List of Lessons
Lesson 2-3. LABORATORY EQUIPMENT
MICROSCOPES A modern microscope for use in the hematology laboratory is equipped with an illuminator system, a substage condenser system, an objective system, a projector (eyepiece or ocular system), an iris diaphragm, nicol prisms, a tubular barrel (monocular or binocular bodies), and a mechanical stage (figure 2-10). A compound microscope or bright field microscope uses a combination of lenses, the objective lens (lens closer to the object) and the ocular lens (lens closer to the eye) to project the image to the retina of the eye. The objective lens acts much like a small projection lens which projects an enlarged primary image near the top of the tubular barrel. This image, formed in air, is known as an "aerial image”. This object is viewed through the projector or eyepiece that acts like a magnifier except that it magnifies an aerial object instead of an actual object. The final image projected on the retina of the eye is called a "virtual image" because the light rays appear to come from the image. The rays are actually created by an increase in magnification by the lens system.
Magnification. Magnification in a microscope is limited to the useful magnification that can be achieved, that is, the ability to obtain fine detail of the object being examined. This ability to render visible the fine detail is the resolving power of the microscope. The resolving power of a microscope is dependent on the numerical aperture (N.A.) of the objective lens and condenser lens. Therefore, proper adjustment of these lenses is essential in order to obtain useful magnification.
Microscopes in general use in medical laboratories are provided with three objectives with focal lengths of 10x, 40x, and 100x, respectively. Microscopes are usually provided with 10X (most common) ocular. Multiplying the power of the ocular by the power of the objective gives the degree of magnification of the object under observation. The degree of magnification is expressed in diameters (refers to an increase in diameter). The ocular magnification, the millimeter length of the objective, its magnification power, and the total apparent increase obtained using oculars and objectives of the powers shown are given:
Magnification is increased in practice by using a higher power objective. Most microscopes are equipped with a revolving nosepiece, and selection of an objective lens is done with ease.
| Ocular || Objective || Magnification |
- 100 diameters
- 400 diameters
- 1000 diameters
Figure 2-10. Compound microscope. Illumination. Compound microscopes are dependent on electricity as the primary source for illumination power. Correct illumination of the object under study is an extremely important detail. Incorrect lighting of the object can lead to inaccurate results and conclusions. Correct illumination can be obtained from an iris diaphragm or substage light (abbe condenser).
Illumination entering the microscope in which the light source is imaged at the specimen resulting in increased but uneven brightness is considered to be critical illumination. The Koehler illumination is a field of evenly distributed brightness across the specimen
Regulation of the amount of light admitted is accomplished by the abbe condenser on the substage. The size of the opening in the diaphragm is controlled by a lever on the side of the condenser. The lever of the abbe condenser should never be forced to the full limit in either direction. Generally, when observing liquid preparations under low power, the condenser opening should be partially closed. Under the high dry objective, the condenser is generally opened to a greater degree to allow more light to pass through the material. When observing stained preparations under the oil immersion objective, the abbe condenser is usually opened wide.
The substage condenser functions to direct a light beam of the desired numerical aperture (N.A.) and field size onto the specimen. The size of the opening in the condenser together with its position up or down controls the light entering the system. When the condenser is close to the stage, concentration of light is greater; as the condenser is moved downward, less light passes upward through the object under observation.
(5) Improper illumination is indicated when: (1) dark points or shadows appear in the field; (2) the outline of an object is bright on one side and dark on the other; or (3) the object appears to be in a glare of light. This can usually be corrected by changing the position of the iris diaphragm , by reducing the amount of light by adjusting the size of the opening in the iris diaphragm, or by raising or lowering the condenser.
Focusing. Focusing can be defined as the adjustment of the relationship between the optical system and the object so that a clear image is obtained. Several important rules to be observed when focusing the microscope on the preparation are:
- After the object is mounted on the stage, the objective to be used is turned into line with the eyepiece.
- Movement of the objective is accomplished by revolving the nosepiece. The nosepiece is provided in order to enable rapid, convenient substitution of one objective for another. This change is effected by grasping two of the objectives between the thumb and forefinger of the right hand and rotating them until the desired objective is brought into line with the axis of the body tube. It is very important that exact alignment be obtained. The correct setting is indicated by a slight "click" as the objective comes into position.
- Whenever the nosepiece is revolved, its movements should be observed to make certain that the objectives do not come into contact with the object. Some microscopes are not parfocal; that is, objects in focus under low power will not be in focus when the nosepiece is rotated to a higher power of magnification. It may, therefore, be necessary to refocus when changing to higher magnification. In microscopes that are parfocal, it is possible to swing other objectives into place without touching the coarse adjustment and with only a slight turn of the fine adjustment knob required to restore perfect focusing.
- To bring an object into focus, watch from the side and use the coarse adjustment to lower the objective until it is below the point at which the object would normally be expected to come into view.
| NOTE: To avoid damage to slide or microscope, view from side for preliminary focusing. Then, using the coarse adjustment and at the same time looking through the ocular, raise the objective very slowly until the field comes into view. Further adjust to the best image, using only the fine adjustment. |
- In focusing upward with the fine adjustment, the object will first appear in faint outline, then gradually more distinctly, and finally, sharply defined. If the adjustment goes beyond the point of sharp definition, return to the point of greatest clarity by using the fine adjustment.
- Never move an objective downward while looking through the eyepiece. When the objective is moved downward, always observe the downward motion with the eye held level with the microscope stage. Failure to observe these precautions can result in damage to the lens of the objective or the object under study.
Care of the Microscope. The microscope is an instrument of precision with many delicate parts, and it must be handled with the utmost care. Care should not be confined to the optical elements alone. The microscope is a combination of optical and mechanical excellence, one complementing the other. The following precautions should always be observed in the care of the microscope:
- No unauthorized person should manipulate the microscope.
- Keep the microscope as free from dirt and dust as possible. Dusty lenses produce foggy images, while dust in the focusing mechanisms causes excessive wear of those parts.
- The microscope should be always covered when not in use.
- Care should be taken to prevent all parts of the microscope from coming into contact with acid, alkali, chloroform, alcohol, or other substances that corrode metal or dissolve the cementing substance by means of which the lenses are secured into the objectives and oculars.
- Always carry the microscope with two hands by the arm and base.
- Avoid sudden jars I such as placing the microscope on the table with undue force.
- No dust should be permitted to settle on the lenses nor should the finger come in contact with any of the surfaces.
- The lens system should never be separated, as the lenses are liable to become decentered and dust can enter.
- Avoid all violent contact of the objective lens and the cover glass.
- Keep eyepieces in the microscope at all times to keep free of dust.
- To remove dust, brush the lenses with a soft brush, or a burst of air. Avoid hard wiping, as dust is often hard and abrasive.
- Ethanol or methanol can be used in cleaning lenses or removing oil from objectives. Only a small amount is necessary and should be used with lens paper.
- The microscope should be protected against direct sunlight and moisture.
- In very warm, humid climates, microscopes should be stored in dry cabinets when not in use. Such cabinets should be reasonably airtight, equipped with a light bulb to supply heat, and several cloth bags containing a hygroscopic salt, such as calcium chloride, to absorb moisture. In warm, humid climates, the lenses of unprotected microscopes can be attacked by certain fungi that etch glass and ruin the lenses.
- After use, always turn the nosepiece to a position, which brings the low power objective into direct line with the opening in the substage condenser. If this precaution is not taken, the longer, higher-powered objectives can accidentally come into contact with the condenser lens
- The entire microscope should be cleaned frequently to remove dust, finger marks, oil, grease, and remnants of specimens. All parts of the microscope should be kept scrupulously clean at all times.
- Never tamper with any of the parts of the microscope. If the instrument does not seem to be functioning properly, immediately call the matter to the attention of the laboratory supervisor.
- Maintenance of the microscope should be done in accordance with the manufacturer's booklet of instruction.
- Immediately after use, the oil immersion objective must be wiped clean of oil with a soft, absorbent lens paper.
- Oil immersion. This type of microscope is extensively for Erythrocyte morophology, estimated platelet counts, and differentiate leukocytes.
- Phase microscopy. Phase microscopy is becoming increasingly prevalent in platelet counting. In bright-field illumination, a completely transparent specimen is difficult to see in any detail. By using phase contrast, transparent living objects can be studied. Phase microscopy operates on the principle that if a portion of light is treated differently from the rest, and caused to interfere with the rest, it produces a visible image of an otherwise invisible transparent specimen. Phase contrast accessories are available from the standard optical companies.
- Fluorescence microscopy. In hematology, used primarily for antinuclear antibody, T-cell and B-cell studies.
These are laboratory devices or units that apply a relatively high centrifugal force (up to 25,000 g) to a specimen, causing its separation into different fractions according to their specific gravities. Centrifugation is the process of separating components of a mixture (away from a center as in centrifugal force) on the basis of differences in densities of the different components using a centrifuge.
Table Top Models. These units are mounted on rubber feet that absorb vibration. The speed is controlled by means of a rheostat on the front panel. Top speeds of centrifuges will vary and the top speed of a particular instrument should be known in order to use the speed control device. Those centrifuges have adapters to hold 6 tubes and adapters for 12 tubes.
Floor-Mounted Models. The heavier floor-mounted models accommodate a large number of tubes at one time. The top speed of these instruments is higher than that of table models. Because of their increased inertia, they are equipped with a brake to facilitate stopping. In these units, the tubes are placed in balanced receptacles that are mounted on spokes emanating from a central hub.Microhematocrit Centrifuge. This centrifuge is a special type of high-speed centrifuge employed to spin capillary tubes. The circular tube holder on this centrifuge is flat and surrounded by a rubber ring. It has a capacity of 24 capillary tubes. After a capillary tube is filled with blood, it is closed with a commercial clay sealing material. During centrifugation the sealed end is always placed in position facing toward the outside of the holder plate. Most centrifuges of this type spin the tubes at 10,000 rpm.
Precautions. In all instances where centrifugation is required, careful attention must be given to balancing the units. This means that tubes must be placed exactly opposite each other, they must be of identical weight, and they must contain the same amount of fluid. If at all possible, centrifuges should be equipped with tachometers so that speed nay be checked and controlled. Certain procedures, such as hematocrits, require a critical relative centrifugal force (RCF or g). Relative centrifugal force is the weight of a particle in a centrifuge relative to its normal weight, the centrifugal force per unit mass in gravities (g). To determine the RCF (or g) for these procedures, consult the serology manual or a monograph. The inside of the centrifuges should occasionally be cleaned to prevent dust particles from being blown into specimens. The lid on the centrifuge should be closed and locked before and during operation. Only open the lid when the centrifuge has stopped rotating.