for illumination. The shorter the wavelength, the greater the resolution. The maximum resolving power of the best light microscope is 0.2 mm. This is sufficient to observe the general morphology of a prokaryotic cell but too low to distinguish a particle the size of a virus.

To obtain maximum resolution when using certain highpower objectives such as the 100x lens, oil is used to displace the air between the lens and the specimen. This avoids the bending of light rays, or refraction, that occurs when light passes from glass to air (figure 3.3). Refraction can prevent those rays from entering the relatively small openings of higher-power objective lenses. The oil has nearly the same refractive index as glass. Refractive index is a measure of the relative velocity of light as it passes through a medium. As light travels from a medium of one refractive index to another, those rays are bent. When oil displaces air at the interface of the glass slide and glass lens, light rays pass with little refraction occurring.

Contrast

Contrast reflects the number of visible shades in a specimen— high contrast being just two shades, black and white. Different specimens require various degrees of contrast to reveal the most information. One example is bacteria, which are essentially transparent against a bright colorless background. The lack of contrast presents a problem when viewing objects (see figure 11.19). One way to overcome this problem is to stain the bacteria with any one of a number of dyes. The types and characteristics of these stains will be discussed shortly.

Specimen stage.

Iris diaphragm. Controls the amount of light that enters the objective lens.

Objective lens.

A number of lenses that provide different magnifications. The total magnification is the product of the magnification of the eyepiece lens and the objective lenses.

Light source with means to control amount of light.

Knob to control intensity of light.

Figure 3.1 A Modern Light Microscope The compound microscope employs a series of magnifying lenses.

3.1 Microscopic Techniques: The Instruments

Objective lens.

A number of lenses that provide different magnifications. The total magnification is the product of the magnification of the eyepiece lens and the objective lenses.

Light source with means to control amount of light.

Knob to control intensity of light.

Figure 3.1 A Modern Light Microscope The compound microscope employs a series of magnifying lenses.

Light Microscopes that Increase Contrast

Special light microscopes that increase the contrast between microorganisms and their surroundings overcome some of the difficulties of observing unstained bacteria. Staining kills microbes; therefore, some of these microscopes are invaluable when the goal is to examine characteristics of living organisms such as motility.

The Phase-Contrast Microscope

The phase-contrast microscope exploits the difference between the refractive index of cells and the surrounding medium,

Light microscope (450x)

Figure 3.2 Comparison of the Resolving Power of the Light Microscope and an Electron Microscope In this case, an onion root tip was magnified 450x. Note the difference in the degree of detail that can be seen at the same magnification.

Light microscope (450x)

Figure 3.2 Comparison of the Resolving Power of the Light Microscope and an Electron Microscope In this case, an onion root tip was magnified 450x. Note the difference in the degree of detail that can be seen at the same magnification.

Chapter 3 Microscopy and Cell Structure

Chapter 3 Microscopy and Cell Structure

(a)

Light source

Figure 3.3 Refraction As light passes from one medium to another, the light rays may bend, depending on the refractive index of the two media. (a)The pencil in water appears bent because the refractive index of water is different from that of air. (b) Light rays bend as they pass from air to glass because of the different refractive indexes of these two media; some rays are lost to the objective. Oil and glass have the same refractive index, and therefore the light rays are not bent.

Light source

Figure 3.3 Refraction As light passes from one medium to another, the light rays may bend, depending on the refractive index of the two media. (a)The pencil in water appears bent because the refractive index of water is different from that of air. (b) Light rays bend as they pass from air to glass because of the different refractive indexes of these two media; some rays are lost to the objective. Oil and glass have the same refractive index, and therefore the light rays are not bent.

resulting in a darker appearance of the denser material (figure 3.4). As light passes through cells, it is refracted slightly differently than when it passes through its surroundings. Special optical devices amplify those differences, thereby increasing the contrast.

The Interference Microscope

The interference microscope causes the specimen to appear as a three-dimensional image (figure 3.5). This microscope, like the phase-contrast microscope, depends on differences in refractive index as light passes through different materials. The most frequently used microscope of this type is the Nomarski differential interference contrast (DIC) microscope, which has a device for separating light into two beams that pass through

Figure 3.4 Phase-Contrast Photomicrograph Filaments of a species of the cyanobacterium Lyngbya.

Figure 3.4 Phase-Contrast Photomicrograph Filaments of a species of the cyanobacterium Lyngbya.

Figure 3.5 Nomarski Differential Interference Contrast (DIC) Microscopy

Protozoan (Paracineta) attached to green algae (Spongomorpha) (320x).

Figure 3.5 Nomarski Differential Interference Contrast (DIC) Microscopy

Protozoan (Paracineta) attached to green algae (Spongomorpha) (320x).

the specimen and then recombine. The light waves are out of phase when they recombine, thereby yielding the three-dimensional appearance of the specimen.

The Dark-Field Microscope

Organisms viewed through a dark-field microscope stand out as bright objects against a dark background (figure 3.6). The microscope operates on the same principle that makes dust visible when a beam of bright light shines into a dark room. A special mechanism directs light toward the specimen at an angle, so that only light scattered by the specimen enters the objective lens. Dark-field microscopy can detect members of the genus

Filamentous alga (Spirogyra)

Colonial alga ( Volvox)

Figure 3.6 Dark-Field Photomicrograph

(filaments), both of which are eukaryotes.

Filamentous alga (Spirogyra)

Colonial alga ( Volvox)

Figure 3.6 Dark-Field Photomicrograph

(filaments), both of which are eukaryotes.

Volvox (sphere) and Spirogyra

3.1 Microscopic Techniques: The Instruments

3.1 Microscopic Techniques: The Instruments

Figure 3.7 Fluorescence Photomicrograph A rod-shaped bacterium tagged with a fluorescent marker.

Figure 3.7 Fluorescence Photomicrograph A rod-shaped bacterium tagged with a fluorescent marker.

Treponema. These thin, spiral-shaped organisms stain poorly and are difficult to see via bright-field microscopy (see figure 11.27).

The Fluorescence Microscope

The fluorescence microscope is used to observe cells or other material that are either naturally fluorescent or have been stained or tagged with fluorescent dyes. A fluorescent molecule absorbs light at one wavelength (usually ultraviolet light) and then emits light of a longer wavelength.

The fluorescence microscope projects ultraviolet light through a specimen, but then captures only the light emitted by the fluorescent molecules to form the image. This allows fluorescent cells to stand out as illuminated objects against a dark background (figure 3.7). The color that the cells will appear depends on the type of dye and the light filters used. The types and characteristics of fluorescent dyes and tags will be discussed shortly. ■ fluorescent dyes and tags, p. 49

A common variation of the standard fluorescence microscope is the epifluores-cence microscope, which projects the ultraviolet light through the objective lens and onto the specimen. Because the light is not transmitted through the specimen, cells can be observed attached to soil particles or other opaque materials.

The Confocal Scanning Laser Microscope

The confocal scanning laser microscope is used to construct a three-dimensional image of a thick structure such as a community of microorganisms (figure 3.8). The instrument can also provide detailed sectional views of the interior of an intact cell. In confocal microscopy, lenses focus a laser beam to illuminate a given point on one vertical plane of a specimen. Mirrors then scan the laser beam across the specimen, illuminating successive regions and planes until the entire specimen has been scanned. Each plane corresponds to an image of one fine slice of the specimen. A

computer then assembles the data and constructs a three-dimensional image, which is displayed on a screen. In effect, this microscope is a miniature CAT scan for cells.

Frequently, the specimens are first stained or tagged with a fluorescent dye. By using certain fluorescent tags that bind specifically to a given protein or other compound, the precise cellular location of that compound can be determined. In some cases, multiple different tags that bind to specific molecules are used, each having a distinct color.

Electron Microscopes

Electron microscopy is in some ways comparable to light microscopy. Rather than using glass lenses, visible light, and the eye to observe the specimen, the electron microscope uses electromagnetic lenses, electrons, and a fluorescent screen to produce the magnified image (figure 3.9). That image can be

Figure 3.8 Confocal Microscopy This technique can be used to produce a clear image of a single plane in a thick structure. (a) Confocal photomicrograph of fava bean mitosis. (b) Regular photomicrograph.

44 Chapter 3 Microscopy and Cell Structure

Light Microscope

Lamp

Condenser lens

Specimen

Objective lens

Eyepiece

Final image seen by eye

Light Microscope

Lamp

Condenser lens

Specimen

Objective lens

Eyepiece

Transmission Electron Microscope

Electron gun

Transmission Electron Microscope

Electron gun

Condenser lens magnet

Specimen

Objective lens magnet

Condenser lens magnet

Specimen

Objective lens magnet

Projector lens magnet

Final image on fluorescent screen or

Final image on photographic film when screen is lifted aside

Figure 3.9 Comparison of the Principles of Light and the Electron Microscopy For the sake of comparison, the light source for the light microscope has been inverted (the light is shown at the top and the eyepiece, or ocular lens, at the bottom).

(a)

Figure 3.10 Transmission Electron Photomicrograph A rod-shaped bacterium prepared by (a) thin section; (b) freeze etching.

Figure 3.10 Transmission Electron Photomicrograph A rod-shaped bacterium prepared by (a) thin section; (b) freeze etching.

captured on photographic film to create an electron photomicrograph. Sometimes, the black and white images are artificially enhanced with color to add visual clarity. ■ electrons, p. 18

Since the electrons have a wavelength about 1,000 times shorter than visible light, the resolving power increases about 1,000-fold, to about 0.3 nanometers (nm) or 0.3 x 10:3 mm. Consequently, considerably more detail can be observed due to the much higher resolution. These instruments can clearly magnify an image 100,000x. One of the biggest drawbacks of the microscope is that the lenses and specimen must all be in a vacuum. Otherwise, the molecules composing air would interfere with the path of the electrons. This results in an expensive, bulky unit and requires substantial and complex specimen preparation.

The Transmission Electron Microscope

The transmission electron microscope (TEM) is used to observe fine details of cell structure, such as the number of layers that envelop a cell. The instrument directs a beam of electrons at a specimen. Depending on the density of a particular region in the specimen, electrons will either pass through or be scattered to varying degrees. The darker areas of the resulting image correspond to the denser portions of the specimen (figure 3.10).

Transmission electron microscopy requires elaborate and painstaking specimen preparation. To view details of internal structure, a process called thin sectioning is used. Cells are carefully treated with a preservative and dehydrated in an organ ic solvent before being embedded in a plastic resin. Once embedded, they can be cut into exceptionally thin slices with a diamond or glass knife and then stained with heavy metals. Even a single bacterial cell must be cut into slices this way to be viewed via TEM. Unfortunately, the procedure can severely distort the cells. Consequently, a major concern in using TEM is distinguishing actual cell components from artifacts occurring as a result of specimen preparation.

A process called freeze fracturing is used to observe the shape of structures within the cell. The specimen is rapidly frozen and then fractured by striking it with a knife blade. The cells break open, usually along the middle of internal membranes. Next, the surface of the section is coated with a thin layer of carbon to create a replica of the surface. This replica is then examined in the electron microscope. A variation of freeze fracturing is freeze etching. In this process, the frozen surface exposed by fracturing is dried slightly under vacuum, which allows underlying regions to be exposed.

The Scanning Electron Microscope

The scanning electron microscope (SEM) is used for observing surface details, but not internal structures of cells. The SEM does not transmit electrons through the specimen; instead, a beam of electrons scans back and forth over the surface of a specimen coated with a thin film of metal. As those beams move, electrons are released from the specimen and reflected

Figure 3.11 Scanning Electron Photomicrograph A rod-shaped bacterium.

3.2 Microscopic Techniques: Dyes and Staining and its surroundings. The fluorescence microscope is used to observe microbes stained with special dyes. The confocal scanning laser microscope is used to construct a three-dimensional image of a thick structure. Electron microscopes can magnify images 100,000x. Scanning probe microscopes map images on an atomic scale.

■ Why might oil be used when using the 100x lens?

■ Why are microscopes that enhance contrast used to view live rather than stained specimens?

■ If an object being viewed under the phase-contrast microscope has the same refractive index as the background material, how would it appear?

Figure 3.11 Scanning Electron Photomicrograph A rod-shaped bacterium.

Figure 3.12 Scanning Probe Photomicrograph DNA-protein complex.

Figure 3.12 Scanning Probe Photomicrograph DNA-protein complex.

back into the viewing chamber. This reflected radiation is observed with the microscope. Relatively large specimens can be viewed, and a dramatic three-dimensional effect is observed with the SEM (figure 3.11).

Scanning Probe Microscopes

Scanning probe microscopes make it possible to view images at an atomic scale (figure 3.12). Their resolving power is much greater than the electron microscope, and the samples do not need special preparation as they do for electron microscopy. One such microscope, developed in the early 1980s, is the scanning tunneling microscope. This instrument has a sharp metallic probe, the tip of which is the size of a single atom. Electrons tunnel between the probe and a conductive surface. By scanning across a surface, this instrument produces a map, which shows the bumps and valleys of the atoms on that surface. A more recent instrument is the atomic force microscope, which has a tip that can bend in response to even the slightest force between the tip and the sample. By monitoring the motion of the tip as it scans across a surface, a map of the surface is produced. Unlike the scanning tunneling microscope, the atomic force microscope does not rely on a vacuum and thus can operate in air and in liquids.