7-3 THE MICROSCOPE

LEARNING OBJECTIVE: Identify the parts of the microscope, and determine their functions.

Before any attempts are made to view blood smears, urinary sediments, bacteria, parasites, etc., it is absolutely essential that beginners know the instrument with which they will be spending considerable time—the microscope. The microscope is a precision instrument used extensively in clinical laboratories to make visible objects too small to be seen by the unaided eye. Most laboratories are equipped with binocular (two-eyepiece) microscopes, but monocular microscopes are also commonly used. The type of microscope most often used in the laboratory is referred to as the compound microscope. See figure 7-5. A compound microscope contains a system of lenses of sufficient magnification and resolving power (ability to show, separate, and distinguish) so that small elements lying close together in a specimen appear larger and distinctly separated. In the following sections, the compound microscope’s framework, illumination system, magnification system, and focusing system will be discussed.

Figure 7-5.—Compound microscope.

FRAMEWORK

The framework of the compound microscope consists of four parts: the arm, the stage, the mechanical stage, and the base (fig. 7-5).

Arm. The arm is the structure that supports the magnification and focusing system. It is the handle by which the microscope is carried.

Stage. The stage is the platform on which a specimen is placed for examination. In the center of the stage is an aperture or hole that allows the passage of light from the condenser.

Mechanical Stage. The mechanical (movable) stage holds the specimen in place and is the means by which the specimen may be moved about on the stage.

Base. The base is the structure on which the microscope rests.

ILLUMINATION SYSTEM

Ideal illumination of a specimen viewed under the microscope requires even light distribution. The objectives must also be entirely filled with light from the condenser. To fulfill these requirements, the illumination system of the compound microscope consists of three parts: an internal light source, a condenser, and an iris diaphragm. See figure 7-5.

Internal Light Source. The internal light source is built into the base of the microscope. It provides a precise and steady source of light into the microscope.

Condenser. The condenser is composed of a compact lens system and is located between the light source and stage. The condenser concentrates and focuses light from the light source directly through the specimen.

Iris Diaphragm. An iris diaphragm located on the condenser controls the diameter of the light source’s beam. To improve resolution, the operator should adjust the opening of the iris diaphragm to approximately the same size as the face of the objective lens. In addition to the diaphragm on the condenser, an iris diaphragm may be located on the internal light source. This iris diaphragm controls the amount of light sent to the condenser from the internal light source.

MAGNIFICATION SYSTEM

The magnification system of the compound microscope contains at least two lens systems. The two lens systems are mounted on either end of a tube called the body tube. The lens nearest the object is called the objective lens, and the lens nearest the eye is the ocular lens or eye piece. See figure 7-5.

Objective Lenses. On a compound microscope, there is usually a set of three objective lenses (or “objectives”). This set of objectives is the component most responsible for the magnification and resolution of detail in a specimen. Each objective lens has a different focus distance and magnification power. A set of objectives normally consists of a low-power lens (approximate focus 16 mm, magnification 10X), a high-power lens (approximate focus 4 mm, magnification 45X), and an oil-immersion lens (approximate focus 1.8 mm, magnification 100X). Objective lenses are color coded for easy recognition: 16 mm-10X (green), 4 mm-45X (yellow), and 1.8 mm-100X (red).

Revolving Nosepiece. The revolving nosepiece contains openings into which objective lenses are fitted, and revolves objectives into desired position.

Body Tube. The body tube is a tube that permits light to travel from the objective to the ocular lens.

Ocular Lenses. Ocular lenses, or eyepieces, are located on top of the body tube and usually have a magnification power of 10X. To calculate the total magnification of a specimen, you multiply the magnification power of the objective by the magnification power of the ocular lens. Examples of total magnifications are provided in table 7-1.

Table 7-1.—Examples of Total Magnifications

Objective
Lens
Color
Code
10X
Ocular
Total
Magnification
16 mm -10X
4 mm - 45X
1.8 mm -100X
Green
Yellow
Red
10X
10X
10X
100X
450X
1000X

FOCUSING SYSTEM

Focusing is accomplished by moving the stage up or down with the coarse and fine control knob (fig. 7-5). Whether the stage needs to be raised or lowered depends on the focal length of the objective being used. For example, the high-powered objective of short focal length (4 mm) will need the stage raised so the objective is very close to the specimen, while the low-powered objective of a longer focal length (16 mm) will need the stage lowered so the objective is farther from the specimen.

The coarse control knob is used initially to bring the specimen’s image into approximate focus. Once this is accomplished, the fine control knob sharpens the image.

Coarse Control Knob. The coarse control knob is the larger and inner knob. Rotating the coarse control knob allows the image to appear in approximate focus.

Fine Control Knob. The fine control knob is the smaller and outer knob. Rotating this control knob renders the image clear and well-defined.

FOCUSING THE MICROSCOPE

The process of focusing consists of adjusting the relationship between the optical system of the microscope and the object to be examined so that a clear image of the object is obtained. The distance between the upper surface of the glass slide on the microscope stage and the faces of the objective lens varies depending upon which of the three objectives is in the focusing position. It is a good practice to obtain a focus with the low-power objective first, then change to the higher objective required to avoid accidentally damaging the objective lens, the specimen, or both. Most modern microscopes are equipped with parfocal objectives (meaning that if one objective is in focus, the others will be in approximate focus when the nosepiece is revolved). With the low-power objective in focusing position, observe the following steps in focusing.

  1. Seat yourself behind the microscope, then lower your head to one side of the microscope until your eyes are approximately at the level of the stage.
  2. Using the coarse adjustment knob, lower the body tube until the face of the objective is within 1/4 inch of the object. Most microscopes are constructed in such a way that the low-power (green) objective cannot be lowered and make contact with the object on the stage. 
  3. While you are looking through the ocular, you should use the coarse adjustment knob to elevate the body tube until the image becomes visible. Then use the fine adjustment knob to obtain a clear and distinct image. Do not move the focusing knob while changing lenses.
  4. If the high-power objective (yellow) is to be used next, bring it into position by revolving the nosepiece (a distinct “click” indicates it is in proper alignment with the body tube). Use the fine adjustment knob only to bring the object into exact focus.
  5. If specimen is too dark, you can increase lighting by opening the iris diaphragm of the condenser.
  6. The oil-immersion objective (red) is used for detailed study of stained blood and bacterial smears. Remember that the distance between objective lens and object is very short, and great care must be employed so the specimen is not damaged. After focusing with the high-power objective and scanning for well-defined cells, raise the objective, place a small drop of immersion oil, free of bubbles, on the slide, centering the drop in the circle of light coming through the condenser. Next, revolve the nosepiece to bring the oil-immersion objective into place, and, by means of the coarse adjustment knob, slowly lower the body tube until the lens just makes contact with the drop of oil on the slide. The instant of contact is indicated by a flash of light illuminating the oil. The final step in focusing is done with the fine adjustment knob. It is with this lens in particular that lighting is important. The final focus, clear and well-defined, will be obtained only when proper light adjustment is made.

CARE OF THE MICROSCOPE

The microscope is an expensive and delicate instrument that should be given proper care.

Moving or transporting microscopes should be accomplished by grasping the arm of the scope in one hand and supporting the weight of the scope with the other hand. Avoid sudden jolts and jars.

Keep the microscope clean at all times; when not in use, microscopes should be enclosed in a dustproof cover or stored in their case. Remove dust with a camel hair brush. Lenses may be wiped carefully with lens tissue. When the oil-immersion lens is not being used, remove the oil with lens tissue. Use oil solvents (such as xylene) on lenses only when required to remove dried oil and only in the minimal amount necessary. Never use alcohol or similar solvents to clean lenses.