Knowing the Different Types of Microscopes and Their Functions

The human eye, a telescope, a pair of binoculars, a magnifying glass, and a microscope can all be thought of as various types of optical instruments. A microscope is an optical instrument that is used to observe tiny objects, often objects that cannot be seen at all with the unaided human eye (the “naked eye”). Each optical instrument has a limit as to what can be seen using that instrument. This limit is referred to as the resolving power or resolution of the instrument. Table 2-2 contains the resolving powers for various optical instruments.
Table: Characteristics of Various Types of Microscopes
Table: Characteristics of Various Types of Microscopes

Simple Microscopes 

A simple microscope is defined as a microscope containing only one magnifying lens. Actually, a magnifying glass could be considered a simple microscope. Images seen when using a magnifying glass usually appear about 3 to 20 times larger than the object’s actual size. During the late 1600s, Anton van Leeuwenhoek, who was discussed in 3 Pioneers in the Science of Microbiology, used simple microscopes to observe many tiny objects, including bacteria and protozoa (Fig.1). Because of his unique ability to grind glass lenses, scientists believe that Leeuwenhoek’s simple microscopes had a maximum magnifying power of about x300 (300 times).
Leeuwenhoek’s microscopes. A. Leeuwenhoek’s microscopes were very simple devices. Each had a tiny glass lens, mounted in a brass plate. The specimen was placed on the sharp point of a brass pin, and two screws were used to adjust the position of the specimen. The entire instrument was about 3 to 4 in long. It was held very close to the eye. (Courtesy of Biomed Ed, Round Rock, TX.) B. Although his microscopes had a magnifying capability of only around x200 to x300, he was able to create remarkable drawings of different types of bacteria that he observed. (From Volk WA, Gebhardt BM, Hammarskjold ML, et al. Essentials of Medical Microbiology. 5th ed. Philadelphia, PA: Lippincott-Raven; 1996.)
Fig.1 : Leeuwenhoek’s microscopes. A. Leeuwenhoek’s microscopes were very simple devices. Each had a tiny glass lens, mounted in a brass plate. The specimen was placed on the sharp point of a brass pin, and two screws were used to adjust the position of the specimen. The entire instrument was about 3 to 4 in long. It was held very close to the eye. (Courtesy of Biomed Ed, Round Rock, TX.) B. Although his microscopes had a magnifying capability of only around x200 to x300, he was able to create remarkable drawings of different types of bacteria that he observed. (From Volk WA, Gebhardt BM, Hammarskjold ML, et al. Essentials of Medical Microbiology. 5th ed. Philadelphia, PA: Lippincott-Raven; 1996.)

Compound Microscopes 

A compound microscope is a microscope that contains more than one magnifying lens. Although the first person to construct and use a compound microscope is not known with certainty, Hans Jansen and his son Zacharias are often given credit for being the first (see the following “Historical Note”). Compound light microscopes usually magnify objects about 1,000 times. Photographs taken through the lens system of compound microscopes are called photomicrographs. A simple microscope contains only one magnifying lens, whereas a compound microscope contains more than one magnifying lens.

Because visible light (from a built-in light bulb) is used as the source of illumination, the compound microscope is also referred to as a compound light microscope. It is the wavelength of visible light (approximately 0.45 μm) that limits the size of objects that can be seen using the compound light microscope. When using the compound light microscope, objects cannot be seen if they are smaller than half of the wavelength of visible light (i.e., smaller than about 0.225 μm). A compound light microscope is shown in Figure 2, and the functions of its various components are described in Table 2-3.
A modern compound light microscope.
Fig. 2: A modern compound light microscope.
Table: Components of the Compound Light Microscope
Table: Components of the Compound Light Microscope

The compound light microscopes used in today’s laboratories contain two magnifying lens systems. Within the eyepiece or ocular is a lens called the ocular lens; it usually has a magnifying power of x10. The second magnifying lens system is in the objective, which is positioned immediately above the object to be viewed. The four objectives used in most laboratory compound light microscopes are x4, x10, x40, and x100 objectives. As shown in Table 2-4, total magnification is calculated by multiplying the magnifying power of the ocular (x10) by the magnifying power of the objective that you are using.
Table: Magnifications Achived Using the Compound Light Microscope
Table: Magnifications Achived Using the Compound Light Microscope

The x4 objective is rarely used in microbiology laboratories. Usually, specimens are first observed using the x10 objective. Once the specimen is in focus, the highpower or “high-dry” objective is then swung into position. This lens can be used to study algae, protozoa, and other large microorganisms. However, the oil-immersion objective (total magnification = x1,000) must be used to study bacteria, because they are so tiny. To use the oilimmersion objective, a drop of immersion oil must first be placed between the specimen and the objective; the immersion oil reduces the scattering of light and ensures that the light will enter the oil-immersion lens. The oilimmersion objective cannot be used without immersion oil. Oil is not required when using the other objectives.

Total magnification of the compound light microscope is calculated by multiplying the magnifying power of the ocular lens by the magnifying power of the objective being used. For optimal observation of the specimen, the light must be properly adjusted and focused. The condenser, located beneath the stage, focuses light onto the specimen, adjusts the amount of light, and shapes the cone of light entering the objective. Generally, the higher the magnification, the more light that is needed.

As magnification is increased, the amount of light striking the object being examined must also be increased. There are three correct ways to accomplish this: (a) by opening the iris diaphragm in the condenser, (b) by opening the field diaphragm, and (c) by increasing the intensity of light being emitted from the microscope’s light bulb, by turning the rheostat knob clockwise. Turning the knob that raises and lowers the condenser is an incorrect way to adjust lighting.

Magnification alone is of little value unless the enlarged image possesses increased detail and clarity. Image clarity depends on the microscope’s resolving power (or resolution), which is the ability of the lens system to distinguish between two adjacent objects. If two objects are moved closer and closer together, there comes a point when the objects are so close together that the lens system can no longer resolve them as two separate objects (i.e., they are so close together that they appear to be one object). That distance between them, at which they cease to be seen as separate objects, is referred to as the resolving power of the optical instrument. Knowing the resolving power of an optical instrument also defines the smallest object that can be seen with that instrument. For example, the resolving power of the unaided human eye is approximately 0.2 mm. Thus, the unaided human eye is unable to see objects smaller than 0.2 mm in diameter.

The resolving power of the compound light microscope is approximately 1,000 times better than the resolving power of the unaided human eye. In practical terms, this means that objects can be examined with the compound microscope that are as much as 1,000 times smaller than the smallest objects that can be seen with the unaided human eye. Using a compound light microscope, we can see objects down to about 0.2 μm in diameter.

The resolving power of the compound light microscope is approximately 0.2 μm, which is approximately one half the wavelength of visible light. Additional magnifying lenses could be added to the compound light microscope, but this would not increase the resolving power. As stated earlier, as long as visible light is used as the source of illumination, objects smaller than half of the wavelength of visible light cannot be seen. Increasing magnification without increasing the resolving power is called empty magnification. It does no good to increase magnification without increasing resolving power.

Because objects are observed against a bright background (or “bright field”) when using a compound light microscope, that microscope is sometimes referred to as a brightfield microscope. If the regularly used condenser is replaced with what is known as a darkfield condenser, illuminated objects are seen against a dark background (or “dark field”), and the microscope has been converted into a darkfield microscope. In the clinical microbiology laboratory, darkfield microscopy is routinely used to diagnose primary syphilis (the initial stage of syphilis). The etiologic (causative) agent of syphilis—a spiral-shaped bacterium, named Treponema pallidum—cannot be seen with a brightfield microscope because it is thinner than 0.2 μm and, therefore, is beneath the resolving power of the compound light microscope. T. pallidum can be seen using a darkfield microscope, however, much in the same way that you can “see” dust particles in a beam of sunlight. Dust particles are actually beneath the resolving power of the unaided eye and, therefore, cannot really be seen. What you see in the beam is sunlight being reflected off the dust particles. With the darkfield microscope, laboratory technologists do not really see the treponemes—they see the light being reflected off the bacteria, and that light is easily seen against the dark background (Fig. 3).
Spiral-shaped T. pallidum bacterium. The causative agent of syphilis, as seen by darkfield microscopy. (Courtesy of the Centers for Disease Control and Prevention [CDC].)
Fig.3 : Spiral-shaped T. pallidum bacterium. The causative agent of syphilis, as seen by darkfield microscopy. (Courtesy of the Centers for Disease Control and Prevention [CDC].)

Other types of compound microscopes include phasecontrast microscopes and fluorescence microscopes. Phase-contrast microscopes can be used to observe unstained living microorganisms. Because the light refracted by living cells is different from the light refracted by the surrounding medium, contrast is increased, and the organisms are more easily seen. Fluorescence microscopes contain a built-in ultraviolet (UV) light source. When UV light strikes certain dyes and pigments, these substances emit a longer wavelength light, causing them to glow against a dark background (Fig.4). Fluorescence microscopy is often used in immunology laboratories to demonstrate that antibodies stained with a fluorescent dye have combined with specific antigens; this is a type of immunodiagnostic procedure.
Photomicrograph of T. pallidum spirochetes using immunofluorescence. A fluorescent dye is first attached to anti-T. pallidum antibodies. The antibodies are then attached to the surface of the bacteria. When examined under UV light, the fluorescent dye emits a greenish light. (Courtesy of Russell and the CDC.)
Fig.4 : Photomicrograph of T. pallidum spirochetes using immunofluorescence. A fluorescent dye is first attached to anti-T. pallidum antibodies. The antibodies are then attached to the surface of the bacteria. When examined under UV light, the fluorescent dye emits a greenish light. (Courtesy of Russell and the CDC.)

Early Compound Microscopes 

Hans Jansen, an optician in Middleburg, Holland, is often given credit for developing the first compound microscope, sometime between 1590 and 1595. Although his son, Zacharias, was only a young boy at the time, Zacharias apparently later took over production of the Jansen microscopes. The Jansen microscopes contained two lenses and achieved magnifications of only 33 to 39. Compound microscopes having a three lens system were later used by Marcello Malpighi in Italy and Robert Hooke in England, both of whom published papers between 1660 and 1665 describing their microscopic findings. In his 1665 book entitled Micrographia, Hooke described a fossilized shell of a foraminiferana type of protozoan and two species of microscopic fungi. Some scientists consider these to be the first written descriptions of microorganisms and feel that Hooke (rather than Leeuwenhoek) should be given credit for discovering microbes. Some early compound microscopes are shown in Figure 5.
A simple Leeuwenhoek microscope (center), surrounded by examples of early compound light microscopes.
Fig.5 : A simple Leeuwenhoek microscope (center), surrounded by examples of early compound light microscopes.

Electron Microscopes 

Although extremely small infectious agents, such as rabies and smallpox viruses, were known to exist, they could not be seen until the electron microscope was developed. It should be noted that electron microscopes cannot be used to observe living organisms. Organisms are killed during the specimen-processing procedures. Even if they were not, they would be unable to survive in the vacuum created within the electron microscope.

Electron microscopes use an electron beam as a source of illumination and magnets to focus the beam. Because the wavelength of electrons traveling in a vacuum is much shorter than the wavelength of visible light—about 100,000 times shorter—electron microscopes have a much greater resolving power than compound light microscopes. There are two types of electron microscopes: transmission electron microscopes (TEMs) and scanning electron microscopes (SEMs).
A CDC intern using a TEM. (Courtesy of Cynthia Goldsmith, James Gathany, and the CDC.)
Fig.6 : A CDC intern using a TEM. (Courtesy of Cynthia Goldsmith, James Gathany, and the CDC.)

A TEM (Fig. 6) has a tall column, at the top of which an electron gun fires a beam of electrons downward. When an extremely thin specimen (less than 1 μm thick) is placed into the electron beam, some of the electrons are transmitted through the specimen, and some are blocked. An image of the specimen is produced on a phosphor-coated screen at the bottom of the microscope’s column. The object can be magnified up to approximately 1 million times. Thus, using a TEM, a magnification is achieved that is about 1,000 times greater than the maximum magnification achieved using a compound light microscope. Even very tiny microbes (e.g.,viruses) can be observed using a TEM (Fig. 7). Because thin sections of cells are examined, transmission electron microscopy enables scientists to study the internal structure of cells. Special staining procedures are used to increase contrast between different parts of the cell. The first TEMs were developed during the late 1920s and early 1930s, but it was not until the early 195s that electron microscopes began to be used routinely to study cells.
Transmission electron micrograph of influenza virus A. (From Winn WC Jr, Allen S, Janda W, et al. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2006.)
Fig.7 : Transmission electron micrograph of influenza virus A. (From Winn WC Jr, Allen S, Janda W, et al. Koneman’s Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2006.)

The resolving power of a TEM is approximately 0.2 nm, which is about 1 million times better than the resolving power of the unaided human eye and 1,000 times better than the resolving power of the compound light microscope. An SEM (Fig. 8) has a shorter column, and instead of being placed into the electron beam, the specimen is placed at the bottom of the column. Electrons that bounce off the surface of the specimen are captured by detectors, and an image of the specimen appears on a monitor. SEMs are used to observe the outer surfaces of specimens (i.e., surface detail). Although the resolving power of SEMs (about 20 nm) is not quite as good as the resolving power of TEMs (about 0.2 nm), it is still possible to observe extremely tiny objects using an SEM. SEMs became available during the late 1960s.
Scanning electron microscope. (Courtesy of Jim Yost and the National Renewable Energy Institute.)
Fig.8 : Scanning electron microscope. (Courtesy of Jim Yost and the National Renewable Energy Institute.)

Both types of electron microscopes have built-in camera systems. The photographs taken using TEM and SEM are called transmission electron micrographs and scanning electron micrographs, respectively. They are black and white images. If you ever see electron micrographs in color, they have been artificially colorized. Figures 9 to 11 show the differences in magnification and detail between photomicrographs and electron micrographs. Note that Figures 9 to 11 all depict Staphylococcus aureus bacteria, but each of these photographs was made using a different type of microscope. Refer back to  Table  2-2 for the characteristics of various types of microscopes.
A grapelike cluster of blue-stained S. aureus bacteria and red blood cells, as seen by light microscopy. (From Marler LM, Siders JA, Allen SD. Direct Smear Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 2001.)
Fig.9 : A grapelike cluster of blue-stained S. aureus bacteria and red blood cells, as seen by light microscopy. (From Marler LM, Siders JA, Allen SD. Direct Smear Atlas. Philadelphia, PA: Lippincott Williams & Wilkins; 2001.)

Transmission electron micrograph of S. aureus showing S. aureus cells in various stages of binary fission. (From Volk WA, Gebhardt BM, Hammarskjold ML, et al. Essentials of Medical Microbiology. 5th ed. Philadelphia, PA: Lippincott-Raven; 1996.)
Fig.10 : Transmission electron micrograph of S. aureus showing S. aureus cells in various stages of binary fission. (From Volk WA, Gebhardt BM, Hammarskjold ML, et al. Essentials of Medical Microbiology. 5th ed. Philadelphia, PA: Lippincott-Raven; 1996.)

Scanning electron micrograph of S. aureus. (Courtesy of Janice Carr, Matthew J. Arduino, and the CDC.)
Fig.11 : Scanning electron micrograph of S. aureus. (Courtesy of Janice Carr, Matthew J. Arduino, and the CDC.)


Photographs taken using compound light microscopes are called photomicrographs. Those taken using TEMs and SEMs are called transmission electron micrographs and scanning electron micrographs, respectively.

Atomic Force Microscopes 

Neither TEMs nor SEMs enable scientists to observe live microbes because of the required specimen-processing procedures and subjection of the specimens to a vacuum. Atomic force microscopy (AFM) enables scientists to observe living cells at extremely high magnification and resolution under physiological conditions. Using AFM, it is possible to observe single live cells in aqueous solutions where dynamic physiological processes can be observed in real time. Unlike the SEM, which provides a two-dimensional image of a sample, the AFM provides a true three-dimensional surface profile.
Atomic force microscope. See text for details. PZT, lead zirconate titanate. (Courtesy of Askewmind and Wikipedia.)
Fig.12 : Atomic force microscope. See text for details. PZT, lead zirconate titanate. (Courtesy of Askewmind and Wikipedia.)

Figure 12 is a diagrammatic representation of an AFM. A silicon or silicon nitride cantilever having a sharp tip (probe) at its end is used to scan the specimen surface. When the tip is brought in proximity to a sample surface, forces between the tip and the sample lead to a deflection of the cantilever. Typically, the deflection is measured using a laser spot reflected from the top surface of the cantilever into an array of photodiodes, creating an image on a monitor screen. Additional information regarding AFM can be found using an Internet search engine.

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