Bernadette F. Rodak
Principles of Microscopy
Component Parts and Their Functions
Operating Procedure with Koehler Illumination
Immersion Oil and Types
Care of the Microscope
Other Microscopes Used in the Clinical Laboratory
Polarized Light Microscope
After completion of this chapter, the reader will be able to:
1. Given a diagram of a brightfield light microscope, identify the component parts.
2. Explain the function of each component of a brightfield light microscope.
3. Define achromatic, plan achromatic, parfocal, and parcentric as applied to lenses and microscopes; explain the advantages and disadvantages of each; and recognize examples of each from written descriptions of microscope use and effects.
4. Explain the purpose of and proper order of steps for adjusting microscope light using Koehler illumination.
5. Describe the proper steps for viewing a stained blood film with a brightfield light microscope, including use of oil immersion lenses, and recognize deviations from these procedures.
6. Describe the proper care and cleaning of microscopes and recognize deviations from these procedures.
7. Given the magnification of lenses in a compound microscope, calculate the total magnification.
8. Given a problem with focusing a blood film using a brightfield light microscope, suggest possible causes and their correction.
9. For each of the following, describe which components of the microscope differ from those of a standard light microscope, what the differences accomplish, and what are the uses and benefits of each type in the clinical laboratory:
• Phase-contrast microscope
• Polarized light microscope
• Darkfield microscope
After studying the material in this chapter, the reader should be able to respond to the following case study:
A Wright-stained peripheral blood film focuses under 10× and 40× but does not come into focus under the 100× oil objective. What steps should be taken to identify and correct this problem?
Microscopes available today reflect improvement in every aspect from the first microscope of Anton van Leeuwenhoek (1632-1723).1 Advanced technology as applied to microscopy has resulted in computer-designed lens systems, sturdier stands, perfected condensers, and built-in illumination systems. Microscopes can be fitted with multiple viewing heads for teaching or conferences, or they can be attached to a computer to allow an object to be projected onto a monitor or a large screen. Regular care and proper cleaning ensure continued service from this powerful diagnostic instrument. The references listed at the end of this chapter address the physical laws of light and illumination as applied to microscopy.
Principles of microscopy
In the compound microscope, a magnified intermediate image of the illuminated specimen is formed in the optical tube by each objective lens. This image is then magnified again and viewed through the eyepiece as an enlarged virtual image that appears to be located about 10 inches from the eye (). Microscopists must focus their eyes in that more distant plane, rather than trying to focus at the distance of the microscope stage. Figure 4-1
FIGURE 4-1 Compound microscope. Two separate lens systems are used (objective and eyepiece). Each objective lens forms a magnified image of the illuminated specimen in the optical tube. The eyepiece lenses further magnify this image so that the microscopist sees an enlarged virtual image that appears to be approximately 10 inches from the eye. Source: (From Abramowitz M: The microscope and beyond, vol 1, Lake Success, NY, 1985, Olympus Corp, p. 2. Reprinted courtesy Eastman Kodak Company, Rochester, NY.)
An example of a simple microscope is a magnifying lens that enlarges objects that are difficult to view with the unaided eye. Movie theater projection units incorporate this system efficiently.
The compound microscope employs two separate lens systems, objective and eyepiece, the product of which produces the final magnification. Standard microscopes use brightfield illumination in which light passes through the thin specimen.
Component parts and their functions
Component parts and the function of each part of the microscope are summarized as follows (): Figure 4-2
1. The eyepieces, or oculars, usually are equipped with 10× lenses (degree of magnification is 10×). The lenses magnify the intermediate image formed by the objective lenses in the optical tube; they also limit the area of visibility. Microscopes may have either one or two adjustable eyepieces. All eyepieces should be used correctly for optimal focus (see section on operating procedure). Eyepieces should not be interchanged with the eyepieces of the same model or other models of microscopes, because the eyepieces in a pair are optically matched.
2. The interpupillary control is used to adjust the lateral separation of the eyepieces for each individual. When it is properly adjusted, the user should be able to focus both eyes comfortably on the specimen and visualize one clear image.
3. The optical tube connects the eyepieces with the objective lens. The intermediate image is formed in this component. The standard length is 160 mm, which, functionally, is the distance from the real image plane (eyepieces) to the objective lenses.
4. The neck, or arm, provides a structural site of attachment for the revolving nosepiece.
5. The stand is the main vertical support of the microscope. The stage assembly, together with the condenser and base, is supported by the stand.
6. The revolving nosepiece holds the objectives and allows for easy rotation from one objective lens to another. The working distance (WD) between the objectives and the slide varies with the make and model of the microscope.
7. There are usually three or four objective lenses (Figure 4-3), each with a specific power of magnification. Engraved on the barrel of each objective lens is the power of magnification and the numerical aperture (NA). The NA is related to the angle of light collected by the objective; in essence, it indicates the light-gathering ability of the objective lens. Functionally, the larger the NA, the greater the resolution or the ability to distinguish between fine details of two closely situated objects.
Four standard powers of magnification and NA used in the hematology laboratory are 10×/0.25 (low power), 40×/0.65 or 45×/0.66 (high power, dry), 50×/0.90 (oil immersion), and 100×/1.25 (oil immersion). The smaller the magnification, the larger the viewing field; the larger the magnification, the smaller the viewing field. Total magnification is calculated by multiplying the magnification of the eyepiece by the magnification of the objective lens; for example, 10× (eyepiece) multiplied by 100× (oil immersion) is 1000× total magnification.
Microscopes employed in the clinical laboratory are used with achromatic or plan achromatic objective lenses, whose function is to correct for chromatic and spheric aberrations. Chromatic aberrations are caused by the spheric surface of the lens, which acts as a prism. As the various wavelengths pass through the lens, each focuses at a different point, which gives rise to concentric rings of color near the periphery of the lens. Spheric aberrations result as light waves travel through the varying thicknesses of the lens, blurring the image. The achromatic objective lens brings light of two colors into focus, partially correcting for the aberrations. When achromatic objective lenses are used, the center of the field is in focus, whereas the periphery is not. A plan achromatic lens provides additional corrections for curvature of the field, which results in a flat field with uniform focus.2 Plan achromatic lenses sometimes are referred to as flat field lenses. Critical microscopy applications may require a plan apochromatic lens, which brings light of three colors into focus and almost completely corrects for chromatic aberration. This type of objective lens is more expensive and is rarely needed for routine laboratory use.
A set of lenses with corresponding focal points all in the same plane is said to be parfocal. As the nosepiece is rotated from one magnification to another, the specimen remains in focus, and only minimal fine adjustment is necessary.
8. The stage supports the prepared microscope slide to be reviewed. A spring assembly secures the slide to the stage.
9. The focus controls (or adjustments) can be incorporated into one knob or can be two separate controls. When a single knob is used, moving it in one direction engages the coarse control, whereas moving it in the opposite direction engages the fine control. One gradation interval of turning is equivalent to 2 μm. Many microscopes are equipped with two separate adjustments: one coarse and one fine. The order of usage is the same: engage the coarse adjustment first and then fine-tune with the fine adjustment.
10. The condenser, consisting of several lenses in a unit, may be permanently mounted or vertically adjustable with a rack-and-pinion mechanism. It gathers, organizes, and directs the light through the specimen. Attached to and at the bottom of the condenser is the aperture diaphragm, an adjustable iris containing numerous leaves that control the angle and amount of the light sent through the specimen. The angle, also expressed as an NA, regulates the balance between contrast (ability to enhance parts within a cell) and resolution (ability to differentiate fine details of two closely situated objects). The best resolution is achieved when the iris is used fully open, but there is some sacrifice of image contrast. In practice, this iris is closed only enough to create a slight increase in image contrast. Closing it beyond this point leads to a loss of resolution.
Some microscopes are equipped with a swing-out lens immediately above or below the main condenser lens. This lens is used to permit a wider field of illumination when the NA of the objective lens is less than 0.25 (e.g., the 4×/0.12 objective lens).3 If the swing-out lens is above the main condenser, it should be out for use with the 4× objective lens and in for lenses with magnification of 10× and higher. If it is below the condenser, it should be in for use with the 4× objective lens and out for lenses of magnification of 10× and higher. The 4× objective is not used routinely for examination of peripheral blood films.
FIGURE 4-2 Components of a compound microscope. Source: (Courtesy Nikon Instruments, Inc., Melville, NY.)
FIGURE 4-3 Microscope objective lens. The numerical aperture (NA) indicates the light-gathering ability of the objective lens and reflects its ability to distinguish between fine details of two closely situated objects. The working distance (WD) is the distance in millimeters between the lens of the objective and the cover glass when the specimen is in focus. Source: (Courtesy Nikon Instruments, Inc., Melville, NY.)
The stage and condenser () consist of a swing-out lens, an aperture diaphragm, a control for vertical adjustment of the condenser, and two centering screws for adjustment of the condenser. Figure 4-4
11. The condenser top lens can swing out of position.
12. The stage controls located under the stage move it along an x- or a y-axis.
13. The field diaphragm is located below the condenser within the base. When it is open, it allows a maximally sized circle of light to illuminate the slide. Almost closing the diaphragm, when low power is used, assists in centering the condenser apparatus by the use of two centering screws. Some microscopes have permanently centered condensers, whereas in others the screws are used for this function. The glass on top of the field diaphragm protects the diaphragm from dust and mechanical damage.
14. Microscopes depend on electricity as the primary source for illumination power. There are two types of brightfield illumination: (1) critical illumination, in which the light source is focused at the specimen, which results in increased but uneven brightness; and (2) the Koehler (or Köhler) system, in which the light source and the condenser are properly aligned. The end result of Koehler illumination is a field of evenly distributed brightness across the specimen. This is especially important when using the oil objectives or when taking photomicrographs. Tungsten-halogen light bulbs are used most frequently as the illumination source. They consist of a tungsten filament enclosed in a small quartz bulb that is filled with a halogen gas. Tungsten possesses a high melting point and gives off bright yellowish light. A blue (daylight) filter should be used to eliminate the yellow color produced by tungsten.4, 5 The rheostat or light control knob or lever turns on the light and should be used to regulate the brightness of the light needed to visualize the specimen. The aperture diaphragm control lever should never be used for this purpose, because closing it reduces resolving ability.3
FIGURE 4-4 Condenser. Source: (Courtesy Nikon Instruments, Inc., Melville, NY.)
Operating procedure with koehler illumination
The procedure outlined here applies to microscopes with a nonfixed condenser. The following steps should be performed at the start of each laboratory session in which the oil objectives will be used:
1. Connect the microscope to the power supply.
2. Turn on the light source with the power switch.
3. Open the condenser aperture and field diaphragms.
4. Revolve the nosepiece until the 10× objective lens is directly above the stage.
5. Place a stained blood film on the stage and focus on it, using the fixed eyepiece, while covering the other eye. (Do not simply close the other eye, because this would necessitate adjustment of the pupil when you focus with the other eyepiece.)
6. Adjust the interpupillary control so that looking through both eyepieces yields one clear image.
7. Using the adjustable eyepiece and covering the opposite eye, focus on the specimen. Start with the eyepiece all the way out, and adjust inward. If using two adjustable eyepieces, focus each individually.
8. Raise the condenser to its upper limit.
9. Focus the field so that the cells become sharp and clear. Concentrate on one cell and place it in the center of the field.
10. Close the field (lower) diaphragm. Look through the eyepieces. A small circle of light should be seen. If the light is not in the center of the field, center it by using the two centering screws located on the condenser. This step is essential, because an off-center condenser will result in uneven distribution of light. Adjust the vertical height of the substage condenser so that you see a sharp image of the field diaphragm, ringed by a magenta halo. If the condenser is raised too much, the halo is orange; if it is lowered too far, the halo is blue.
11. Reopen the field diaphragm until it is nearly at the edge of the field, and fine-tune the centering process.
12. Open the field diaphragm slightly until it just disappears from view.
13. Remove one eyepiece and, while looking through the microscope (without the eyepiece), close the condenser aperture diaphragm completely. Reopen the condenser aperture diaphragm until the leaves just disappear from view. Replace the eyepiece.
14. Rotate the nosepiece until the 40× objective lens is above the slide. Adjust the focus (the correction should be minimal) and find the cell that you had centered. If it is slightly off center, center it again with the stage x-y control. Note the greater amount of detail that you can see.
15. Move the 40× objective out of place. Place a drop of immersion oil on top of the slide. Rotate the nosepiece until the 100× objective lens is directly above the slide. Avoid moving a non–oil immersion objective through the drop of oil. Adjust the focus (the correction should be minimal) and observe the detail of the cell: the nucleus and its chromatin pattern; the cytoplasm and its color and texture. The objective lens should dip into the oil slightly.
1. When revolving the nosepiece from one power to another, rotate it in such a direction that the 10× and 40× objective lenses never come into contact with the oil on a slide. If oil inadvertently gets onto the high dry objective, clean the objective immediately.
2. Parcentric refers to the ability to center a cell in question in the microscopic field and rotate from one magnification power to another while retaining the cell close to the center of the viewing field. Recentering of the cell at each step is minimal. Most laboratory microscopes have this feature.
3. In general, when the 10× and 40× objective lenses are used, the light intensity should be low. When the 50× and 100× objective lenses are used, increase the intensity of the light by adjusting only the rheostat (light control knob or lever) or by varying neutral density filters. Neutral density filters are used to reduce the amplitude of light and are available in a variety of densities.3
4. Do not change the position of the condenser or the aperture diaphragm control lever to regulate light intensity when viewing specimens with the oil immersion objectives. The condenser should always be in its upward position as set during the Koehler illumination adjustment. The aperture diaphragm may be adjusted to achieve proper contrast of the features of the specimen being viewed.
5. After setting the Koehler illumination, when a new slide is to be examined, always bring the specimen into focus with the 10× objective first, and then move to the higher magnifications.
Immersion oil and types
Immersion oil is required to increase the refractive index when either the 50× or the 100× oil immersion objective lens is used. The refractive index is the speed at which light travels in air divided by the speed at which light travels through a substance. This oil, which has the same properties as glass, allows the objective lens to collect light from a wide NA, which provides high resolution of detail.
Three types of immersion oil, differing in viscosity, are employed in the clinical laboratory:
1. Type A has very low viscosity and is used in fluorescence and darkfield studies.
2. Type B has high viscosity and is used in brightfield and standard clinical microscopy. In hematology, this oil is routinely used.
3. Type C has very high viscosity and is used with inclined microscopes with long-focus objective lenses and wide condenser gaps.
Bubbles in the oil tend to act as prisms and consequently reduce resolution. Bubbles may be created when oil is applied to the slide. They are caused by lowering the objective immediately into the oil. Sweeping the objective from right to left in the oil eliminates bubbles.5
Care of the microscope
Care of the microscope involves the following details:
1. When not in use for an extended period of time, always cover the microscope to protect it from dust.
2. Before use, inspect the component parts. If dust is found, use an air syringe, a camel hair brush, or a soft lint-free cloth to remove it. Using lens paper directly on a dirty lens without first removing the dust may scratch the lens. Do not use laboratory wipes or facial tissue to clean the lenses.6
3. Avoid placing fingers on the lens surface. Fingerprints affect the contrast and resolution of the image.
4. Use solvent sparingly. The use of xylene is discouraged, because it contains a carcinogenic component (benzene). Xylene is also a poor cleaning agent, leaving an oily film on the lens. Lens cleaner or 70% isopropyl alcohol employed sparingly on a cotton applicator stick can be used to clean the objective lenses. Alcohol should be kept away from the periphery of the lenses, because alcohol can dissolve the cement and seep into the back side of the lens.
5. When fresh oil is added to residual oil on the 100× objective lens, there may be loss of contrast. Clean off all residual oil first.
6. Do not use water to clean lenses. If no lens cleaner is available, use a clean microfiber cloth.
7. When transporting the microscope, place one hand under the base as support and one hand firmly around the arm.
In addition to daily care of the microscope, semiannual or annual maintenance with thorough cleaning should be done by a professional. Microscope professionals may recognize and correct problems with mechanics or optics before they are detected by the microscope user. They can correct problems such as sticking of stage controls or incorrect optical alignment that can lead to physical problems like carpal tunnel syndrome and headaches.
Most common problems are related to inability to focus. Once the operator has ensured that he or she is not trying to obtain a “flat field” using an objective lens that is not plan achromatic, the following checklist can aid in identifying the problem:
• Objective lens
Screwed in tightly?
Dry objective free of oil?
Adjusted to proper height?
Free of oil?
Correct side up?
Correct side of blood film?
Only one coverslip on slide?
Free of mounting media?
• Light source
Fingerprints on bulb?
Bulb in need of changing?
Light source aligned correctly?
Other microscopes used in the clinical laboratory
The ability to view a stained specimen by the use of brightfield microscopy is affected by two features: (1) the ability of the specimen to absorb the light hitting it, and (2) the degree to which light waves traveling through the specimen remain in phase ( ).
Specimens that are transparent or colorless, such as unstained cells, are not clearly visualized with brightfield microscopy. Phase-contrast microscopy, through the installation of an annular diaphragm in the condenser, together with a phase-shifting element, creates excellent contrast of a cell against its surrounding background.
The principle of phase contrast is related to the index of refraction and the thickness of a specimen, which produce differences in the optical path. Light passing through a transparent specimen travels slightly slower than light that is unobstructed. The difference is so small that it is not noticeable to the viewer. When a transparent phase plate is placed into the microscope, however, the change in phase can be increased to half a wavelength, which makes the otherwise transparent objective visible ( ).
This phase difference produces variation in light intensity from bright to dark, creating contrast in the image. Often the objects appear to have “haloes” surrounding them.
In hematology, phase-contrast microscopy is employed in counting platelets in a hemacytometer, since they are difficult to visualize and count using brightfield microscopy. It also can be used to view formed elements in unstained urine sediments.
Polarized light microscope
Polarized light microscopy is another contrast-enhancing technique used to identify substances such as crystals in urine and other body fluids (Chapter 18). With brightfield microscopy, light vibrates in all directions. If a polarizer (filter) is placed in the light path, the light vibrates in only one direction or plane, which creates polarized light. To convert a brightfield microscope to a polarizing one, two filters are needed. One filter (the polarizer) is placed below the condenser and allows only light vibrating in the east-west direction perpendicular to the light path to pass through the specimen. The second filter (the analyzer) is placed between the objective and the eyepiece and allows only light vibrating in a north-south direction to pass to the eyepiece. When the transmission axes of these two filters are oriented at right angles, no light can pass through the pair to the eyepieces. When polarized light (vibrating in an east-west direction) passes through an optically active substance such as a monosodium urate crystal, however, the light is refracted into two beams, one vibrating in the original direction (east-west) and one vibrating in a plane 90 degrees to it (i.e., north-south). The refracted light vibrating in the north-south direction can pass through the second filter (the analyzer) and is visible at the eyepiece. The magnified crystal appears white against a black background. If a first-order red compensator filter also is placed in the light path below the stage, the background becomes pink-red, and the crystal appears yellow or blue, depending on its physical orientation relative to the incident light path (east-west). Some crystals can be specifically identified based on their unique birefringent (doubly refractive) characteristics when polarizing microscopy is used (Figures 18-22 and 18-23).
Darkfield microscopy is a contrast-enhancing technique that employs a special condenser. The condenser sends light up toward the specimen in a hollow cone. Because of the high angle of this cone, none of the illuminating rays enters the objective lens. Without the specimen in place, the field would appear black because of the absence of light. When the specimen is in place, and if fine detail exists in the specimen, light is diffracted in all directions. This diffracted light is picked up by the objective lens and appears as bright detail on a black background. Darkfield microscopy is helpful in microbiology in the identification of spirochetes.
• The compound microscope, through the use of an objective lens in the optical tube, forms an intermediate image of the illuminated specimen. The image is then magnified and viewed through the eyepiece lenses.
• The numerical aperture or NA, which is engraved on the barrel of objective lenses, designates the light-gathering ability of the lens. The larger the NA, the greater the resolution.
• Achromatic lenses maintain the center of the field in focus, whereas plan achromatic lenses correct for the curvature of a field, providing a flat field uniform focus.
• The condenser gathers the light and directs it through a thin specimen.
• Koehler illumination establishes a field of evenly distributed brightness across the specimen; the microscope should be adjusted for proper Koehler illumination with each use.
• Only the rheostat (light control knob or lever) should be used to regulate the light intensity needed to visualize a specimen. Light intensity should not be regulated by adjusting the position of the aperture diaphragm or the height of the condenser when using the oil immersion lenses.
• The aperture diaphragm control lever may be adjusted to achieve proper contrast of the features of the specimen being viewed.
• The use of oil immersion for the 50× and 100× oil immersion objectives improves the resolution of the image; type B oil is typically used with brightfield microscopy in hematology.
• Microscopes should be carefully handled and maintained. Solvents should not be used to clean lenses; lens cleaner or 70% isopropyl alcohol is recommended.
• Phase-contrast microscopy relies on the effect of index of refraction and the thickness of the specimen; these two features affect light by retarding a fraction of the light waves, resulting in a difference in phase. This allows transparent or colorless objects to become visible.
• Polarizing microscopes use two polarizing filters to cancel the light passing through the specimen. If the object is able to polarize light, as are some crystals, the light passing through is rotated and the object becomes visible.
• Darkfield microscopes use condensers that send light to the specimen at a high angle, directing the light away from the objective lens. If the specimen has fine detail, it causes the light to bend back toward the objective, which allows it to be viewed against an otherwise dark background.
Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the question presented.
Answers can be found in the Appendix.
1. Use of which one of the following type of objective lens causes the center of the microscope field to be in focus, whereas the periphery is blurred?
a. Plan achromatic
c. Plan apochromatic
d. Flat field
2. Which of the following gathers, organizes, and directs light through the specimen?
b. Objective lens
d. Optical tube
3. After focusing a specimen by using the 40× objective, the laboratory professional switches to a 10× objective. The specimen remains in focus at 10×. Microscopes with this characteristic are described as:
4. Which objective has the greatest degree of color correction?
b. Plan apochromatic
d. Plan achromatic
5. In adjusting the microscope light using Koehler illumination, which one of the following is true?
a. Condenser is first adjusted to its lowest position
b. Height of the condenser is adjusted by removing the eyepiece
c. Image of the field diaphragm iris is used to center the condenser
d. Closing the aperture diaphragm increases the resolution of the image
6. The total magnification obtained when a 10× eyepiece and a 10× objective lens are used is:
7. After a microscope has been adjusted for Koehler illumination, and the specimen is being viewed with an oil immersion objective lens, light intensity should never be regulated by adjusting the:
b. Neutral density filter
c. Light control knob
8. The recommended cleaner for removing oil from objectives is:
a. 70% alcohol or lens cleaner
9. Which of the following types of microscopy is valuable in the identification of crystals that are double refractive?
a. Compound brightfield
10. A laboratory science student has been reviewing a hematology slide using the 10× objective to find a suitable portion of the slide for examination. He moves the 10× objective out of place, places a drop of oil on the slide, rotates the nosepiece so that the 40× objective passes through the viewing position, and continues to rotate the 100× oil objective into viewing position. This practice should be corrected in which way?
a. The stage of a parfocal microscope should be lowered before the objectives are rotated.
b. The 100× oil objective should be in place for viewing before the oil is added.
c. The drop of oil should be in place and the 100× objective lowered into the oil, rather than swinging the objective into the drop.
d. The objectives should be rotated in the opposite direction so that the 40× objective does not risk entering the oil.
11. Darkfield microscopes create the dark field by:
a. Using two filters that cancel each other out, one above and the other below the condenser
b. Angling the light at the specimen so that it misses the objective unless something in the specimen bends it backward
c. Closing the condenser diaphragm entirely, limiting light to just a tiny ray in the center of the otherwise dark field
d. Using a light source above the specimen and collecting light reflected from the specimen, rather than transmitted through the specimen, so that when there is no specimen in place, the field is dark
1. http://www.microscopyu.com/ Nikon Microscopy U.
2. 3rd ed. Chapter 1. Brunzel NA.Microscopy. In Fundamentals of Urine and Body Fluid Analysis. St Louis: Saunders. 2013.
3. Köhler illumination. Gill GW.Lab Med. 2005; 36(9):530.
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