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Chapter 2 : Microscopy

Author: Danny L. Wiedbrauk
Content Type: Reference
Publication Year: 2011

Category: Clinical Microbiology

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Microscopy, Page 1 of 2

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Abstract:

This chapter describes the basic concepts of light microscopy as they are practiced in the microbiology laboratory. Immersion fluids are used between the condenser and the microscope slide in transmitted light fluorescence microscopy and dark-field microscopy to minimize refraction, increase the numerical aperture of the objective, and improve optical resolution. As the refractive index of a material increases, light beams entering or leaving a material are deflected to a greater extent. Field diaphragm is located in the light path between the light source and the substage condenser. The resolving power of a microscope is the most important feature of the optical system because it defines one's ability to distinguish fine details in a specimen. Reducing the voltage will alter the color of the incoming light, and voltage changes are not recommended for photomicroscopy. Phase microscopy is an important tool for examining living and/or unstained material in wet mounts and cell cultures. In phase-contrast microscopy, structures within living cells appear as hills or craters, depending upon their optical thickness. Today, fluorescence microscopy is used in conjunction with nucleic acid hybridization to visualize the location of fluorescent in situ hybridization and multicolor fluorescent in situ hybridization probes. The availability of digital photomicroscopy has significantly enhanced the microbial identification process, and it has helped to standardize microbe identification. Microscopy still has a central role in the detection of infectious agents despite highly publicized advances in DNA and RNA detection systems.

Citation: Wiedbrauk D. 2011. Microscopy, p 5-14. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch2
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Microscopy
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Citation: Wiedbrauk D. 2011. Microscopy, p 5-14. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch2
/content/10.1128/9781555816728.chap2.fig2-1
FIGURE 1

Objective lens labeling. Objective lenses are labeled with information on the manufacturer, correction factors, NA, tube length, coverslip thickness, working distance (WD), and expected immersion medium. Objectives without a listed aberration correction are considered achromats. Objectives without a listed immersion medium (Oil, Oel, W, Gly) are considered dry objectives and are meant to operate with air between the lens and the specimen.

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10.1128/9781555816728/fig2-1.gif
Image of FIGURE 1
FIGURE 1

Objective lens labeling. Objective lenses are labeled with information on the manufacturer, correction factors, NA, tube length, coverslip thickness, working distance (WD), and expected immersion medium. Objectives without a listed aberration correction are considered achromats. Objectives without a listed immersion medium (Oil, Oel, W, Gly) are considered dry objectives and are meant to operate with air between the lens and the specimen.

Citation: Wiedbrauk D. 2011. Microscopy, p 5-14. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch2
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FIGURE 2

Typical configuration for bright-field microscopy. The column of light generated by the field lens and the field diaphragm enters the bottom of the condenser and is focused on the slide by the condenser lens. The condenser diaphragm controls the angle of the light, the NA of the condenser, and the amount of contrast in the image. The working distance is the vertical distance from the top of the specimen to the leading edge of the objective lens. The semiangle of the objective aperture (θ) is used to calculate NA. Modified from reference .

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Image of FIGURE 2
FIGURE 2

Typical configuration for bright-field microscopy. The column of light generated by the field lens and the field diaphragm enters the bottom of the condenser and is focused on the slide by the condenser lens. The condenser diaphragm controls the angle of the light, the NA of the condenser, and the amount of contrast in the image. The working distance is the vertical distance from the top of the specimen to the leading edge of the objective lens. The semiangle of the objective aperture (θ) is used to calculate NA. Modified from reference .

Citation: Wiedbrauk D. 2011. Microscopy, p 5-14. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch2
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FIGURE 3

Anatomy of a typical clinical microscope with an integral camera.

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Image of FIGURE 3
FIGURE 3

Anatomy of a typical clinical microscope with an integral camera.

Citation: Wiedbrauk D. 2011. Microscopy, p 5-14. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch2
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FIGURE 4

Dark-field illumination. The central light path interacts with the silvered dome located at the bottom of the condenser and is reflected away from the specimen. Peripheral light is reflected into the condenser and is reflected again by the internal condenser surfaces to produce a cone of light that is directed obliquely away from the objective.

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10.1128/9781555816728/fig2-4.gif
Image of FIGURE 4
FIGURE 4

Dark-field illumination. The central light path interacts with the silvered dome located at the bottom of the condenser and is reflected away from the specimen. Peripheral light is reflected into the condenser and is reflected again by the internal condenser surfaces to produce a cone of light that is directed obliquely away from the objective.

Citation: Wiedbrauk D. 2011. Microscopy, p 5-14. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch2
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References

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Tables

Microscopy
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Citation: Wiedbrauk D. 2011. Microscopy, p 5-14. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch2
/content/10.1128/9781555816728.chap2.tab2-1
TABLE 1

Resolving power of selected lenses with different NA

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Generic image for table
TABLE 1

Resolving power of selected lenses with different NA

Citation: Wiedbrauk D. 2011. Microscopy, p 5-14. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch2
/content/10.1128/9781555816728.chap2.tab2-2
TABLE 2

Excitation and emission wavelengths of commonly used fluorochromes

Excitation and emission wavelengths can vary depending upon the solvent and the pH of the solution.

TRITC, tetramethylrhodamine isothiocyanate.

10.1128/9781555816728/tab2-2_thmb.gif
10.1128/9781555816728/tab2-2.gif
Generic image for table
TABLE 2

Excitation and emission wavelengths of commonly used fluorochromes

Excitation and emission wavelengths can vary depending upon the solvent and the pH of the solution.

TRITC, tetramethylrhodamine isothiocyanate.

Citation: Wiedbrauk D. 2011. Microscopy, p 5-14. In Versalovic J, Carroll K, Funke G, Jorgensen J, Landry M, Warnock D (ed), Manual of Clinical Microbiology, 10th Edition. ASM Press, Washington, DC. doi: 10.1128/9781555816728.ch2

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