# Microscopy

section epub:type=”chapter” id=”c0018″ role=”doc-chapter”>

# Microscopy

Learning Objectives

## Brightfield Microscope

A brightfield microscope (Fig. 18.1) produces a magnified specimen image that appears dark against a brighter background. A simple brightfield microscope consisting of only one lens is known as a magnifying glass. In the clinical laboratory, however, compound brightfield microscopes predominate and consist of two lens systems. The first lens system, located closest to the specimen, is the objective mounted in the nosepiece. The objective produces the primary image magnification and directs this image to the second lens system, the eyepiece, or ocular.

The eyepiece further magnifies the image received from the objective lens. Total magnification of a specimen is the product of these lens systems—that is, multiplication of the objective lens magnification by the eyepiece lens magnification. For example, a ×10 objective with a ×10 eyepiece produces a ×100 magnification. In other words, the viewed image is 100 times larger than its actual size.

The eyepiece also determines the diameter of the field of view (FOV) observed. This diameter is established by a round baffle or ridge inside the eyepiece and is indicated by the field number assigned to the eyepiece. Field numbers that predominate in clinical laboratory microscopes typically range from 18 to 26. This number indicates the diameter of the FOV, in millimeters (mm), when a ×1 objective is used. To determine the diameter of any FOV the following equation is used:

FOV=Field numberM Equation 18.1

Equation 18.1

where M is the magnification of the objective and any additional optics. (Note: This sum does not include the eyepiece magnification.)

For example, an eyepiece with a field number of 18 has a diameter of 18 mm when a ×1 objective is used, a diameter of 1.8 mm when a ×10 objective is used, and a diameter of 0.18 mm when a ×100 objective is used. Some manufacturers engrave the field number on the eyepiece along with the eyepiece magnification. For those that do not, a small ruler can be placed on the stage to measure the diameter, or the manufacturer can be contacted. As the field number increases, so does the cost of the eyepiece. Before about 1990, microscopes used in the clinical laboratory had eyepieces with a field number of 18. Most microscopes purchased after that time have field numbers of 20 or 22.

In areas of the laboratory where results are reported as the number of elements observed per FOV (e.g., urinalysis, number per high-power or low-power field), performing a microscopic examination on the same or equivalent microscopes is crucial. If this is not done, results depend on the microscope used and can be significantly different even with evaluation of the same specimen. In other words, two microscopes with the same objective magnification and the same eyepiece magnification (e.g., 10×) but with different field numbers will have different diameters for their FOVs. Therefore standardizing microscopic examinations reported as the number of elements observed per FOV requires that microscopes with the same eyepiece field number are used.

R=0.612×λNA Equation 18.2

Equation 18.2

where R is the resolving power or the resolvable distance in microns, λ is the wavelength of light, and NA is the numerical aperture of the objective.

Because the light source on a microscope remains constant, as the NA of the objective lens increases, the resolution distance decreases. In other words, one can distinguish a smaller distance between two distinct points.

A numerical aperture is a designation engraved on objective lenses and condensers that indicates the R of each specific lens. The NA is derived mathematically from the refractive index (n) of the optical medium (e.g., air has an n value of 1.0) and the angle of light made by the lens (μ) (i.e., the aperture angle).

NA=n×Sin μ Equation 18.3

Equation 18.3

The NA of a lens can be increased by changing the refractive index of the optical medium or by increasing the aperture angle. For example, immersion oil has a greater refractive index (n = 1.515) than air, and it increases the magnitude of the aperture angle (Fig. 18.2). As a result, use of immersion oil effects a greater NA (e.g., ×100, NA = 1.2) than is possible with high-power dry lenses. An increase in NA equates with greater magnification and resolution.

As previously discussed, the ability of a lens to resolve two points increases as the NA increases. However, to achieve the maximal resolution of a microscope, the NA of the microscope condenser must be equal to or slightly greater than the NA of the objective lens used. This requirement is necessary to ensure adequate illumination to the objective lens and can be understood better by reviewing the dynamics involved. Illumination light from the light source is presented to the condenser. The condenser lens system, along with the aperture diaphragm, is adjustable and serves to converge the illumination light into a cone-shaped focus on the specimen for viewing. If the condenser NA is less than the objective NA, the condenser presents inadequate illumination light to the objective lens, and one cannot attain maximal resolution. In contrast, objective lenses with NAs less than the condenser NA are optimal on the microscope. This can be accomplished by making routine condenser and diaphragm adjustments that effectively reduce the condenser NA to match the objective NA (see “Microscope Adjustment Procedure” later in this chapter). Condenser height adjustments serve to focus the light specifically on the specimen plane, thus achieving maximal resolution. Optimal field diaphragm adjustments diminish stray light, thereby increasing image definition and contrast. Adjustment of the aperture diaphragm to approximately 75% of the NA of the objective is necessary to achieve increased image contrast, increased focal depth, and a flatter FOV.

The body or frame of the microscope serves to hold its four basic components in place: (1) the optical tube with its lenses (eyepieces and objectives), (2) a stage on which the specimen is placed for viewing, (3) a condenser to focus light onto the specimen, and (4) an illumination source. Each component and its unique features are discussed next.

### Eyepiece

Whereas some microscopes have only one eyepiece (monocular), those used in most clinical laboratories have two eyepieces (binocular). However, when using a monocular microscope, always view with both eyes open to reduce eyestrain. Initially this may be difficult, but with practice the image seen by the unused eye will be suppressed. With a binocular microscope, adjustments to the oculars are necessary to ensure optimal viewing. The interpupillary distance of the eyepiece tubes is adjusted by simply sliding them together or apart. Because vision in both eyes is usually not the same, each individual eyepiece is adjustable to compensate, using the diopter adjustment. To adjust the eyepieces, first view the image using only the right eye and eyepiece. Look at a specific spot on the specimen, and bring it into sharp focus using the fine adjustment knob. Next, close the right eye, and while looking with the left eye through the left eyepiece, rotate the diopter adjustment ring on this eyepiece until the same spot on the specimen is also in sharp focus (Fig. 18.3). Each technologist must make the interpupillary and diopter adjustments to suit his or her eyes. To eliminate eyestrain or tired eyes when performing microscopic work, always look through the microscope with eyes relaxed, and continually focus and refocus the microscope as needed using the fine adjustment control.

Eyeglass wearers should consider keeping their glasses on when performing microscopic work. Rubber guards are available that fit over the eyepieces and prevent scratching of the eyeglasses. People with only spherical corrections (nearsighted or farsighted) can work at the microscope without their glasses. Focus adjustments of the microscope compensate for these visual defects. However, those with an astigmatism that requires a toric lens for correction should do microscopic work while wearing their eyeglasses, because the microscope cannot compensate for this. To determine whether eyeglasses have any toric correction in them, hold the glasses in front of some lettering at arm’s length. The eyeglass will magnify or reduce the lettering. Now, rotate the glasses 45 degrees. If the lettering changes in length and width, the glasses contain toric correction and should be worn when one uses the microscope; if the lettering does not change, the eyeglasses have only spherical correction and do not need to be worn for microscopic work.

### Mechanical Stage

The microscope mechanical stage is designed to hold firmly in place the slide to be examined. The stage has conveniently located adjustment knobs to move the slide front to back and side to side. When viewing the slide, the user views the image upside down. Moving the slide in one direction causes the image to move in the opposite direction. Some stages have a vernier scale on a horizontal and a vertical edge to facilitate relocation of a particular FOV. By recording the horizontal and vertical vernier scale values, the slide can be removed and at a later time placed back onto the stage and the identical FOV can be found and reexamined.

### Condenser

The condenser, located beneath the mechanical stage, consists of two lenses (Fig. 18.4). The purpose of the condenser is to evenly distribute and optimally focus light from the illumination source onto the specimen. This is achieved by adjusting the condenser up or down using the condenser adjustment knob. The correct position of the condenser is always at its uppermost stop; it is slightly lowered only with Köhler illumination. The aperture diaphragm, located at the base of the condenser, regulates the beam of light presented to the specimen. The aperture diaphragm is usually an iris diaphragm made up of thin metal leaves that can be adjusted to form an opening of various diameters. Some microscopes use a disk diaphragm, consisting of a movable disk with openings of various sizes, for placement in front of the condenser (Fig. 18.5). The purpose of the aperture diaphragm is to control the angle of the illumination light presented to the specimen and the objective lens. When the user properly adjusts the aperture diaphragm, he or she achieves maximal resolution, contrast, and definition of the specimen. One of the most common mistakes is to use the aperture diaphragm to reduce the brightness of the image field; in so doing, resolution is decreased. Instead, the user should decrease the light source intensity or place a neutral density filter over the source.

### Illumination System

Microscopes today usually have a built-in illumination system. The light source is a tungsten or tungsten-halogen lamp located in the microscope base. These lamps often are manufactured specifically to ensure alignment of the lamp filament when a bulb requires changing. Dual controls are usually available: one to turn the microscope on, and another to adjust the intensity of the light. One should adjust the illumination intensity at the light source by turning down the lamp intensity or by placing neutral density filters over the source. Neutral density filters do not change the color of the light but reduce its intensity. The filters are marked to indicate the reduction made; for example, a neutral density of 25 allows 75% of the light to pass. Some microscopes come with a daylight blue filter that makes the light slightly bluish. This color has been found to be restful to the eyes and is desirable for prolonged microscopic viewing.

### Objectives

The objectives are the most important optical components of the microscope because they produce the primary image magnification. The objectives are located on a rotatable nosepiece; only one objective is used at a time. Objectives are easily changed by simple rotation of the nosepiece; however, each objective has a different working distance, that is, the distance between the objective and the coverslip on the slide. This working distance decreases as the magnification of the objective used increases; for example, a ×10 objective has a working distance of 7.2 mm, whereas a ×40 objective has only 0.6-mm clearance. Therefore to prevent damage to the objective or to the slide that is being observed, care must be taken when changing or focusing objectives.

A microscope has coarse and fine focus adjustment knobs. The user can focus the microscope by moving the mechanical stage holding the specimen up and down. Coarse focusing adjustments are made first, followed by any necessary fine adjustments.

Various engravings found on the objective indicate its magnification power, the NA, the optical tube length required, the coverglass thickness to be used, and the lens type (if not an achromat). Most often, the uppermost or largest number inscribed on the objective is the magnification power. After this number is the NA, inscribed on the same line or just beneath it (Fig. 18.6). As already discussed, the objective produces the primary magnification of the specimen, and the NA mathematically expresses the R of the objective. Most objectives, designed for use with air between the lens and the specimen, are called dry objectives. In contrast, some objectives require immersion oil to achieve their designated NA. These objectives are inscribed with the term oil or oel.

The optical tube length—the distance between the eyepiece and the objective in use—can differ depending on the microscope. If the microscope has a fixed optical tube length (usually 160 mm), the objectives used should have “160” engraved on them (see Fig. 18.6). On some microscopes, one can change the tube length when placing devices such as a polarizer or a Nomarski prism between the objective and the eyepiece. Use of these devices requires objectives designed for infinity correction or lenses corrected to maintain the tube length of 160 mm optically. Objectives designed for infinity correction have an infinity symbol (∞) engraved on them.

Some objectives are designed to be used with a coverglass. If a coverglass is required, its thickness is engraved on the lens after the optical tube length (e.g., 160/0.17). Objectives that do not use a coverglass are designated with a dash (e.g., ∞/− or 160/−). A third type of objective is designed to be used with or without a coverglass. These objectives have no inscription for coverglass thickness; rather, they have a correction collar on them with which to adjust and fine focus the lens appropriately for either application.

Objectives are corrected for two types of aberrations: chromatic and spherical. Chromatic aberration occurs because different wavelengths of light bend at different angles after passing through a lens (Fig. 18.7). This results in a specimen image with undesired color fringes. Objectives corrected to bring the red and blue components of white light to the same focus are called achromats and may not have a designation engraved on them. Objectives that bring red, blue, and green light to a common focus are termed apochromats and are identified by the inscription “apo.” Spherical aberration occurs when light rays pass through different parts of the lens and therefore are not brought to the same focus (Fig. 18.8). As a result, the specimen image appears blurred and cannot be focused sharply. Objectives are corrected to bring all light entering the lens, regardless of whether the light is at the center or the periphery, to the same central focus. Achromat objectives are corrected spherically for green light, whereas apochromats are corrected spherically for green and blue light.

Other abbreviations may be engraved on the objective to indicate specific lens types. For example, “Plan” indicates that the lens is a plan achromat, achromatically corrected and designed for a flat FOV over the entire area viewed. “Ph” indicates that the objective lens is for phase-contrast microscopy. Regardless of the manufacturer, the same basic information is engraved on all objective lenses, with only the format varying slightly. To ensure a compatible system, use of objectives and eyepieces designed by the same manufacturer that designed the microscope is advisable.

Two final features of objective lenses need to be discussed. The first characteristic is termed parcentered and relates to the ability of objective lenses to retain the same central FOV when the user switches from one objective to another. In other words, when an objective is changed to one of higher magnification for a closer look, the object does not move from the center of the FOV. The second feature, termed parfocal, refers to the ability of objectives to remain in focus regardless of the objective used. This allows initial focusing at low power; changing to other magnifications requires only minimal fine focus adjustment. The parcentered and parfocal features of objectives today are taken for granted, whereas in the past, each objective required individual centering and focusing.

When using a microscope, adjustments must be made with each objective to produce optimal viewing. These adjustments strive to equate the NA of the objective lens in use (e.g., ×10, NA 0.25) with the condenser NA (NA 0.9), thereby achieving maximal magnification and resolution. On current microscopes that use Köhler illumination, once the condenser height adjustment is made, it remains unchanged regardless of the objective used. The user lowers the effective NA of the condenser by decreasing the light the condenser receives (i.e., closing the field diaphragm) and by adjusting the aperture diaphragm for the objective. On microscopes with which Köhler illumination is not possible, use of low-power objectives may require (1) reducing the illumination source light, if possible; (2) slightly lowering (by approximately 1.0 mm) the condenser from its uppermost position; or (3) minimally closing the aperture diaphragm. An adjustment error that users frequently make is to lower the condenser too much, resulting in loss of resolution as contrast is increased.

When high-power dry objectives are used (e.g., ×40, NA 0.65), the NA is closer to that of the condenser (NA 0.9). Therefore the condenser NA needs less reduction to achieve maximal viewing. Because going from a low-power to a high-power objective means changing from a low NA to a higher NA, more illumination is required. Microscopes using Köhler illumination require only field and aperture diaphragm adjustments with each objective change. When high-power objectives are used on a microscope without Köhler illumination, the user should put the condenser all the way up and close the aperture diaphragm just enough to attain effective contrast. Never use the condenser or the aperture diaphragm to reduce image brightness; rather, decrease the illumination intensity or use neutral density filters.

## Ocular Field Number

The eyepieces or oculars together with the objective lenses perform two important functions. They determine (1) the diameter of the field of view (FOV) and (2) the total magnification of a specimen. The diameter of the FOV is determined by the round baffle or ridge inside each ocular, and its numerical value is known as the ocular field number. Before 1990, most laboratory microscopes had an ocular field number of 18, which means that the diameter of the FOV when a ×1 objective is used is 18 mm (or 1.8 mm with a ×10 objective). In other words, if a metric ruler were placed on the stage and a ×1 objective used, the diameter of the circle of view observed when looking through the eyepieces would measure 18 mm. Typically, the higher the field number, the more expensive the microscope. Areas of high microscope use, such as hematology and pathology laboratories, may be able to justify the expense of microscopes with even higher ocular field numbers of 22 to 26 or larger.

When multiple microscopes are used in the laboratory for urine sediment examination, it is of paramount importance that their FOVs are the same, because clinically significant sediment components are reported as the number present per low-power field or per high-power field. The larger the ocular field number, the larger the FOV, and the greater the number of components that may be observed. Note that two microscopes with equivalent magnifying power (e.g., ×100 and ×400) can have FOVs that differ! In other words, the magnification of the oculars on both microscopes is the same, but their field numbers are not. Unfortunately, most microscope manufacturers do not engrave the field number on the oculars, and if needed, it must be obtained from the original purchase information by measuring the diameter using a ruler and a ×1 objective, or by contacting the manufacturer.

## Microscope Adjustment Procedure

Clinical microscopes today primarily use Köhler illumination. With this type of illumination, the light source image (light filament) is focused onto the front focal plane of the substage condenser at the aperture diaphragm by a lamp condenser, located just in front of the light source. The substage condenser then focuses this image onto the back of the objective in use (see Fig. 18.1). As a result, this illumination system produces bright, uniform illumination at the specimen plane even when a coil filament light source is used. Proper use of this illuminating system is just as important as selection of a microscope and its objectives. To use a microscope with Köhler illumination, the microscopist must know how to set up and optimally adjust the condenser and the field and aperture diaphragms. Manufacturers supply instructions with the microscope that are clear and easy to follow. In addition, online interactive tutorials are available that demonstrate the improved optical performance of a microscope when adjusted to achieve Köhler illumination.1 Box 18.1 gives a basic procedure for adjustment of a typical binocular microscope with a Köhler illumination system. Whereas initially these steps may feel cumbersome, with use they become routine. When using other types of microscopy, additional adjustment procedures may be necessary to ensure optimal viewing. For example, phase-contrast microscopy requires that the phase rings be checked and aligned, if necessary.

Each day, before setting up and adjusting the microscope, the user should check it to ensure that it is clean. The microscopist should look for dust or dirt on the illumination source port, the filters, and the upper condenser lens. The user should check the eyepiece and the objectives, especially any oil immersion objective, to be sure that they are clean and free of oil and fingerprints. By routinely inspecting the microscope and optical surfaces before adjustment, valuable time can be saved in microscope setup and in troubleshooting problems. In laboratories in which the entire staff uses the same microscope, inspection before use helps identify people who need to be reminded of proper microscope care and maintenance.

## Care and Preventive Maintenance

The microscope is a precision instrument. Therefore ensuring long-term mechanical and optical performance requires care, including routine cleaning and maintenance. Dust is probably the greatest cause of harm to the mechanical and optical components of a microscope. Dust settles in mechanical tracks and on lenses. Although dust can be removed from lenses by cleaning, the less the lenses are cleaned, the better. To remove dust, dirt, or other particulate matter, the microscopist should use a grease-free brush (camel hair) or an air syringe (e.g., an infant’s ear syringe). If compressed air is used, the air should be filtered (e.g., with cotton wool) to remove any contaminating residues or moisture. Using a microscope dust cover when the instrument is not in use or placing it in a storage cabinet eliminates dust buildup.

On microscopes, all mechanical parts are lubricated with special long-lasting lubricants. Therefore the user should never use grease or oils to lubricate the microscope. When mechanical parts are dirty, cleaning and regreasing should be performed by the manufacturer or by a professional service representative.

In climates in which the relative humidity is consistently greater than 60%, precautions must be taken to prevent fungal growth on optical surfaces. In these areas, a dust cover or a storage cabinet may reduce ventilation and enhance fungal growth. Therefore microscopes may require storage with a desiccant or in an area with controlled temperature and air circulation. In addition to high humidity, microscopes should be protected from direct sunlight and high temperatures.

When handling the microscope—for example, when removing it from a storage cupboard or when changing work areas—the user must always carry it firmly, using both hands, and must avoid abrupt movements. The counter on which the microscope is placed should be vibration free. This eliminates undesired movement in the FOV when viewing wet preparations, as well as the detrimental effects that long-term vibration can have on precision equipment.

All optical surfaces must be clean to provide crisp, brilliant images. Because the nosepiece is rotated by hand, the objectives are constantly in danger of becoming smeared with skin oils. The user should avoid all handling of optical surfaces with the fingers. Should a lens need cleaning, the user should follow the manufacturer’s suggested cleaning protocol. Optical lenses are easily scratched; therefore one must remove all particulate matter from the lens before cleaning. Some residues may be removed simply by breathing on the lens surface and polishing with lens paper. Others may require a commercial lens cleaner. The microscopist should never use gauze, facial tissue, or lint-free tissue to clean optical surfaces. After using oil immersion objective lenses, the microscopist should remove the oil carefully using a dry lens paper and should repeat this procedure using a lens paper moistened with lens cleaner. The microscopist must store oil immersion objectives dry, because oil left on the lens surface can impair its optical performance. Whereas some manufacturers suggest the use of xylene to clean oil immersion lenses, this practice is not recommended for several reasons. If residual xylene is left on the objective, it destroys the adhesive that holds the lens in place. In addition, xylene fumes are toxic and should be avoided.

The eyepiece is particularly susceptible to becoming dirty, especially when the user wears mascara. Therefore people should avoid wearing mascara when performing microscopy. If an eyepiece is removed for cleaning, care must be taken to prevent dust from entering the microscope tube and settling on the back lens of the objective.

When the specimen image shows a visual aberration and a dirty lens is suspected, the following procedure can help identify which lens needs attention. Specks appearing in the FOV are most often noted on the eyepiece or the coverglass. The user should rotate the eyepiece; if the speck moves, the eyepiece lens requires cleaning. If the speck moves when the slide position is changed, the coverglass is dirty. If the objective lens is dirty, the speck will not be present when a different objective is used. Often the image is blurred or hazy (i.e., decreased sharpness or contrast) when an objective lens is dirty, as with a fingerprint.

Replacement of the light source is easy to perform when the manufacturer’s directions are followed. Use only replacement lamps designated by the manufacturer to ensure compatibility and proper light source alignment. Any other repair that requires microscope disassembly should be performed only by a professional service representative. As with other instrumentation, microscope cleaning, maintenance, and problems should be documented. In addition, service to clean, lubricate, and align components should be performed annually by the manufacturer or a professional service representative.

Box 18.2 lists the dos and don’ts of good microscope care. As with any precision instrument, the microscope will give long-lasting and optimal performance if it is maintained and cared for properly.

Box 18.2

Microscope Dos and Don’ts

Dos