Instruments of Microscopy Quiz

PDF page 63: microscopes, and scanning probe microscopes The early pioneers of microscopy opened a window into the invisible world of microorganisms. But microscopy continued to advance in the centuries that followed. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy, which uses an ultraviolet light source, and electron microscopy, which uses shortwavelength electron beams. These advances led to major improvements in magnification, resolution, and contrast. By comparison, the relatively rudimentary microscopes of van Leeuwenhoek and his contemporaries were far less powerful than even the most basic microscopes in use today. In this section, we will survey the broad range of modern microscopic technology and common applications for each type of microscope.

PDF page 63: microscopes, and scanning probe microscopes The early pioneers of microscopy opened a window into the invisible world of microorganisms. But microscopy continued to advance in the centuries that followed. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes that leveraged nonvisible light, such as fluorescence microscopy, which uses an ultraviolet light source, and electron microscopy, which uses shortwavelength electron beams. These advances led to major improvements in magnification, resolution, and contrast. By comparison, the relatively rudimentary microscopes of van Leeuwenhoek and his contemporaries were far less powerful than even the most basic microscopes in use today. In this section, we will survey the broad range of modern microscopic technology and common applications for each type of microscope.

PDF page 64: Brightfield Microscopes The brightfield microscope, perhaps the most commonly used type of microscope, is a compound microscope with two or more lenses that produce a dark image on a bright background. Some brightfield microscopes are monocular (having a single eyepiece), though most newer brightfield microscopes are binocular (having two eyepieces), like the one shown in Figure 2.12; in either case, each eyepiece contains a lens called an ocular lens. The ocular lenses typically magnify images 10 times (10⨯). At the other end of the body tube are a set of objective lenses on a rotating nosepiece. The magnification of these objective lenses typically ranges from 4⨯ to 100⨯, with the magnification for each lens designated on the metal casing of the lens. The ocular and objective lenses work together to create a magnified image. The total magnification is the product of the ocular magnification times the objective magnification: ocular magnificatio × objective magnificatio For example, if a 40⨯ objective lens is selected and the ocular lens is 10⨯, the total magnification would be

PDF page 64: Brightfield Microscopes The brightfield microscope, perhaps the most commonly used type of microscope, is a compound microscope with two or more lenses that produce a dark image on a bright background. Some brightfield microscopes are monocular (having a single eyepiece), though most newer brightfield microscopes are binocular (having two eyepieces), like the one shown in Figure 2.12; in either case, each eyepiece contains a lens called an ocular lens. The ocular lenses typically magnify images 10 times (10⨯). At the other end of the body tube are a set of objective lenses on a rotating nosepiece. The magnification of these objective lenses typically ranges from 4⨯ to 100⨯, with the magnification for each lens designated on the metal casing of the lens. The ocular and objective lenses work together to create a magnified image. The total magnification is the product of the ocular magnification times the objective magnification: ocular magnificatio × objective magnificatio For example, if a 40⨯ objective lens is selected and the ocular lens is 10⨯, the total magnification would be

PDF page 64: Brightfield Microscopes The brightfield microscope, perhaps the most commonly used type of microscope, is a compound microscope with two or more lenses that produce a dark image on a bright background. Some brightfield microscopes are monocular (having a single eyepiece), though most newer brightfield microscopes are binocular (having two eyepieces), like the one shown in Figure 2.12; in either case, each eyepiece contains a lens called an ocular lens. The ocular lenses typically magnify images 10 times (10⨯). At the other end of the body tube are a set of objective lenses on a rotating nosepiece. The magnification of these objective lenses typically ranges from 4⨯ to 100⨯, with the magnification for each lens designated on the metal casing of the lens. The ocular and objective lenses work together to create a magnified image. The total magnification is the product of the ocular magnification times the objective magnification: ocular magnificatio × objective magnificatio For example, if a 40⨯ objective lens is selected and the ocular lens is 10⨯, the total magnification would be

PDF page 64: The item being viewed is called a specimen. The specimen is placed on a glass slide, which is then clipped into place on the stage (a platform) of the microscope. Once the slide is secured, the specimen on the slide is positioned over the light using the x-y mechanical stage knobs. These knobs move the slide on the surface of the stage, but do not raise or lower the stage. Once the specimen is centered over the light, the stage position can be raised or lowered to focus the image. The coarse focusing knob is used for large-scale movements with 4⨯ and 10⨯ objective lenses; the fine focusing knob is used for small-scale movements, especially with 40⨯ or 100⨯ objective lenses. When images are magnified, they become dimmer because there is less light per unit area of image. Highly magnified images produced by microscopes, therefore, require intense lighting. In a brightfield microscope, this light is provided by an illuminator, which is typically a high-intensity bulb below the stage. Light from the illuminator passes up through condenser lens (located below the stage), which focuses all of the light rays on the specimen to maximize illumination. The position of the condenser can be optimized using the attached condenser focus knob; once the optimal distance is established, the condenser should not be moved to adjust the brightness. If less-than-maximal light

PDF page 64: The item being viewed is called a specimen. The specimen is placed on a glass slide, which is then clipped into place on the stage (a platform) of the microscope. Once the slide is secured, the specimen on the slide is positioned over the light using the x-y mechanical stage knobs. These knobs move the slide on the surface of the stage, but do not raise or lower the stage. Once the specimen is centered over the light, the stage position can be raised or lowered to focus the image. The coarse focusing knob is used for large-scale movements with 4⨯ and 10⨯ objective lenses; the fine focusing knob is used for small-scale movements, especially with 40⨯ or 100⨯ objective lenses. When images are magnified, they become dimmer because there is less light per unit area of image. Highly magnified images produced by microscopes, therefore, require intense lighting. In a brightfield microscope, this light is provided by an illuminator, which is typically a high-intensity bulb below the stage. Light from the illuminator passes up through condenser lens (located below the stage), which focuses all of the light rays on the specimen to maximize illumination. The position of the condenser can be optimized using the attached condenser focus knob; once the optimal distance is established, the condenser should not be moved to adjust the brightness. If less-than-maximal light

PDF page 65: levels are needed, the amount of light striking the specimen can be easily adjusted by opening or closing a diaphragm between the condenser and the specimen. In some cases, brightness can also be adjusted using the rheostat, a dimmer switch that controls the intensity of the illuminator. A brightfield microscope creates an image by directing light from the illuminator at the specimen; this light is differentially transmitted, absorbed, reflected, or refracted by different structures. Different colors can behave differently as they interact with chromophores (pigments that absorb and reflect particular wavelengths of light) in parts of the specimen. Often, chromophores are artificially added to the specimen using stains, which serve to increase contrast and resolution. In general, structures in the specimen will appear darker, to various extents, than the bright background, creating maximally sharp images at magnifications up to about 1000⨯. Further magnification would create a larger image, but without increased resolution. This allows us to see objects as small as bacteria, which are visible at about 400⨯ or so, but not smaller objects such as viruses. At very high magnifications, resolution may be compromised when light passes through the small amount of air between the specimen and the lens. This is due to the large difference between the refractive indices of air and glass; the air scatters the light rays before they can be focused by the lens. To solve this problem, a drop of oil can be used to fill the space between the specimen and an oil immersion lens, a special lens designed to be used with immersion oils. Since the oil has a refractive index very similar to that of glass, it increases the maximum angle at which light leaving the specimen can strike the lens. This increases the light collected and, thus, the resolution of the image (Figure 2.13). A variety of oils can be used for different types of light.

PDF page 65: levels are needed, the amount of light striking the specimen can be easily adjusted by opening or closing a diaphragm between the condenser and the specimen. In some cases, brightness can also be adjusted using the rheostat, a dimmer switch that controls the intensity of the illuminator. A brightfield microscope creates an image by directing light from the illuminator at the specimen; this light is differentially transmitted, absorbed, reflected, or refracted by different structures. Different colors can behave differently as they interact with chromophores (pigments that absorb and reflect particular wavelengths of light) in parts of the specimen. Often, chromophores are artificially added to the specimen using stains, which serve to increase contrast and resolution. In general, structures in the specimen will appear darker, to various extents, than the bright background, creating maximally sharp images at magnifications up to about 1000⨯. Further magnification would create a larger image, but without increased resolution. This allows us to see objects as small as bacteria, which are visible at about 400⨯ or so, but not smaller objects such as viruses. At very high magnifications, resolution may be compromised when light passes through the small amount of air between the specimen and the lens. This is due to the large difference between the refractive indices of air and glass; the air scatters the light rays before they can be focused by the lens. To solve this problem, a drop of oil can be used to fill the space between the specimen and an oil immersion lens, a special lens designed to be used with immersion oils. Since the oil has a refractive index very similar to that of glass, it increases the maximum angle at which light leaving the specimen can strike the lens. This increases the light collected and, thus, the resolution of the image (Figure 2.13). A variety of oils can be used for different types of light.

PDF page 65: levels are needed, the amount of light striking the specimen can be easily adjusted by opening or closing a diaphragm between the condenser and the specimen. In some cases, brightness can also be adjusted using the rheostat, a dimmer switch that controls the intensity of the illuminator. A brightfield microscope creates an image by directing light from the illuminator at the specimen; this light is differentially transmitted, absorbed, reflected, or refracted by different structures. Different colors can behave differently as they interact with chromophores (pigments that absorb and reflect particular wavelengths of light) in parts of the specimen. Often, chromophores are artificially added to the specimen using stains, which serve to increase contrast and resolution. In general, structures in the specimen will appear darker, to various extents, than the bright background, creating maximally sharp images at magnifications up to about 1000⨯. Further magnification would create a larger image, but without increased resolution. This allows us to see objects as small as bacteria, which are visible at about 400⨯ or so, but not smaller objects such as viruses. At very high magnifications, resolution may be compromised when light passes through the small amount of air between the specimen and the lens. This is due to the large difference between the refractive indices of air and glass; the air scatters the light rays before they can be focused by the lens. To solve this problem, a drop of oil can be used to fill the space between the specimen and an oil immersion lens, a special lens designed to be used with immersion oils. Since the oil has a refractive index very similar to that of glass, it increases the maximum angle at which light leaving the specimen can strike the lens. This increases the light collected and, thus, the resolution of the image (Figure 2.13). A variety of oils can be used for different types of light.

PDF page 66: A darkfield microscope is a brightfield microscope that has a small but significant modification to the condenser. A small, opaque disk (about 1 cm in diameter) is placed between the illuminator and the condenser lens. This opaque light stop, as the disk is called, blocks most of the light from the illuminator as it passes through the condenser on its way to the objective lens, producing a hollow cone of light that is focused on the specimen. The only light that reaches the objective is light that has been refracted or reflected by structures in the specimen. The resulting image typically shows bright objects on a dark background (Figure 2.14).

PDF page 67: Darkfield microscopy can often create high-contrast, high-resolution images of specimens without the use of stains, which is particularly useful for viewing live specimens that might be killed or otherwise compromised by the stains. For example, thin spirochetes like Treponema pallidum, the causative agent of syphilis, can be best viewed using a darkfield microscope (Figure 2.15).