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Definition

A microscope is an optical instrument consisting of a lens or combination of lenses for enlarging images of objects. It is typically used in a laboratory to view objects that are not visible to the naked eye.

Purpose

In health care, a microscope is used in a laboratory to determine the amount or number of analytes (measured substances) present in a specimen, such as blood, urine, or stool. Laboratory tests may be ordered for various reasons:

• to detect disease or to quantify the risk of future disease
• to establish or exclude a diagnosis
• to assess the severity of a disease
• to direct the selection of interventions
• to monitor the progress of a disorder
• to monitor the effectiveness of a treatment

Description

In health care, the most commonly used microscope to evaluate laboratory specimens is the compound microscope, a kind of light microscope (also known as an optical microscope). The compound microscope contains several lenses that magnify the image of a specimen. The lens located directly over the object is called the objective lens, and the lens closest to the eye is called the eyepiece. The total magnification is a product of the magnification of these two lenses-if the objective lens magnifies 100-fold, and the eyepiece magnifies 10-fold, then the final magnification will be 1,000-fold. But enlarging the image of a specimen is not the only consideration for selection of a microscope. A key property of a microscope is its power of resolution—its ability to distinguish between two objects, such as two cells, positioned closely together. The resolving power of a microscope is denoted by the numerical aperature value (NA). The larger the number, the greater the resolution of the lens.

In addition to the eyepiece and objective there are several other components of a compound microscope that require adjustment by the user. The condenser is a lens that is located below the stage. Its purpose is to focus the light on the specimen. The iris diaphragm is located beneath the condenser. It can be closed to reduce the amount of peripheral light passing through the specimen. This is useful when viewing unstained cells because a narrow diaphragm adds contrast; however, if closed too much the brightness and resolution are reduced significantly. For most applications the iris diaphragm can be positioned correctly by closing it all the way, and then opening it until the black diaphragm is just beyond the field of view. The type of illumination used by most microscopes is called Koehler illumination. To use Koehler illumination the filament of the microscope lamp should be focused on the iris diaphram by moving the condenser lens. This will evenly distribute the light through the specimen.

In addition to the light microscope, there are several other types that are used for specific purposes. A brief descripition of those used in a clincial laboratory follows:

• Darkfield microscope. A darkfield microscope uses a special condenser that directs the light away from the objective unless it passes through the cell or object from the side. The background appears dark and the object light. The darkfield scope is used when examining unstained cells or objects. The most frequent clincial application is the examination of fluid from a genital chancre for the characteristic corkscrew shaped organism that causes syphilis, Treponema pallidum.
• The fluorescence microscope. Fluorescence is the emission of long wavelength light (visible light of a specific color) by compounds when excited by short wavelength (higher energy) light. Fluorescence microscopes are used to examine cells or objects stained with fluorescent dyes. They use an ultraviolet light source (mercury vapor lamp) to transmit short wavelength light through the specimen. The light passes through a darkfield condenser that blocks all light from the objective except rays that pass through the object. A barrier filter above the objective removes any residual ultraviolet light and transmits the wavelength emitted by the fluorochrome. This technique is used to identify antibodies attached to cell components. Because the background is dark and fluorescence dyes are more sensitive than other stains, it permits the detection of extremely low concentrations of antibody.

• An inverted microscope is one in which the light source is above the stage and the objectives are beneath the specimen rather than above it. This type of microscope is ideal for examining cells in tissue culture and for manipulating cells as is done in artificial reproductive procedures. The cell culture can be placed on the stage and the technologist can manipulate the cells because access to them is unobstructed.
• Phase contrast microscope. This type of microscope uses a condenser with a diaphragm inside that contains an annulus (ring cutout) in the center. The objective is constructed so that it diffracts the light transmitted through the annulus. When this light passes through the specimen, dense objects such as nuclei enhance this effect. Light from dense objects seem to reach the eye a fraction of a second later and the objects appears darker. Phase contrast makes it easier to distinguish different types of unstained cells and is preferred for urinalysis.
• Interference-contrast microscope. One disadvantage of phase contrast is that light is refracted from the edge of objects giving cells a halo. Interference-contrast microscopy uses polarizing filters and prisms to achieve the same effect as the annulus without the halo effect.
• Polarizing microscope. Some objects, such as certain crystals or minerals are able to change the direction (rotate) of light. This property is called birefringence and the object is said to be anisotropic. The polarizing microscope uses a polarizing filter beneath the stage. This transmits all the light from the lamp through the specimen in the same plane. A second polarizing filter called the analyzer is placed before the eyepiece so that it is out of phase with the substage polarizing filter. The analyzer blocks all of the light causing a dark background unless the object on the slide is anisotropic. Birefringent objects rotate the light so that it passes through the analyzer lens and the object appears light (white) against a dark background. This technique is used to identify uric acid needles in joint fluid from a patient with gout, since uric crystals are birefringent.
• The transmission electron microscope uses electromagnetic lenses, not optical lenses, that focus a high-velocity electron beam instead of visible light. A transmission electron microscope directs a beam of electrons through a specimen. Only a small piece of a cell can be observed in any one section. Generally, an electron microscope cannot be used to study live cells because they are too vulnerable to the required conditions and preparatory techniques. However, magnification can be achieved on the order of one thousand fold higher than a compound microscope.

Many medical tests require the use of a compound microscope for evaluation. These include:

• Biopsy. Tissue examined for cancer or other abnormalities.
• Blood cells. Identification of abnormal red and white blood cells, immature cells, and the different types of white cells.
• Bone marrow aspiration. Examination of marrow from hipbone or breastbone under a microscope for abnormalities of blood cell precursors and bone marrow tissue.
• Chorionic villus sampling. Examination of chromosomes of fetal cells under the microscope to determine if an abnormal number are present of if there is structural damage.
• Papanicolaou (Pap) test. Microscopic examination of cells scraped from the cervix to detect cancer.
• Microbiological exam. Microscopic examination of specimens (some normally sterile) for the presence of bacteria, parasites, yeast, and fungi. Most often this involves use of the Gram stain or acid-fast stain.
• Cytological exam of body fluids. Examination of urine, cerebrospinal fluid, pleural, pericardial, and synovial fluid for blood cells, malignant cells, crystals, bacteria, and other cells.
• Seminal fluid exam. The determination of sperm concentration, viability, and morphology (appearance).

Operation

After a specimen is prepared and placed on the microscope, the microscope is adjusted to change the magnification and focus the image. Precise mechanical adjustments are necessary to manipulate the objective and eyepiece, the substage condenser, iris diaphragm, and the object.

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KEY TERMS

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Condenser—A lens or system of lenses to collect light rays and converge them to a focus.

Electron microscope.—A device which beams electrons instead of light beams at and through an object. A powerful magnet is used to bend the electron beam (instead of a glass lens). This type of microscope provides the greatest resolution of extremely small details, such as individual atoms in an object or substance.

Eyepiece—The lens system nearest the eye which magnifies the primary image produced by the objective so as to form a secondary, virtual image 10 in (25 cm) away from the eyepoint.

Light microscope—A device that works by passing visible light through a condenser and an objective lens.

Objective—The lens system near the object which forms the primary inverted image.

Magnification—The apparent increase in size under the microscope.

Resolution—Degree of detail, ranging from low to high, determining the ability to distinguish between two objects positioned closely together.

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Maintenance

The microscope should be kept covered when not in use. It should be cleaned, lubricated, and adjusted by a microscope technician at least once a year to conserve the life of the instrument. Lenses should be cleaned after each use taking care to remove any oil from the lens surface. When cleaning the lenses, use only lens paper to avoid scratching the lenses.

Health care team roles

Collection of a specimen for laboratory evaluation is typically done by a nurse or other health care practitioner. For example, venipuncture (puncture of a vein for the withdrawal of blood) may be performed by various members of the health care team. Although labs employ phlebotomists (individuals who perform venipuncture) to collect blood specimens, nurses must know how to perform this procedure because they routinely perform it in the home, in long-term care settings, and in hospital critical care units.

The nurse may inform the client about the reasons for the test, what to expect during the test, and any associated side effects or risks. The nurse should notify the practitioner of any client or family concerns not alleviated by discussions.

Assessment of the client for symptoms such as post-puncture bleeding or occlusion is the responsibility of a nurse or other allied health professional.

Training

Microscopes are usually used by pathologists, laboratory technologists, and technicians who evaluate specimens. Proper use of a microscope is part of training for nurses and other allied health care professionals.

Jennifer F. Wilson