Microscope and Microscopy

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Microscope and Microscopy

Introduction

The microscope is a powerful tool for investigating the complexity of biological life. This includes looking at the identity and structure of microorganisms, which is essential in the diagnosis of many infectious diseases. Microorganisms are not visible to the human eye, owing to their small size. The light microscope focuses visible light upon a clinical specimen and allow the microbe to be magnified through a series of lenses.

Staining a specimen that may contain microorganisms is an additional aid to identification. Some microbes will absorb a certain stain while others will not, which provides a way of identifying them. Modern microscopic technologies allow for quick and accurate identification of microorganisms involved in human disease. However, microscopic identification alone is only part of the investigations underlying a diagnosis of an infectious disease. The patient's medical history and biochemical tests upon clinical specimens are equally important.

History and Scientific Foundations

The magnifying power of lenses—curved pieces of glass that can bend light—was first mentioned in the writings of the Roman philosophers Seneca and Pliny the Elder during the first century AD. But they were not put to practical use until the development of spectacles towards the end of the thirteenth century. The Dutch spectacle makers Zaccharias Janssen and his son Hans began to experiment with the magnifying properties of combinations of lenses in the late sixteenth century. News of their work spread to Galileo who produced a primitive microscope in 1609. But it was Anthony van Leewenhoek (1632–1723), the Dutch biologist, who first realized the potential of the microscope for the study of the world of microorganisms.

Looking at specimens from many different sources, he described the appearance of what he called animalcules—namely, yeast, bacteria, and protozoa. During his life, he wrote over 100 papers on his discoveries for both the Royal Society of England and the French academy. The English scientist Robert Hooke went on to confirm van Leewenhoek's work and improved on the design of his light microscope. Towards the end of the nineteenth century, there were some major advances in microscope manufacture. The American pioneer Charles A. Spencer founded an industry based upon instruments with fine optical systems, which are similar to today's basic light microscopes.

Magnifications of 1,250 are achievable with ordinary white light and up to 5,000 if blue light is used. The microscope is a compound optical system. A condensing lens focuses a bright beam of light upon the clinical specimen, which is placed on a platform called a stage and covered with a thin sheet of glass called a cover slip. The objective lens, near the specimen, forms an intermediate magnified image, which is magnified again by the eyepiece, which is close to the eye.

The magnification of a light microscope is limited by the wavelength of the light used to illuminate the specimen. It cannot distinguish objects that are smaller than half the wavelength of the light. Thus, white light has an average wavelength of around 0.55 micrometers, so any two lines that are closer together than half of this—0.275 micrometers—will shown up as a single line and an object that is smaller than this in diameter will show up as a blur, or not at all. Smaller objects, such as viruses, can only be seen with the aid of the electron microscope, in which the beam of illuminating light is replaced by a beam of electrons. Electron microscopes were invented in the late 1940s and are much more expensive than light microscopes. However, they have allowed not only the study of viruses but also of so called biological ultrastructure, which is the fine details of cells, tissues, and their activities in health and disease.

WORDS TO KNOW

ELECTRON: A fundamental particle of matter carrying a single unit of negative electrical charge.

LENS: An almost clear, biconvex structure in the eye that, along with the cornea, helps to focus light onto the retina. It can become infected with inflammation, for instance, when contact lenses are improperly used.

MICROORGANISM: Microorganisms are minute organisms. With the single yet-known exception of a bacterium that is large enough to be seen unaided, individual microorganisms are microscopic in size. To be seen, they must be magnified by an optical or electron microscope. The most common types of microorganisms are viruses, bacteria, blue-green bacteria, some algae, some fungi, yeasts, and protozoans.

STAINING: Staining refers to the use of chemicals to identify target components of microorganisms.

WAVELENGTH: A distance of one cycle of a wave; for instance, the distance between the peaks on adjoining waves that have the same phase.

ANTONI VAN LEEUWENHOEK

Antoni van Leeuwenhoek (1632–1723) who, using just a single lens microscope, was able to describe organisms and tissues, such as bacteria and red blood cells, which were previously not known to exist. In his lifetime, Leeuwenhoek built over 400 microscopes, each one specifically designed for one specimen only. The highest resolution he was able to achieve was about 2 micrometers.

Applications and Research

The chemical and dyestuffs industry that began in Germany in the nineteenth century provided microscopists with a range of stains that made the identification of specific microorganisms much easier. Many of these are still used in modern microbiology laboratories. For instance, Gram's stain distinguishes between bacteria on the basis of the thickness and composition of their cell wall. Gram-positive bacteria, such as Corynebacterium, listeria and Bacillus species, which have a more complex cell wall, do absorb the stain, trapping it between the layers of this wall. Gram-negative bacteria, such as Salmonella and Shigella species, do not retain the stain because their walls lack one of the layers.

Ziehl-Nielsen stain is useful for identifying the mycobacteria that cause tuberculosis (TB), and silver methenamine stains chitin, a carbohydrate that is found in the walls of fungi and of Pneumocystis carinii, the microorganism that causes an otherwise rare form of pneumonia among HIV/AIDS patients. Giemsa stain is found useful in identifying malaria and other parasites, such as Leishmania.

Immunofluorescence is a modern microscopy technique that uses antibodies labeled with a fluorescent marker to bind to specific parts of a microbial pathogen. When the specimen is examined under ultraviolet light, the antibody will glow with a green fluorescence, if the pathogen is present.

Microsocopy aids diagnosis by examining the clinical specimens that are likely to be infected with the causative organism. Therefore, sputum is examined for TB, blood for malaria, stool samples for parasites, and urine to detect bacteria causing urinary tract infections. Viruses are detected, although not routinely, with an electron microscope. There are many other laboratory methods for the detection of microorganisms that complement microscopy.

The optics of a light microscope are adjustable depending on the type of result desired. In light field microscopy, the specimen is visualized by light passing from the condenser through the specimen, while dark field microscopy uses oblique illumination that gives higher resolution of detail, if this is needed. Phase contrast microscopy involves modification to the condenser and objective to give an optical interference pattern in the viewed image. This is very valuable for transparent specimens because it makes details appear darker against a light background.

Impacts and Issues

A microscope must be operated by a skilled scientist, if findings are to be of clinical value. Some microorganisms are easy to identify under the microscope, especially if the specimen is given the correct preparation, including staining. However, it is not always possible to distinguish between a pathogen and a harmless organism present within the same specimen.

But sometimes inadequate preparation will give faulty results and an important diagnosis may be missed. Use of the microscope is also relatively insensitive as a diagnostic tool in that many organisms must be present for a positive result to be given. Infections caused by relatively few bacteria may be missed.

It also takes time and experience to come to a correct conclusion based upon microscope findings. If lab technicians are handling a large number of specimens—from a cervical cancer screening program, for example—they may miss positive findings. Cancer cells have different features under the microscope compared to healthy cells. Sometimes these differences may be missed, leading to a false negative. The microscope is just one of many diagnostic tools at the disposal of the pathology laboratory for the diagnosis of infectious and other diseases.

ELECTRON MICROSCOPES

There are two types of electron microscope, the transmission electron microscope (TEM) and the scanning electron microscope (SEM). The TEM transmits electrons through an extremely thin sample. The electrons scatter as they collide with the atoms in the sample and form an image on a photographic film below the sample. This process is similar to a medical x ray, where x rays (very short wavelength light) are transmitted through the body and form an image on photographic film behind the body. By contrast, the SEM reflects a narrow beam of electrons off the surface of a sample and detects the reflected electrons. To image a certain area of the sample, the electron beam is scanned in a back and forth motion parallel to the sample surface, similar to the process of mowing a square section of lawn. The chief differences between the two microscopes are that the TEM gives a two-dimensional picture of the interior of the sample while the SEM gives a three-dimensional picture of the surface of the sample. Images produced by SEM are familiar to the public, as in television commercials showing pollen grains or dust mites.