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Microscope

Microscope

Background

A microscope is an instrument used to produce enlarged images of small objects. The most common kind of microscope is an optical microscope, which uses lenses to form images from visible light. Electron microscopes form images from beams of electrons. Acoustic microscopes form images from high-frequency sound waves. Tunneling microscopes form images from the ability of electrons to "tunnel" through the surface of solids at extremely small distances.

An optical microscope with a single lens is known as a simple microscope. Simple microscopes include magnifying glasses and jeweler's loupes. An optical microscope with two lenses is known as a compound microscope. The basic parts of a compound microscope are the objective, which holds the lens near the specimen, and the eyepiece, which holds the lens near the observer. A modern compound microscope also includes a source of light (either a mirror to catch external light or a light bulb to provide internal light), a focusing mechanism, and a stage (a surface on which the object being examined can be held in place). Compound microscopes may also include a built-in camera for microphotography.

Ancient peoples noted that objects seen through water appeared larger. The first century Roman philosopher Seneca recorded the fact that letters seen through a glass globe full of water were magnified. The earliest simple microscopes consisted of a drop of water captured in a small hole in a piece of wood or metal. During the Renaissance, small glass lenses replaced the water. By the late seventeenth century, the Dutch scientist Antonie van Leeuwenhoek built outstanding simple microscopes using very small, high-quality lenses mounted between thin brass plates. Because of the excellence of his microscopes, and the fact that he was the first to make observations of microscopic organisms, Leeuwenhoek is often incorrectly thought of as the inventor of the microscope.

The compound microscope made its first appearance between the years 1590 and 1608. Credit for this invention is often given to Hans Janssen, his son Zacharias Janssen, or Hans Lippershey, all of whom were Dutch spectacle makers. Early compound microscopes consisted of pairs of lenses held in a small metal tube and looked much like modern kaleidoscopes. Because of the problem of chromatic aberration (the tendency of a lens to focus each color of light at a slightly different point, leading to a blurred image) these microscopes were inferior to well-made simple microscopes of the time.

The earliest written records of microscopic observations were made by the Italian scientist Francesco Stelluti in 1625, when he published drawings of a bee as seen through a microscope. The first drawings of bacteria were made by Leeuwenhoek in 1683. During the seventeenth and eighteenth centuries, numerous mechanical improvements were made in microscopes in Italy, including focusing devices and devices for holding specimens in place. In England in 1733, the amateur optician Chester Moor Hall discovered that combining two properly shaped lenses made of two different kinds of glass minimized chromatic aberration. In 1774, Benjamin Martin used this technique in a microscope. Many advances were made in the building of microscopes in the nineteenth and twentieth centuries. Electron microscopes were developed in the 1930s, acoustic microscopes in the 1970s, and tunneling microscopes in the 1980s.

Raw Materials

An optical microscope consists of an optical system (the eyepiece, the objective, and the lenses inside them) and hardware components which hold the optical system in place and allow it to be adjusted and focused. An inexpensive microscope may have a mirror as a light source, but most professional microscopes have a built-in light bulb.

Lenses are made of optical glass, a special kind of glass which is much purer and more uniform than ordinary glass. The most important raw material in optical glass is silicon dioxide, which must be more than 99.9% pure. The exact optical properties of the glass are determined by its other ingredients. These may include boron oxide, sodium oxide, potassium oxide, barium oxide, zinc oxide, and lead oxide. Lenses are given an antireflective coating, usually of magnesium fluoride.

The eyepiece, the objective, and most of the hardware components are made of steel or steel and zinc alloys. A child's microscope may have an external body shell made of plastic, but most microscopes have an body shell made of steel.

If there is a mirror included, it is usually made of a strong glass such as Pyrex (a trade name for a glass made from silicon dioxide, boron dioxide, and aluminum oxide). The mirror has a reflective coating made of aluminum and a protective coating made of silicon dioxide.

If a light bulb is included, it is made from glass and contains a tungsten filament and wires made of nickel and iron within a mixture of argon and nitrogen gases. The base of the light bulb is made of aluminum.

If a camera is included, it contains lenses made of optical glass. The body of the camera is made of steel or other metals or of plastic.

The Manufacturing
Process

Making the hardware components

  • 1 Metal hardware components are manufactured from steel or steel and zinc alloys using precision metalworking equipment such as lathes and drill presses.
  • 2 If the external body shell of an inexpensive microscope is plastic, it is usually a light, rigid plastic such as acrylonitrile-butadiene-styrene (ABS) plastic. ABS plastic components are made by injection molding. In this process the plastic is melted and forced under pressure into a mold in the shape of the final product. The plastic is then allowed to cool back into a solid. The mold is opened and the product is removed.

Making optical glass

  • 3 The proper raw materials for the type of optical glass desired are mixed in the proper proportions, along with waste glass of the same type. This waste glass, known as cullet, acts as a flux. A flux is a substance which causes raw materials to react at a lower temperature than they would without it.
  • 4 The mixture is heated in a glass furnace until it has melted into a liquid. The temperature varies with the type of glass being made, but is typically about 2550°F (1400°C).
  • 5 The temperature is raised to about 2800°F (1550°C) to force air bubbles to rise to the surface. It is then slowly cooled and stirred constantly until it has reached a temperature of about 1800°F (1000°C). The glass is now an extremely thick liquid, which is poured into molds shaped like the lenses to be made.
  • 6 When the glass has cooled to about 600°F (300°C), it is reheated to about 1000°F (500°C). This process, known as annealing, removes internal stresses which form during the initial cooling period and which weaken the glass. The glass is then allowed to cool slowly to room temperature. The pieces of glass are removed from the molds. They are now known as blanks.

Making the lenses

  • 7 The blank is now placed in a vise and held beneath a rapidly rotating cylindrical cutter with a diamond blade. This cutter, known as a curve generator, trims the surface of the blank until a close approximation of the desired curve is obtained. The cut lens is inspected and cut again if necessary. The difficulty of this process varies widely depending on the type of glass being cut and the exact curvature required. Several cuttings may be required, and the time involved may be a few minutes or more than half an hour.
  • 8 Several cut blanks are placed on the surface of a curved block in such a way that their curved surfaces line up as if they were all part of one spherical surface. This allows many lenses to be ground at the same time. A cast iron grinding surface known as a tool is placed on top on the lenses. The block of lenses rotates while the tool moves at random on top of it. A steady flow of liquid moves between the tool and the lenses. This liquid, known as a slurry, contains water, an abrasive (usually silicon carbide) to do the grinding, a coolant to prevent overheating, and a surfactant to keep the abrasive from settling out of the slurry. The lenses are inspected after grinding and reground if necessary. The grinding process may take one to eight hours.
  • 9 The lenses are moved to a polishing machine. This is similar to the grinding machine, but the tool is made of pitch (a thick, soft resin derived from tar). A pitch tool is made by placing tape around a curved dish, pouring in hot, liquid pitch, and letting it cool back into a solid. A pitch tool can be used about 50 times before it must be reshaped. It works in the same manner as a grinding tool, but instead of an abrasive the slurry contains a polishing substance (usually cerium dioxide). The lenses are inspected after polishing and the procedure is repeated as necessary. Polishing may take from half an hour to five hours. The lenses are cleaned and ready to be coated.
  • 10 The lenses are coated with magnesium fluoride. They are then inspected again, labeled with a date of manufacture and a serial number, and stored until needed.

Making the mirror

  • 11 If a mirror is included, it is made in a way similar to the way in which a lens is made. Unlike a lens, it is cut, ground, and polished to be flat rather than curved. A reflective coating is then applied. Aluminum is heated in a vacuum to produce a vapor. A negative electrostatic charge is applied to the surface of the mirror so that it attracts the positively charged aluminum ions. This allows a thin, even coating of metal to be applied. A protective coating of silicon dioxide is then applied. Like a lens, the mirror is inspected, labeled, and stored.

Assembling the microscope

  • 12 All of the final assembly of the microscope is done by hand. The workers wear gloves, masks, and gowns so that dirt does not damage the lenses or the internal mechanisms of the microscope. First the lenses are placed in the steel tubes, which make up the bodies of the eyepiece and the objective. These tubes are manufactured in standard sizes, which allow them to be assembled into a standard size microscope.
  • 13 The focusing mechanism of most microscopes is a rack and pinion system. This consists of a flat piece of metal with teeth on one side (the rack) and a metal wheel with teeth (the pinion), which controls the movement of the rack. The rack and pinion direct the objective so that its movement toward or away from the object being observed can be controlled. In many microscopes, the rack and pinion are attached to the stage (the flat metal plate on which the object being observed rests) and the objective remains stationary. After the rack and pinion system is installed, the knobs that control it are attached.
  • 14 The external body shell of the microscope is assembled around the internal focusing mechanism. The eyepiece (or two eyepieces, for a binocular microscope) and objective (or a rotating disk containing several different objectives) are screwed into place. Eyepieces and objectives are manufactured in standard sizes that allow many different eyepieces and objectives to be used in any standard microscope.
  • 15 If the microscope contains a mirror, this is attached to the body of the microscope below the opening in the stage. If it contains a light bulb instead, this may be attached in the same place (to shine light through the observed object) or it may be placed to the side of the stage (to shine light on top of the object). Some professional microscopes contain both kinds of light bulbs to allow both kinds of observation. If the microscope contains a camera, it is attached to the top of the body.
  • 16 The microscope is tested. If it functions correctly, the eyepiece and objective are usually unscrewed before packing. The parts of the microscope are packed securely in close-fitting compartments lined with cloth or foam. These compartments are often part of a wood or steel box. The microscope is then placed in a strong cardboard container and shipped to consumers.

Quality Control

The most critical part of quality control for a microscope is the accuracy of the lenses. During cutting and polishing, the size of the lens is measured with a vernier caliper. This device holds the lens between two jaws. One remains stationary while the other is gently moved into place until it touches the lens. The dimensions of the lens are read off a scale, which moves along with the movable jaw.

The curvature of the lens is measured with a spherometer. This device looks like a pocket watch with three small pins protruding from the base. The two outer pins remain in place, while the inner pin is allowed to move in or out. The movement of this pin is connected to a scale on the face of the spherometer. The scale reveals the degree of curvature of the lens. A typical lens should vary no more than about one-thousandth of an inch (25 micrometers).

During polishing, these tests are not accurate enough to ensure that the lens will focus light properly. Optical tests must be used. One typical test, known as an autocollimation test, involves shining a pinpoint light source through a lens in a dark room. A diffraction grating (a surface containing thousands of microscopic parallel grooves per inch) is placed at the point where the lens should focus the light. The grating causes a pattern of light and dark lines to form around the true focal point. It is compared with the theoretical focal point and the lens is repolished if necessary.

The mechanical parts of the microscope are also tested to ensure that they function correctly. The eyepiece and the objective must screw firmly into their proper places and must be perfectly centered to form a sharp image. The rack and pinion focusing mechanism is tested to ensure that it moves smoothly and that the distance between the objective and the stage is controlled precisely. Rotating disks containing multiple objectives are tested to be sure that they rotate smoothly and that each objective remains firmly in place during use.

The Future

Amateur observers may soon be able to purchase microscopes with small, built-in video cameras, which allow the movements of microscopic organisms to be recorded. Computers may be built into the internal control mechanisms of the microscope to provide automatic focusing.

Where to Learn More

Books

Bradbury, Savile. An Introduction to the Microscope. Oxford University Press, 1984.

Jacker, Corrine. Window on the Unknown: A History of the Microscope. Charles Scribner's Sons, 1966.

Rochow, Theodore George, and Eugene George Rochow. An Introduction to Microscopy by Means of Light, Electrons, X-rays, or Ultrasound. Plenum Press, 1978.

Periodicals

Bardell, David. "The First Record of Microscopic Observations." Bioscience, January 1983, pp. 36-38.

Other

Ford, Brian J. "History of the Microscope." October 11, 1996. http://www.sciences.demon.co.uk/whistmic.htm

RoseSecrest

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microscopy

microscopy A microscope is an instrument that enables one to observe objects too small to be seen clearly by the naked eye. The microscope that is most familiar and most widely used for biomedical examination is the optical or light microscope, typically using visible light to study a transparent specimen mounted on a glass slide, which produces an optical image directly on the retina of the observer.

History

The use of light microscopes for biological studies was pioneered by the English scientist Robert Hooke (1635–1703), who first used the term ‘cells’ to describe the cavities he saw within sections of cork, and by Antoni van Leeuwenhoek (1632–1723), a Dutchman who created single lens microscopes of extraordinary resolving power unequalled by the later compound microscopes until the mid nineteenth century. Using these, he was the first to describe red blood cells, sperm, protozoa, and even bacteria.

Between the early eighteenth century and the mid nineteenth century, increasingly elegant and sophisticated compound light microscopes were developed; these involved separate lenses at the eye (eyepieces) and above the specimen (objective lenses). But since lens design was an empirical art, with little understanding of the optical principles governing image resolution, these microscopes were of little use for serious scientific research, and largely remained the playthings of gentleman naturalists.

Modern light microscopy can be dated from 1866, when Carl Zeiss (1816–88), a microscope manufacturer in Jena, Germany, invited a young physicist named Ernst Abbe (1840–1905) to join him as research director. Over the next decade, Abbe determined the principles of image formation in compound light microscopes, and the Zeiss workshop started producing rather plain-looking optical microscopes of exceptional optical performance and resolving power, equipped with objective lenses of near theoretical perfection. Subsequent advances in optical microscopy have principally been concerned with the development of novel contrast generation techniques.

The twentieth century also witnessed the invention of many other ingenious forms of microscope, employing electrons, sound, X-rays, surface probes, or electromagnetic radiation outside the visible spectrum to generate their images. Since the human eye can only image visible light, the images generated by these other types of microscope must all be converted into some form of optical image in order to be perceived.

Microscope design and image formation

All microscopes fall into one of two classes. The first, the ‘full field’ microscopes, include conventional light microscopes, ultraviolet and infra-red microscopes, and transmission electron microscopes. In these, the entire field of view of the specimen is simultaneously illuminated by the incident radiation, and this radiation, after being modified in some way by the specimen, is focused to form a real image which is then observed. The other class is that of the ‘point scanning’ microscopes, in which the specimen is interrogated point by point, by moving a focused incident beam of illumination or a physical scanning probe line by line along a regular roster through or across the surface of the specimen. In this way, the resulting image is built up point by point, usually electronically on a video monitor or similar display device. Microscopes in this class include the scanning electron microscope, the acoustic microscope, the confocal light microscope, and a wide variety of scanning probe microscopes, including the scanning tunnelling microscope and the atomic force microscope, in which a physical probe is moved over the surface of the specimen.

The characteristics of microscope imaging systems

Whatever the microscope type, the imaging system has three characteristics that the user must understand in order to appreciate the significance of the image formed, which differs from the specimen itself in significant ways. The first and most obvious of these is the magnification, which can range from as little as ten times in a binocular dissection microscope used to observe structures in the millimetre range, to more than a million times in electron microscopes used to observe individual metal atoms about 0.15 nanometres (1 nm = 10-9 metres) in diameter.

The second characteristic is the minimum resolvable distance (also referred to as the resolution or resolving power) of the microscope, defined as the distance separating two point objects within the specimen that can just be distinguished from one another in the image. In a high quality light microscope employing an oil immersion objective lens of the highest numerical aperture (NA 1.4), with which the image quality approaches theoretical perfection, the minimum resolvable distance is a little less than half the wavelength of the illuminating light employed, thus being about 240 nm for green light of wavelength 546 nm. In contrast, the magnetic lenses of even the best electron microscopes are far from perfect, with low numerical aperture and severe lens aberrations, limiting the minimum resolvable distance to about 0.1 nm, many times the wavelength of the electrons employed. For the scanning electron microscope and the confocal light microscope, the minimum resolvable distance is determined in part by the diameter of the focused electron or light beam striking the specimen, while with scanning probe microscopes the resolution is a function of the sharpness of the scanning tip employed. Magnification and image resolution are related in the sense that the image must be sufficiently enlarged for the eye to be able to appreciate the fine details that are resolved within it. However, magnification beyond this range is termed ‘empty magnification’, since it reveals no further detail in the specimen.

The final characteristic of the imaging system is the most variable, both between different types of microscope and also within a single type. This is the contrast generation mechanism. In the light microscope, for example, images can be formed by absorption contrast using stained or naturally pigmented specimens, by polarization contrast using birefringent specimens, or by fluorescent emission from autofluorescent or fluorescently labelled specimens. They can also be formed by dark ground illumination, by reflection techniques such as reflection interference contrast microscopy, or by a variety of interference techniques. In these, cunning optical tricks are performed with the light that is transmitted by the specimen, enabling refractive index changes in transparent and otherwise almost invisible specimens (including living cells) to be converted into intensity variations within the image. Other forms of microscopy measure different properties of the specimen. The acoustic microscope, for example, can be used to image changes in the mechanical properties of the specimen, including the elastic modulus, viscosity, thickness or density, enabling one, for example, to distinguish bone from cartilage. Contrast within transmission electron microscope images directly reflects the distribution of atoms within the specimen, particularly atoms of high atomic number that are often used to stain regions of the specimen selectively. These scatter electrons beyond the limiting angle of the objective aperture, leaving fewer to contribute to the image of that region, which thus appears dark. While transmission electron microscopy requires an extremely thin specimen, typically only 100 nm thick — one ten thousandth of a millimetre! — scanning electron microscopy and the various scanning probe microscopies measure various characteristics of the surface of the specimen, which may therefore be thick and opaque.

Three-dimensional imaging in microscopy

While most microscopic images are two-dimensional, one of the most rewarding characteristics of many types of microscope is their ability to provide three-dimensional information about specimens. The scanning probe microscopes can measure surface topology to high precision by recording the vertical excursions of the probe as it is scanned at a constant distance above the specimen surface. A transmission electron microscope image is an in-focus projection through the specimen. By recording multiple projection images while tilting the specimen in the electron beam, or alternatively by mathematically combining the images obtained from numerous identical objects observed in different orientations, the 3-D structures of such specimens can be determined. While the light microscope can be used to look within transparent specimens, the images conventionally obtained are combinations of in-focus information arising from the focal plane with out-of-focus information contributed by the regions of the specimen lying above and below this plane. This out-of-focus blur is one of the limiting factors of conventional light microscopy of thick specimens, particularly in fluorescence mode. However, the confocal fluorescence microscope avoids this problem by scanning the image point by point, while excluding light from out-of-focus regions by a simple optical device, the confocal imaging aperture, thus generating a blur-free optical section. By systematically changing the focal plane between successive acquisitions of such digital images, a set of optical sections can be non-invasively obtained which constitute a three-dimensional image of the transparent specimen. If this procedure is repeated at regular time intervals on a living specimen, a sequence of 3-D images may be obtained, forming a four-dimensional image with the dimensions of x, y, z, and time, enabling, for instance, dynamic cellular processes to be observed.

Such techniques, together with cryo electron microscopy — a method whereby thin aqueous specimens are vitrified by ultra-rapid cooling, and then observed in the hydrated vitreous state while maintained at below –100°C on the cold stage of a cryo electron microscope — have revolutionized biomedical microscopy in the last two decades, enabling us to determine the 3-D structures of virus particles, crystalline membrane proteins, and macromolecular complexes such as ribosomes, to study changes in the 3-D distribution of known proteins within living cells, and to image cellular physiological responses such as fluctuations of ionic concentrations and membrane potential. For these reasons, microscopy remains the most powerful and versatile of all biomedical research techniques.

David M. Shotton

Bibliography

Royal Microscopical Society's Microscopy Handbooks Series. Oxford University Press and Bios Scientific Publishers, Oxford. Celis J. E. (ed.) (1997) Cell biology: a laboratory handbook, 2nd edn, Vol. 3. Academic Press, San Diego.


For examples of images see blood vessels; deafness; glycogen; lungs; myelin; neuromuscular junction.

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Microscopes

Microscopes

The ability to view things that are too small to be seen by the unaided eye is important in espionage and security. For example, the diagnosis of an infection often relies in part on the visual examination of the microorganism. Information about how the microbe reacts to certain staining methods (e.g, the bacterial Gram stain), the shape of the microbe, and the reaction of antibodies to the microbe all provide important clues as to the identity of the organism.

As well, microscopic examination of documents can reveal information that cannot otherwise be seen. The high magnification and analysis of the elements that make up a sample that is possible using specialized techniques of scanning and transmission electron microscopy can reveal the presence of material that is of suspicious origin (i.e., missile casing), or the presence of codes on a surface.

A microscope is the instrument that produces the highly magnified image of an object that is otherwise difficult or impossible to see with the unaided eye. A microscope is able to distinguish two objects from one another that could not be distinguished with the eye. The resolving power of a microscope is greater than that of the eye.

History of the microscope. In ancient and classical civilizations, people recognized the magnifying power of curved pieces of glass. By the year 1300, these early crude lenses were being used as corrective eyeglasses.

In the seventeenth century Robert Hooke published his observations of the microscopic examination of plant and animal tissues. Using a simple two-lens compound microscope, he was able to discern the cells in a thin section of cork. The most famous microbiologist was Antoni van Leeuwenhoek. Using a single-lens microscope that he designed, Leeuwenhoek described microorganisms in environments such as pond water. His were the first descriptions of bacteria and red blood cells.

By the mid-nineteenth century, refinements in lens grinding techniques had improved the design of light microscopes. Still, advancement was mostly by trial and error, rather than by a deliberate crafting of a specific design of lens. It was Ernst Abbe who first applied physical principles to lens design. Abbe combined glasses that bent light beams to different extents into a single lens, reducing the distortion of the image.

The resolution of the light microscope is limited by the wavelength of visible light. To resolve objects that are closer together, the illuminating wavelength needs to be smaller. The adaptation of electrons for use in microscopes provided the increased resolution.

In the mid-1920s, Louis de Broglie suggested that electrons, as well as other particles, should exhibit wavelike properties similar to light. Experiments on electron beams a few years later confirmed this hypothesis. This was exploited in the 1930s in the development of the electron microscope.

Electron microscopy. There are two types of electron microscope. They are the transmission electron microscope (TEM) and the scanning electron microscope (SEM). The TEM transmits electrons through a sample that has been cut so that it is only a few molecules thin. Indeed, the sample is so thin that the electrons have enough energy to pass right through some regions of the sample. In other regions, where metals that were added to the sample have bound to sample molecules, the electrons either do not pass through as easily, or are restricted from passing through altogether. The different behaviors of the electrons are detected on special film that is positioned on the opposite side of the sample from the electron source.

The combination of the resolving power of the electrons, and the image magnification that can be subsequently obtained in the darkroom during the development of the film, produces a total magnification that can be in the millions.

Because TEM uses slices of a sample, it reveals internal details of a sample. In SEM, the electrons do not penetrate the sample. Rather, the sample is coated with gold, which causes the electrons to bounce off of the surface of the sample. The electron beam is scanned in a back and forth motion parallel to the sample surface. A detector captures the electrons that have bounced off the surface, and the pattern of deflection is used to assemble a three dimensional image of the sample surface.

Scanning, tunneling, and other microscopy techniques. In the early 1980s, the technique called scanning tunneling microscopy (STM) was invented. STM does not use visible light or electrons to produce a magnified image. Instead, a small metal tip is held very close to the surface of a sample and a tiny electric current is measured as the tip passes over the atoms on the surface. When a metal tip is brought close to the sample surface, the electrons that surround the atoms on the surface can actually "tunnel through" the air gap and produce a current through the tip. The current of electrons that tunnels through the air gap is dependent on the width of the gap. Thus, the current will rise and fall as the tip encounters different atoms on the surface. This current is then amplified and fed into a computer to produce a three dimensional image of the atoms on the surface.

Without the need for complicated magnetic lenses and electron beams, the STM is far less complex than the electron microscope. The tiny tunneling current can be simply amplified through electronic circuitry much like that used in other equipment, such as a stereo. In addition, the sample preparation is usually less tedious. Many samples can be imaged in air with essentially no preparation. For more sensitive samples that react with air, imaging is done in vacuum. A requirement for the STM is that the samples be electrically conductive.

Scanning tunneling microscopes can be used as tools to physically manipulate atoms on a surface. This holds out the possibility that specific areas of a sample surface can be changed.

Other forces have been adapted for use as magnifying sources. These include acoustic microscopy, which involves the reflection of sound waves off a specimen; xray microscopy, which involves the transmission of x rays through the specimen; near field optical microscopy, which involves shining light through an opening smaller than the wavelength of light; and atomic force microscopy, which is similar to scanning tunneling microscopy but can be applied to materials that are not electrically conductive, such as quartz.

FURTHER READING:

BOOKS:

Aebi, Engel. Atlas of Microscopy Techniques. San Diego: Plenum Press, 2002.

Hayat, M. Arif. Microscopy, Immunohistochemistry, and Antigen Retrieval Methods for Light and Electron Microscopy. New York: Plenum Publishing, 2002.

Murphy, Douglas, B. Fundamentals of Light Microscopy and Electronic Imaging. New York: Wiley-Liss, 2001.

SEE ALSO

Biological Warfare
Chemical and Biological Detection Technologies

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Microscopes

Microscopes

A microscope is the instrument that produces the high magnification image of an object that is otherwise difficult or impossible to see with the unaided eye. A microscope's resolving power allows the user to differentiate two objects from one another that could not be distinguished with the naked eye.

Microscopes assume a central role in forensic science . Forensic evidence , particularly trace evidence , is often so tiny as to escape detection with the naked eye. But the magnified examination of samples can reveal a great deal of detail. For example, examination of gunshot residue using a scanning electron microscope can allow an investigator to determine the shape of the spent residue and even its elemental composition, both of which are critical to the identification of the gunpowder used. The microscope can aid in matching the residue on a victim to residue present on a suspect. As another example, examination and identification of fibers would be impossible without the use of light microscopy.

Microscopic examination of documents can reveal information that cannot otherwise be seen. The high magnification and analysis possible using specialized techniques of scanning and transmission electron microscopy can reveal the presence of material that is otherwise undetectable in the elements that make up a sample.

Today's sophisticated use of microscopes in forensic analysis had its beginnings hundreds of years ago. In ancient and classical civilizations, people recognized the magnifying power of curved pieces of glass. By the year 1300, these early crude lenses were being used as corrective eyeglasses.

In the seventeenth century Robert Hooke published his observations of the microscopic examination of plant and animal tissues. Using a simple two-lens compound microscope, he was able to discern the cells in a thin section of cork. The most famous microbiologist of this century was Antony van Leeuwenhoek (16321723). Using a single lens microscope that he designed, Leeuwenhoek described microorganisms in environments such as pond water. His were the first descriptions of bacteria and red blood cells.

By the mid-nineteenth century, refinements in lens grinding techniques had improved the design of light microscopes. Still, advancement was mostly by trial and error, rather than by a deliberate crafting of a specific design of lens. It was Ernst Abbe who first applied physical principles to lens design. Abbe combined glasses that bent light beams to different extents into a single lens, reducing the distortion of the image.

The resolution of the light microscope is limited by the wavelength of visible light. To resolve objects that are closer together, the illuminating wavelength needs to be smaller. The adaptation of electrons for use in microscopes provided the increased resolution.

In the mid-1920s, Louis de Broglie suggested that electrons, as well as other particles, should exhibit wavelike properties similar to light. Experiments on electron beams a few years later confirmed this hypothesis. This was utilized in the 1930s in the development of the electron microscope.

There are two types of electron microscope: the transmission electron microscope (TEM) and the scanning electron microscope (SEM). The TEM transmits electrons through a sample that has been cut so that it is only a few molecules thin. Indeed, the sample is so thin that the electrons have enough energy to pass right through some regions of the sample. In other regions, where metals that were added to the sample have bound to sample molecules, the electrons either do not pass through as easily, or are restricted from passing through altogether. The different behaviors of the electrons are detected on special film that is positioned on the opposite side of the sample from the electron source.

The combination of the resolving power of the electrons, and the image magnification that can be subsequently obtained in the darkroom during the development of the film, produces a total magnification that can be in the millions.

Because TEM uses slices of a sample, it reveals internal details of a sample. In SEM, the electrons do not penetrate the sample. Rather, the sample is coated with gold, which causes the electrons to bounce off of the surface of the sample. The electron beam is scanned in a back and forth motion parallel to the sample surface. A detector captures the electrons that have bounced off the surface, and the pattern of deflection is used to assemble a three dimensional image of the sample surface.

In the early 1980s, the technique called scanning tunneling microscopy (STM) was invented. STM does not use visible light or electrons to produce a magnified image. Instead, a small metal tip is scanned very close to the surface of a sample and a tiny electric current is measured as the tip passes over the atoms on the surface. When a metal tip is brought close to the sample surface, the electrons that surround the atoms on the surface can actually "tunnel through" the air gap and produce a current through the tip. The current of electrons that tunnel through the air gap is dependent on the width of the gap. Thus, the current will rise and fall as the tip encounters different atoms on the surface. This current is then amplified and fed into a computer to produce a three dimensional image of the atoms on the surface.

Without the need for complicated magnetic lenses and electron beams, the STM is far less complex than the electron microscope. The tiny tunneling current can be simply amplified through electronic circuitry similar to circuitry that is used in other electronic equipment, such as a stereo. In addition, the sample preparation is usually less tedious. Many samples can be imaged in air with essentially no preparation. For more sensitive samples that react with air, imaging is done in vacuum. A requirement for the STM is that the samples be electrically conducting, such as a metal.

Scanning tunneling microscopes can be used as tools to physically manipulate atoms on a surface. This holds out the possibility that specific areas of a sample surface can be changed.

Other forces have been adapted for use as magnifying sources. These include acoustic microscopy, which involves the reflection of sound waves off a specimen; x-ray microscopy, which involves the transmission of x rays through the specimen; near field optical microscopy, which involves shining light through a small opening smaller than the wavelength of light; and atomic force microscopy, which is similar to scanning tunneling microscopy but can be applied to materials that are not electrically conducting, such as quartz.

see also Fibers; Fluorescence; Microscope, comparison; Polarized light microscopy; Scanning electron microscopy; Scanning electron microscopy; Trace evidence.

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microscope

microscope, optical instrument used to increase the apparent size of an object.

Simple Microscopes

A magnifying glass, an ordinary double convex lens having a short focal length, is a simple microscope. The reading lens and hand lens are instruments of this type. When an object is placed nearer such a lens than its principal focus, i.e., within its focal length, an image is produced that is erect and larger than the original object. The image is also virtual; i.e., it cannot be projected on a screen as can a real image.

Compound Microscopes

The compound microscope consists essentially of two or more double convex lenses fixed in the two extremities of a hollow cylinder. The lower lens (nearest to the object) is called the objective; the upper lens (nearest to the eye of the observer), the eyepiece. The cylinder is mounted upright on a screw device, which permits it to be raised or lowered until the object is in focus, i.e., until a clear image is formed. When an object is in focus, a real, inverted image is formed by the lower lens at a point inside the principal focus of the upper lens. This image serves as an "object" for the upper lens which produces another image larger still (but virtual) and visible to the eye of the observer.

Computation of Magnifying Power

The magnifying power of a lens is commonly expressed in diameters. For example, if a lens magnifies an object 5 times, the magnification is said to be 5 diameters, commonly written simply "5x." The total magnification of a compound microscope is computed by multiplying the magnifying power of the objective by the magnifying power of the eyepiece.

Development and Uses

The invention of the microscope is variously accredited to Zacharias Janssen, a Dutch spectaclemaker, c.1590, and to Galileo, who announced his invention in 1610. Others are known for their discoveries made by the use of the instrument and for their new designs and improvements, among them G. B. Amici, Nehemiah Grew, Robert Hooke, Antony van Leeuwenhoek, Marcello Malpighi, and Jan Swammerdam. The compound microscope is widely used in bacteriology, biology, and medicine in the examination of such extremely minute objects as bacteria, other unicellular organisms, and plant and animal cells and tissue—fine optical microscopes are capable of resolving objects as small as 5000 Angstroms. It has been extremely important in the development of the biological sciences and of medicine.

Modified Compound Microscopes

The ultramicroscope is an apparatus consisting essentially of a compound microscope with an arrangement by which the material to be viewed is illuminated by a point of light placed at right angles to the plane of the objective and brought to a focus directly beneath it. This instrument is used especially in the study of Brownian movement in colloidal solutions (see colloid). The phase-contrast microscope, a modification of the compound microscope, makes transparent objects visible; it is used to study living cells. The television microscope uses ultraviolet light. Since this light is not visible, the apparatus is used with a special camera and may be connected with a television receiver on which the objects (e.g., living microorganisms) may be observed in color.

Electron Microscopes

The electron microscope, which is not limited by the powers of optical lenses and light, permits greater magnification and greater depth of focus than the optical microscope and reveals more details of structure. Instead of light rays it employs a stream of electrons controlled by electric or magnetic fields. The image may be thrown on a fluorescent screen or may be photographed. It was first developed in Germany c.1932; James Hillier and Albert Prebus, of Canada, and V. K. Zworykin, of the United States also made notable contributions to its development. The scanning electron microscope, introduced in 1966, gains even greater resolution by reading the response of the subject material rather than the direct reflection of its beam. Using a similar approach, optical scanning microscopes achieve a resolution of 400 Angstroms, less than the wavelength of the light being used. Finally, the scanning tunnelling microscope, invented in 1982, uses not a beam but an electron wave field, which by interacting with a nearby specimen is capable of imaging individual atoms; its resolution is an astounding one Angstrom.

Bibliography

See C. Marmasse, Microscopes and Their Uses (1980).

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microscope

microscope A device for forming a magnified image of a small object. The simple microscope consists of a biconvex magnifying glass or an equivalent system of lenses, either hand-held or in a simple frame. The compound microscope uses two lenses or systems of lenses, the second magnifying the real image formed by the first (see illustration). The lenses are usually mounted at the opposite ends of a tube that has mechanical controls to move it in relation to the object. An optical condenser and mirror, often with a separate light source, provide illumination of the object. The widely used binocular microscope consists of two separate instruments fastened together so that one eye looks through one while the other eye looks through the other. This gives stereoscopic vision. See Chronology. See also electron microscope; Nomarski microscope; phase-contrast microscope; ultraviolet microscope.

MICROSCOPY

c.1590

Dutch spectacle-makers Hans and Zacharias Janssen invent the compound microscope.

1610

German astronomer Johannes Kepler (1571–1630) invents the modern compound microscope.

1675

Anton van Leeuwenhoek invents the simple microscope.

1826

British biologist Dames Smith (d. 1870) constructs a microscope with much reduced chromatic and spherical aberrations.

1827

Italian scientist Giovanni Amici (1786–1863) invents the reflecting achromatic microscope.

1861

British chemist Joseph Reade (1801–70) invents the kettledrum microscope condenser.

1912

British microscopist Joseph Barnard (1870–1949) invents the ultramicroscope.

1932

Dutch physicist Frits Zernike (1888–1966) invents the phase-contrast microscope.

1936

German-born US physicist Erwin Mueller (1911–77) invents the field-emission microscope.

1938

German engineer Ernst Ruska (1906–88) develops the electron microscope.

1940

Canadian scientist James Hillier (1915– ) makes a practical electron microscope.

1951

Erwin Mueller invents the field-ionization microscope.

1978

US scientists of the Hughes Research Laboratory invent the scanning ion microscope.

1981

Swiss physicists Gerd Binning (1947– ) and Heinrich Rohrer (1933– ) invent the scanning tunnelling microscope.

1985

Gerd Binning invents the atomic force microscope.

1987

James van House and Arthur Rich invent the positron microscope.


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microscope

microscope Optical device for producing an enlarged image of a minute object. Anton van Leeuwenhoek made the first simple microscope in 1668. The modern compound microscope has two converging lens systems, the objective and the eyepiece, both of short focal length. The objective produces a magnified image, which is further magnified by the eyepiece to give the image seen by the observer. Because of the nature of the visible spectrum of light, an optical microscope can magnify objects only up to 2000 times. For extremely small objects, an electron microscope is used. An atomic force microscope can magnify by up to a million times using a probe with a tip the size of a single atom.

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microscopic

mi·cro·scop·ic / ˌmīkrəˈskäpik/ • adj. 1. so small as to be visible only with a microscope: microscopic algae. ∎ inf. extremely small: a microscopic skirt. ∎  concerned with minute detail: such a vision is as microscopic as his is panoramic. 2. of or relating to a microscope: microscopic analysis of the soil. DERIVATIVES: mi·cro·scop·i·cal adj. (in sense 2). mi·cro·scop·i·cal·ly / -ik(ə)lē/ adv.

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microscope

mi·cro·scope / ˈmīkrəˌskōp/ • n. an optical instrument used for viewing very small objects, such as mineral samples or animal or plant cells, typically magnified several hundred times. PHRASES: under the microscope under critical examination.

microscope

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microscope

microscope (my-krŏ-skohp) n. an instrument for producing a greatly magnified image of an object, which may be so small as to be invisible to the naked eye. See also electron microscope, operating microscope.
microscopical adj. —microscopy (my-kros-kŏ-pi) n.

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microscopy

mi·cros·co·py / mīˈkräskəpē/ • n. the use of the microscope. DERIVATIVES: mi·cros·co·pist / -pist/ n.

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microscopic

microscopic (my-krŏ-skop-ik) adj.
1. too small to be seen clearly without the use of a microscope.

2. of, relating to, or using a microscope.

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microscope

microscopeaslope, cope, dope, elope, grope, hope, interlope, lope, mope, nope, ope, pope, rope, scope, slope, soap, taupe, tope, trope •myope • telescope • periscope •stereoscope • bioscope • stroboscope •kaleidoscope • CinemaScope •gyroscope • microscope • horoscope •stethoscope • antelope • envelope •zoetrope • skipping-rope • tightrope •towrope • heliotrope • lycanthrope •philanthrope • thaumatrope •misanthrope •isotope, radioisotope

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microscopic

microscopic •priapic • aspic • epic •philippic, prototypic, stereotypic •Olympic • nitpick •ectopic, gyroscopic, heliotropic, horoscopic, isotopic, isotropic, kaleidoscopic, macroscopic, microscopic, misanthropic, myopic, philanthropic, phototropic, telescopic, topic, tropic •Ethiopic • biopic •Inupik, Yupik •toothpick

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microscopy

microscopycroupy, droopy, goopy, groupie, loopy, pupae, roupy, snoopy, soupy, Tupi •whoopee •duppy, guppy, puppy, yuppie •gulpy, pulpy •bumpy, clumpy, dumpy, frumpy, grumpy, humpy, jumpy, lumpy, plumpy, rumpy-pumpy, scrumpy, stumpy •hiccupy • chirrupy • calliope •pericope • syncope •colonoscopy, horoscopy, microscopy, stereoscopy •Penelope • canopy • satrapy •lycanthropy, misanthropy, philanthropy •aromatherapy, chemotherapy, hypnotherapy, physiotherapy, psychotherapy, radiotherapy, therapy •entropy • syrupy (US sirupy) • chirpy

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Microscope

Microscope

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.

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.

KEY TERMS

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.

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 postpuncture 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.

Resources

BOOKS

Berkow, R., M. H. Beers, A. J. Fletcher, and R. M. Bogin, eds. The Merck Manual of Medical Information—Home Edition. Whitehouse Station, NJ: Merck & Co, 2001.

White, Lois, ed. Foundations of Nursing: Caring for the Whole Person. Albany, N.Y.: Delmar Thomson Learning, 2001.

ORGANIZATIONS

American Society of Clinical Pathologists. 2100 West Harrison Street, Chicago, IL 60612. (312) 738-1336. 〈http://www.ascp.org〉.

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Microscope

Microscope

A brief history of microscopy

Various types of optical microscopes

Electron microscope

Other types of microscopes

Resources

A microscope magnifies and resolves the image of an object that otherwise would be invisible to the naked eye, or whose detail could not be resolved using the unaided eye. These objects include such items as human skin, the eye of a fly, cells of a living organism, microorganisms such as bacteria, protozoa and viruses, individual molecules, and atoms.

Some of the above objects are large enough to be visible using the magnifying power of a light microscope. Examples include skin cells, parts of insects, and bacteria. Bacteria appear just as tiny objects. They

are so small that they approach the detection limits of the light microscope. In order to make out details of microorganisms such as bacteria, and to be able to visualize viruses, much higher magnification is required.

All light moves as a wave. The wavelength of visible light is too large to resolve bacterial detail to any degree. Viruses are invisible. An analogy would be to place a small pebble in the path of an oncoming wave at an ocean side beach. The wave will pass right over the pebble, as if the pebble were not there. However, if the same pebble is placed in a stream, where the waves are much smaller in size, the pebble can disrupt the waves path.

The smaller wave in microscopy (the study and use of microscopes) is achieved by the use of electrons instead of visible light. The wavelength of an electron beam is extremely small. Thus, objects like bacteria and viruses can be visualized. Indeed, versions of microscopes that rely on electrical repulsion between surfaces can now visualize molecules, including the constituents of deoxyribonucleic acid (DNA).

A brief history of microscopy

The first generally accepted use of microscopy was through optical microscopes, which produced images through the use of visible light. These microscopes used drops of water captured in a small hole to function as a magnifying lens. Later, magnifying glasses consisting of a single lens that was bowed outward on either side (biconvex lens) were developed. The lens was capable of enlarging an object up to 20 times its original size as seen by the naked eye. (Scientists describe the power of magnification by writing a number followed by a times sign x. Thus, a 20 times magnification would be expressed as 20x.)

The first record describing an artificial lens being used for magnification dates from 1267. The work of English philosopher Roger Bacon (c. 12141292) entitled Perspectiva described viewing minute objects through a lesser segment of a sphere of glass or crystal to enlarge them. Spectacles were in use shortly after this period to correct vision and enlarge objects, but it was not until 1595 that the first device that could truly be considered a microscope was made. This microscope was prepared by optician and lens grinder Zacharias Jansen (1580c.1638), along with his father Hans, in Holland. This was the first compound microscope in that it employed two separate lenses that could be moved relative to each other by the means of a sliding tube. This mechanism allowed the microscope to zoom (to change its magnification) from 3x (three times normal) to 9x (nine times normal).

This system was eventually improved by English scientist Robert Hooke (16351703) who added a third lens attached to the viewing (eyepiece) lens. This improvement was carried out using an eyepiece from a telescope, an optical instrument with a longer pedigree than the microscope. Once the first Jansen microscopes had been made, word spread rapidly throughout the world. As a result, the seventeenth century saw many microscope manufacturers and users appear. It was at this time that the word microscope was first used by an Italian scientific society, which included astronomer and physicist Galileo Galilei (15641642).

Some of the early work that was carried out using these primitive microscopes is still highly regarded today. In 1660, Italian physiologist Marcello Malpighi (16281694) was able to prove the blood circulation theories of English physician William Harvey (1578 1657) by discovering the presence of capillaries connecting arteries and veins in the body as well as identifying many microscopic structures in the human body. Some five years later, English scientist Robert Hooke (1635 1703) published the first pictures of the cells making up living organisms. Prior to these works, it had been assumed that the microscope was nothing more than a toy. During this period, some microscopes were 2 feet (0.6 meter) in length and illuminated by oil lamps.

One of the early enthusiasts of microscopy was Dutch scientist Anton van Leeuwenhoek (16321723), full name Thonius Philips van Leeuwenhoek). During the sixteenth century, he constructed over 500 single lens microscopes. While crude by todays standards, van Leeuwenhoeks microscopes revealed an array of microbial life. For example, his descriptions of organisms found in lake water were the first observations of the green algae called Spirogyra and another microbe that came to be known as Vorticella. Finally, his observation of animacules in tooth plaque was the first visual detection of bacteria.

Various types of optical microscopes

The familiar monocular compound optical microscopes (i.e., microscopes that have a single lens as the eyepiece, or ocular, lens) are being replaced in many laboratories with binocular styles. These microscopes have a single objective lens, but two ocular ones, each in its own eyepiece. Light coming through the objective lens is split into two beams by a prism. Each eye sees the exact same image, so there is no three dimensional effect.

For a three-dimensional view, scientists use a stereoscopic binocular microscope. This instrument consists of two separate sets of objective lenses as well as two separate ocular lenses. Prisms alter the angle of light coming through each pair of lenses, so each eye sees a slightly different image.

Living or stained specimens often yield poor images when viewed in bright-field illumination. To help with this, scientists developed a phase-contrast microscope that alters the phase differences (the position in space of the waves and troughs) in light waves as they pass through the specimen. This makes some parts of the object brighter and others darker than normal, allowing for a better view of the structural details of the object. Closely related to this type is the interference microscope that superimposes one field of view over a second to improve contrast.

Another optical version of a microscope is called the dark field microscope. This microscope has proven to be particularly useful for biological studies. The dark-field microscope uses a specialized illumination technique that capitalizes on indirect illumination to enhance contrast in specimens. An opaque disk is set in a condenser under the stage of the microscope (the solid support that the object being studied rests on). The disc is also known as the stop. The stop prevents light from shining directly on the specimen. Instead, light passes around the stop and is reflected off the condensers walls.

All the microscopes described so far produce black and white images. To observe color in cells, scientists use polarizing microscopes. The microscope aligns the vibrations of a light wave by directing it through a specially cut prism. If two beams of polarized light are transmitted through the cell, as in a differential polarization microscope, researchers can make quantitative measurements of things such as cell depth and the quantity of certain cell constituents.

Electron microscope

Prior to 1930, all microscopes were optical. In 1931, German physicist Ernst Ruska (19061988) developed the electron microscope that used a beam of moving electrons to illuminate an object instead of light. Magnetic lenses or electric coils produce magnetic fields to deflect the electrons in the same manner that glass lenses bend light rays. The specimen has to be in a vacuum, however, because electrons cannot travel through air. Electron microscopes give point-to-point resolutions of less than 0.2 nanometers (where one nanometer is equal to one-billionth of a meter). This high resolution permits the direct visualization of many molecules and some atoms.

The transmission electron microscope (TEM) images specimens a fraction of a micrometer or less in thickness. In a TEM, the beam passes through the specimen so that some of the electrons are absorbed and some scattered. The remaining electrons are focused onto a fluorescent screen or special photographic plates via the use of magnetic lenses. The resulting image is in black and white.

In the scanning electron microscope (SEM), a narrowly focused electron beam is scanned over the surface of a solid object and used to build up an image of the details of the surface structure through reflection. Researchers use this type to study minute details on a surface of an object. These microscopes created 3-D (three-dimensional) images that are magnified up to 50,000x.

Although there are several other special types of electron microscopes, perhaps the most valuable is the electron-probe microanalyzer, which allows a researcher to make a chemical analysis of the composition of materials. This type of microscope uses the incident electron beam to excite the emission of characteristic x-ray radiation by the various elements composing the specimen. Spectrometers built into the instrument detect and analyze the x rays. Viewing the resulting image, the researcher can easily correlate the structure and composition of the material.

Other types of microscopes

Scanning-tunneling microscopes do not look like conventional microscopes at all. These are used to resolve individual atoms, identifying details down to one-tenth of an angstrom (where one angstrom equals 0.1 nanometer) in height and less than two angstroms in width. Instead of lenses or mirrors, this microscope sports a tungsten rod with a tip is made up of a pyramid of atoms and three pieces of piezoelectric crystal, which compress and stretch in response to changes in the voltage of an electric charge. Electrons tunnel, or flow, through the vacuum or water from the tungsten tip to the atoms on an objects surface, creating a current that reacts with the crystal. Its inventors, German physicist Gerd Karl Binnig (1947) and Swiss physicist Heinrich Rohrer (1933), won the Nobel Prize in Physics, in 1986, for this development. They shared the prize with Ruska.

In 1986, the atomic force microscope (AFM) debuted. The AFM produces three-dimensional images of surfaces both in air and under liquids at a resolution of nanometers, or billionths of a meter. In its contact mode, the AFM lightly touches a tip at the end of a 50 to 300 micrometer long leaf spring (the cantilever) to the sample. As the tip is scanned over the sample, a detector measures the vertical deflection of the cantilever, yielding the precise height of the sample at local points. The deflections of the cantilever are monitored by a laser beam that is reflected off the cantilever and into a position-sensitive detector. If the tip and sample are coated with two types of molecules, an AFM can measure force of attraction or repulsion between them, potentially at the level of a single hydrogen bond. Since its invention, the atomic force microscope has permitted high-resolution imaging at the subnanometer level. More recently, scientists introduced the microscope to a liquid environment and the resolution improved to the atomic level.

Resources

BOOKS

Becker, Wayne M. Guide to Microscopy. San Francisco, CA: Benjamin Cummings Publishing Company, 2003.

Murphy, Douglas B. Fundamentals of Light Microscopy and Electronic Imaging. New York: Wiley-Liss, 2001.

Sluder, Greenfield, and David E. Wolf, eds. Digital Microscopy : A Second Edition of Video Microscopy. Amsterdam, Netherlands, and Boston, MA: Elsevier Academic Press, 2003.

Spector, David L., and Robert D. Goldman, eds. Basic Methods in Microscopy: Protocols and Concepts from Cells (A Laboratory Manual). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2006.

Torok, Peter, and Fu-Jen Keo, eds. Optical Imaging and Microscopy: Techniques and Advanced Systems. Berlin, Germany, and New York: Springer-Verlag, 2003.

Laurie Toupin

Brian Hoyle

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Microscope

Microscope

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.


KEY TERMS


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.


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.

Resources

BOOKS

Berkow, R., M. H. Beers, A. J. Fletcher and R. M. Bogin, eds. The Merck Manual of Medical Information—Home Edition. Whitehouse Station, NJ: Merck & Co, 2001.

White, Lois, ed. Foundations of Nursing: Caring for the Whole Person. Albany, NY: Delmar Thomson Learning, 2001.

ORGANIZATIONS

American Society of Clinical Pathologists. 2100 West Harrison Street, Chicago, IL 60612. (312) 738-1336. <http://www.ascp.org>.

Jennifer F. Wilson

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Microscope

Microscope

A microscope magnifies and resolves the image of an object that otherwise would be invisible to the naked eye , or whose detail could not be resolved using the unaided eye. These objects include such items as human skin, the eye of a fly, cells of a living organism , microorganisms such as bacteria , protozoa and viruses, individual molecules, and atoms .

Some of the above objects are large enough to be visible using the magnifying power of a light microscope. Examples include skin cells, parts of insects , and bacteria. Bacteria appear just as tiny objects. They are so small that they approach the detection limits of the light microscope. In order to make out details of microorganisms such as bacteria, and to be able to visualize viruses, much higher magnification is required.

All light moves as a wave. The wavelength of visible light is too large to resolve much bacterial detail. Viruses are invisible. An analogy would be to place a small pebble in the path of an oncoming wave at an oceanside beach. The wave will pass right over the pebble, as if the pebble were not there. However, if the same pebble is placed in a stream, where the waves are much smaller in size, the pebble can disrupt the wave's path.

The 'smaller wave' in microscopy is achieved by the use of electrons instead of visible light. The wavelength of an electron beam is extremely small. Thus, objects like bacteria and viruses can be visualized. Indeed, versions of microscopes that rely on electrical repulsion between surfaces can now visualize molecules, including the constituents of deoxyribonucleic acid (DNA) .


A brief history of microscopy

The first optical microscopes produced images through the use of visible light. These microscopes used drops of water captured in a small hole to function as a magnifying lens . Later, magnifying glasses consisting of a single lens that was bowed outward on either side (biconvex lens) were developed. The lens was capable of enlarging an object up to 20 times its original size as seen by the naked eye. (Scientists describe the power of magnification by writing a number followed by a times sign "x". Thus, a 20 times magnification would be expressed as 20x.)

Using several lenses in conjunction with each other is the secret of a microscope's success, however. Dutch spectacle maker, Zacharias Janssen, devised the first compound microscope (a microscope that contains more than one magnifying lens) in 1590. Today's basic optical compound microscopes consist of a two-lens system with an objective and an ocular lens, and can magnify up to 2,000x. The specimen sits close to the objective lens, which magnifies the images as an ordinary magnifying glass would. The lens forms an enlarged real image of the specimen near the ocular or eyepiece lens. The eyepiece works in conjunction with the objectives to correct for aberration and further magnify the image, creating the virtual image seen in a microscope. Light reflected from a mirror and concentrated by the condenser lenses located under the specimen stage, illuminates the object.

One of the early enthusiasts of microscopy was Antoni van Leeuwenhoek. During the sixteenth century he constructed over 500 single lens microscopes. While crude by today's standards, van Leeuwenhoek's microscopes revealed an array of microbial life. For example, his descriptions of organisms found in lake water were the first observations of the green algae called Spirogyra and another microbe that came to be known as Vorticella. Finally, his observation of "animacules" in tooth plaque was the first visual detection of bacteria.


Various types of optical microscopes

The familiar monocular compound optical microscopes (i.e., microscopes that have a single lens as the eyepiece, or ocular, lens) are being replaced in many laboratories with binocular styles. These microscopes have a single objective lens, but two ocular ones, each in its own eyepiece. Light coming through the objective lens is split into two beams by a prism . Each eye sees the exact same image, so there is no three dimensional effect.

For a three-dimensional view, scientists use a stereoscopic binocular microscope. This instrument consists of two separate sets of objective lenses as well as two separate ocular lenses. Prisms alter the angle of light coming through each pair of lenses, so each eye sees a slightly different image.

Living or stained specimens often yield poor images when viewed in bright-field illumination. To help with this, scientists developed a phase-contrast microscope that alters the phase differences (the position in space of the waves and troughs) in light waves as they pass through the specimen. This makes some parts of the object brighter and others darker than normal, allowing for a better view of the structural details of the object. Closely related to this type is the interference microscope that superimposes one field of view over a second to improve contrast.

Another optical version of a microscope is called the darkfield microscope. This microscope has proven to be particularly useful for biological studies. The dark-field microscope uses a specialized illumination technique that capitalizes on indirect illumination to enhance contrast in specimens. An opaque disk is set in a condenser under the stage of the microscope (the solid support that the object being studied rests on). The disc is also known as the stop. The stop prevents light from shining directly on the specimen. Instead, light passes around the stop and is reflected off the condenser's walls.

All the microscopes described so far produce black and white images. To observe color in cells, scientists use polarizing microscopes. The microscope aligns the vibrations of a light wave by directing it through a specially cut prism. If two beams of polarized light are transmitted through the cell , as in a differential polarization microscope, researchers can make quantitative measurements of things such as cell depth and the quantity of certain cell constituents.


Electron microscope

Prior to 1930, all microscopes were optical. In 1931, German physicist Ernst Ruska developed the electron microscope that used a beam of moving electrons to illuminate an object instead of light. Magnetic lenses or electric coils produce magnetic fields to deflect the electrons in the same manner that glass lenses bend light rays. The specimen has to be in a vacuum , however, because electrons cannot travel through air. Electron microscopes give point-to-point resolutions of less than 0.2 nanometers. This high resolution permits the direct visualization of many molecules and some atoms.

The transmission electron microscope (TEM) images specimens a fraction of a micrometer or less in thickness. In a TEM, the beam passes through the specimen so that some of the electrons are absorbed and some scattered. The remaining electrons are focused onto a fluorescent screen or special photographic plates via the use of magnetic lenses. The resulting image is in black and white.

In the scanning electron microscope (SEM), a narrowly focused electron beam is scanned over the surface of a solid object and used to build up an image of the details of the surface structure through reflection. Researchers use this type to study minute details on a surface of an object. These microscopes created 3-D images that are magnified up to 50,000x.

Although there are several other special types of electron microscopes, perhaps the most valuable is the electron-probe microanalyzer, which allows a researcher to make a chemical analysis of the composition of materials. This type of microscope uses the incident electron beam to excite the emission of characteristic x radiation by the various elements composing the specimen. Spectrometers built into the instrument detect and analyze the x rays . Viewing the resulting image, the researcher can easily correlate the structure and composition of the material.


Other types of microscopes

Scanning-tunneling microscopes do not look like conventional microscopes at all. These are used to resolve individual atoms, identifying details down to one-tenth of an angstrom in height and less than two angstroms in width. Instead of lenses or mirrors , this microscope sports a tungsten rod with a tip is made up of a pyramid of atoms and three pieces of piezoelectric crystal , which compress and stretch in response to changes in the voltage of an electric charge . Electrons "tunnel" or flow through the vacuum or water from the tungsten tip to the atoms on an object's surface, creating a current that reacts with the crystal. Its inventors, Gerd Binnig of West Germany, and Heinrich Rohrer of Switzerland, won the Nobel Prize in 1986 for this development. They shared the prize with Ernst Ruska.

In 1986, the atomic force microscope (AFM) debuted. The AFM produces three-dimensional images of surfaces both in air and under liquids at a resolution of nanometers, or billionths of a meter. In its contact mode, the AFM lightly touches a tip at the end of a 50–300 micrometer long leaf spring (the cantilever ) to the sample. As the tip is scanned over the sample, a detector measures the vertical deflection of the cantilever, yielding the precise height of the sample at local points. The deflections of the cantilever are monitored by a laser beam that is reflected off the cantilever and into a position-sensitive detector. If the tip and sample are coated with two types of molecules, an AFM can measure force of attraction or repulsion between them, potentially at the level of a single hydrogen bond. Since its invention, the atomic force microscope has permitted high-resolution imaging at the subnanometer level. More recently, scientists introduced the microscope to a liquid environment and the resolution improved to the atomic level.


Resources

books

Rogers, Kirsteen, and Paul Dowswell. The Usborne Complete Book of the Microscope. Tulsa: EDC Publishing, 1999.

Yarris, Lynn. The New Book of Popular Science. Bethel, CT: Grolier Educational, 1998.


Laurie Toupin Brian Hoyle

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Microscope

Microscope


A microscope is a scientific instrument that magnifies objects that are too small to be seen by the naked eye. As one of the most important scientific tools ever invented, it is especially significant to the life sciences, since it made possible the discovery of an entirely unseen world of microorganisms. Today's increasingly powerful and highly specialized microscopes can achieve magnifications of a million times or more.

The earliest types of magnifiers were probably globes of water-filled glass or chips of transparent rock crystal used by the Romans. The first microscope could not be invented, however, until the first lenses were devised for use in eyeglasses sometime around the year 1300. These first eyeglasses or spectacles were made with convex lenses (curved inward) that helped farsightedness (the inability to see objects up close). By 1500, it is known that concave lenses (curving outward) were crafted to help with myopia, or nearsightedness (the inability to see object far away). Lenscrafters had learned that by grinding any clear glass or crystal into a certain shape, usually with the edges thinner than the center, a magnifying effect was achieved. The first real microscope was therefore a single, handheld lens, and it is called a simple microscope. Today, we would call it a magnifying glass. The individual most identified with the improvement and use of the simple microscope is the Dutch naturalist Anton van Leeuwenhoek (1632–1723), whose secret grinding, polishing, and mounting techniques allowed him to achieve possibly as much as 270 times magnification. Beginning in the 1670s, he examined mainly biological specimens and was the first to observe spermatozoa (male sex cells), red blood cells, and bacteria.

The typical microscope used today is a tubelike instrument with a lens at its top and bottom. It is called a compound microscope because it has more than one lens. This device is believed to have come about in Holland near the end of the sixteenth century when the telescope was invented there at the same time. Apparently, it was soon realized that a telescope could be used as a microscope when reversed. The first compound microscopes were, therefore, two lenses housed in a long tube (in which the enlarged image produced by the first lens is further magnified by the second one). The first scientist to improve the compound microscope and to put it to real scientific use was the English physicist, Robert Hooke (1635–1703). In 1665, Hooke published Micrographia, which contained excellent drawings of what he had observed with his improved microscope. Hooke was the first to use a microscope to observe the structure of plants (actually thin slices of cork), finding that they consisted of tiny walled chambers that he called "cells." After Hooke, there were minor improvements in microscopy until the mid-1800s, when the German physicist Ernst Abbe (1840–1905) collaborated with the German optician Carl Zeiss (1816–1888), and produced high-quality lenses with no blurring or distortions. Later developments resulted in the basic microscope with built-in illumination (lighting) that is used today in schools and small laboratories. These generally have a magnification of up to 400 power (times).

Today's school and lab microscopes are called compound light microscopes because they let light pass through the object being studied and then through two or more lenses. The lenses enlarge the image and bend the light toward the eye. Such a microscope has two lenses: an objective lens and an ocular lens. The ocular is also called the eyepiece and is what you look through. The objective lens, sometimes only called the objective, magnifies the object just below it on a slide. If the objective lens has a power of 50X (magnifying an object 50 times), and the ocular has a power of 10X, then together they have a total magnification of 500X (10X times 50X). Such a magnifying power would allow a cell to be easily observed.

ROBERT HOOKE

English physicist (a person specializing in the study of energy and matter and their interactions) Robert Hooke (1635–1703) was one of the earliest and greatest of the microscope pioneers. His microscopic studies of insects, feathers, and fish scales are both beautiful and accurate, and he published the first book dedicated to microscopy. He is responsible for first using the word "cell," which later would become the cornerstone of microbiology (the study of microorganisms).

Born on the Isle of Wight in England, Robert Hooke was born sickly and with a backbone that did not grow straight. As a child prodigy (exceptionally smart) Hooke impressed everyone with his mechanical gifts. He built elaborately complicated toys as a child, and as a young man he attended Westminster School and later Oxford University. At Oxford, his abilities caught the eye of the great English physicist and chemist, Robert Boyle (1627–1691), and Hooke quickly was made Boyle's assistant. It is known that it was Hooke who designed and built an improved air pump that Boyle used to establish his gas laws. When Hooke was made a member of the Royal Society in 1663, he also became its "curator of experiments," which allowed him access to the society's facilities. He remained in this position for the rest of his life, and was able to pursue whatever interested him scientifically.

As a man of wide talents, Hooke's scientific interests were even broader, and he went on to make contributions in physics, astronomy, architecture, microscopy, and biology, among other fields. Although Hooke was not the first to experiment using a microscope, he was the first to dedicate an entire book to microscopy. In 1665, he published his Micrographia, which was written in English despite its Latin title. This work contains descriptions and illustrations of the structures of insects, fossils, and plants in never-before-seen detail. His drawings of tiny insects, parts of bird feathers, and even fish scales are both artistically beautiful and scientifically accurate. The biological discovery for which he is best remembered is the porous (having pores or holes) structure of cork. When he took a thin slice of cork and put it under the compound microscope that he had built himself, he noticed that it was made up of tiny rectangular holes that he called "cells." It was an appropriate name for these little boxes, or empty rectangular structures, since the word usually meant a small room (like a jail cell). In fact, what he was seeing were the now-dead remnants of once-living structures that had been filled with fluid. That is, he actually was viewing what had been cells. Hooke's word "cell" came to be adopted by biologists once they were able to observe living structures under a microscope. Ever since, the word and the concept it stands for has become one of the cornerstones of biology. Hooke's Micrographia is recognized today as containing some of the best microscopic views of nature. In addition to his microscopic studies of insects and plants, he studied fossils a great deal, which led him to offer some early ideas about what is now realized to be evolution (the process by which living things change over generations). Hooke was described by some as quarrelsome, miserly, and a hypochondriac (someone who believes that are always sick), and he engaged some of the best minds of his time in some terrible feuds. Yet his contributions to biology are, in the full range of his life, but a small part of what he accomplished and contributed to other fields of science.

While such a microscope is adequate for schools and modest labs, greater magnification is often needed for more advanced research. In 1931 the electron microscope was invented. Using the knowledge that beams of electrons (particles that make up a single atom) could be focused using a magnetic field the same way a glass lens focuses light, scientists built an electron microscope a thousand times more powerful than a regular compound microscope. Today there are two types of electron microscopes: the transmission electron microscope (TEM) and the scanning electron microscope (SEM). In a TEM, electrons pass through the object and cannot be used to magnify living things. In a SEM, electrons are bounced off the surface of the object, meaning that it is possible to examine things that are alive. Although the SEM cannot magnify things as greatly as the TEM, it produces a more three-dimensional image. Both microscopes display their magnified images on a video screen.

Besides the TEM and SEM, other more advanced type of microscopes include the phase contrast and dark-field microscopes. The phase microscope contrasts light waves that pass through a specimen with those that do not, making for a sharper contrast. The dark field microscope makes an object appear bright against a dark background. Finally, a scanning optical microscope uses a laser to obtain pinpoint illumination, while the atomic-force microscope uses a diamond-tipped probe that moves across the surface of the specimen and is able to "see" individual living cells without damaging them. A microscope extends the sense of sight to an incredible degree, and while it has been an important instrument to all of science, it is an essential tool for the life sciences.

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Microscopy

Microscopy

The light microscope

History of light microscopy

Electron microscopy

Scanning tunneling microscopy

Recent developments in microscopy

Resources

Microscopy is the science of producing and observing images of objects that cannot be seen by the unaided eye. A microscope is an instrument the produces the image within microscopy. The primary function of a microscope is to resolve, that is distinguish, two closely spaced objects as separate. The secondary function of a microscope is to magnify. Microscopy has developed into an exciting field with numerous applications in biology, geology, chemistry, physics, and in various other areas of technology.

The light microscope

The most common, inexpensive, and easy to use microscope is the light microscope, which produces a magnified image of the object by bending and focusing light rays. The light microscope uses a variety of glass lenses to produce a magnified image that is focused before the eye. The magnifying properties of a converging lens, like that which is used in a typical magnifying glass or camera. Light from the object is bent, or refracted, as it passes through the lens producing an image that is inverted and magnified. In the simplest compound microscope, two converging lenses are used. The image from the first lens (objective) becomes the object for the second lens (eyepiece). The final image is much larger than either lens could produce independently. With a little effort, one can reproduce this effect by using two magnifying glasses.

The wavelength of visible light ultimately limits the resolving power of the light microscope. Therefore, two objects separated by distances significantly less than about 0.4 micrometers (the smallest wavelength of visible light) cannot be distinguished as separate. This is because the light microscope produces its images by reflecting from or transmitting visible light through a specimen. An analogy can be made to ocean waves at the beach, with wavelengths of a few meters. If two people were wading into the surf only a few inches apart (a separation much less than the wavelength of the ocean waves), it would be impossible to distinguish them as separate by analyzing the ocean waves that reflected from them. Despite these limitations, the resolution of the light microscope is sufficient to produce excellent images of many of the important cell structures and organelles, and consequently still has many applications, chiefly in biology.

History of light microscopy

Since the time of the Romans, it was realized that certain shapes of glass had properties that could magnify objects. By the year 1300, these early crude lenses were being used as corrective eyeglasses. It was not until the late 1500s, however, that the first compound microscopes were developed.

There are several different forms of microscopes available to the researcher in a modern laboratory but all owe their origin to the microscopes that used optical lenses to enlarge objects. The first record describing an artificial lens being used for magnification dates from 1267. The work of English philosopher Roger Bacon (c. 12141292) entitled Perspectiva described viewing minute objects through a lesser segment of a sphere of glass or crystal to enlarge them. Spectacles were in use shortly after this period to correct vision and enlarge objects, but it was not until 1595 that the first device that could truly be considered a microscope was made. This microscope was prepared by lens grinder Zacharias Jansen (1580c.1638), along with his father Hans, in Holland. This was the first compound microscope in that it employed two separate lenses that could be moved relative to each other by the means of a sliding tube. This mechanism allowed the microscope to zoom (to change its magnification) from 3x (three times normal) to 9x (nine times normal).

This system was eventually improved by English scientist Robert Hooke (16351703) who added a third lens attached to the viewing (eyepiece) lens. This improvement was carried out using an eyepiece from a telescope, an optical instrument with a longer pedigree than the microscope. Hooke also was the first to publish results on the microscopy of plants and animals. Using a simple two-lens compound microscope, he was able to discern the cells in a thin section of cork. Once the first Jansen microscopes had been made, word spread rapidly throughout the world. As a result, the seventeenth century saw many microscope manufacturers and users appear. It was at this time that the word microscope was first used by an Italian scientific society, which included Italian astronomer and physicist Galileo Galilei (15641642) as a member.

Some of the early work that was carried out using these primitive microscopes is still highly regarded today. In 1660, Italian physiologist Marcello Malpighi (16281694) was able to prove the blood circulation theories of English physician William Harvey (15781657) by discovering the presence of capillaries connecting arteries and veins in the body as well as identifying many microscopic structures in the human body. Some five years later, English scientist Robert Hooke (16351703) published the first pictures of the cells making up living organisms. Prior to these works, it had been assumed that the microscope was nothing more than a toy. During this period, some microscopes were 2 feet (0.6 meter) in length and illuminated by oil lamps.

The most famous microbiologist was Antoni van Leeuwenhoek (16321723) 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.

By the mid-nineteenth century, significant improvements had been made in the light microscope design, mainly due to refinements in lens grinding techniques. However, most of these lens refinements were the result of trial and error rather than inspired through principles of physics. Ernst Abb´ (18401905) was the first to apply physical principles to lens design. Combining glasses with different refracting powers into a single lens, he was able to reduce image distortion significantly. Despite these improvements, the ultimate resolution of the light microscope was still limited by the wavelength of light. To resolve finer detail, something with a smaller wavelength than light would have to be used.

Electron microscopy

In the mid-1920s, French physicist Louis Victor de Broglie (18921966) suggested that electrons, as well as other particles, should exhibit wave like properties similar to light. Experiments on electron beams a few years later confirmed de Broglies hypothesis. Electrons behave like waves. Of importance to microscopy was the fact that the wavelength of electrons is typically much smaller than the wavelength of light. Therefore, the limitation imposed on the light microscope of 0.4 micrometers could be significantly reduced by using a beam of electrons to illuminate the specimen. This fact was exploited in the 1930s in the development of the electron microscope.

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.

For the light microscope, light can be focused and bent using the refractive properties of glass lenses. To bend and focus beams of electrons, however, it is necessary to use magnetic fields. The magnetic lens that focuses the electrons works through the physical

principle that a charged particle, such as an electron, which has a negative charge, will experience a force when it is moving in a magnetic field. By positioning magnets properly along the electron beam, it is possible to bend the electrons in such a way as to produce a magnified image on a photographic film or a fluorescent screen. This same principle is used in a television set to focus electrons onto the television screen to give the appropriate images.

Electron microscopes are complex and expensive. To use them effectively requires extensive training. They are rarely found outside the research laboratory. Sample preparation can be extremely time consuming. For the TEM, the sample must be ground extremely thin, less than 0.1 micrometer, so that the electrons will make it through the sample. For the SEM, the sample is usually coated with a thin layer of gold to increase its ability to reflect electrons. Therefore, in electron microscopy, the specimen cant be living. Today, the best TEMs can produce images of the atoms in the interior of a sample. This is a factor of a 1,000 better than the best light microscopes. The SEM, on the other hand, can typically distinguish objects about 100 atoms in size.

Scanning tunneling microscopy

In the early 1980s, a new technique in microscopy was developed which did not involve beams of electrons or light to produce an image. Instead, a small metal tip is scanned very close to the surface of a sample and a tiny electric current is measured as the tip passes over the atoms on the surface. The microscope that works in this manner is the scanning tunneling microscope (STM). When a metal tip is brought close to the sample surface, the electrons that surround the atoms on the surface can actually tunnel through the air gap and produce a current through the tip. This physical phenomenon is called tunneling and is one of the amazing results of quantum physics. If such phenomenon could occur with large objects, it would be possible for a baseball to tunnel through a brick wall with no damage to either. The current of electrons that tunnel through the air gap is very much dependent on the width of the gap and therefore the current will rise and fall in succession with the atoms on the surface. This current is then amplified and fed into a computer to produce a three dimensional image of the atoms on the surface.

Without the need for complicated magnetic lenses and electron beams, the STM is far less complex than the electron microscope. The tiny tunneling current can be simply amplified through electronic circuitry similar to what is used in other electronic equipment, such as a stereo. In addition, the sample preparation is usually less tedious. Many samples can be imaged in air with essentially no preparation. For more sensitive samples that react with air, imaging is done in vacuum. A requirement for the STM is that the samples be electrically conducting, such as a metal.

Recent developments in microscopy

There have been numerous variations on the types of microscopy outlined so far. A sampling of these is: acoustic microscopy, which involves the reflection of sound waves off a specimen; x-ray microscopy, which involves the transmission of x rays through the specimen; near field optical microscopy, which involves shining light through a small opening smaller than the wavelength of light; and atomic force microscopy, which is similar to scanning tunneling microscopy but can be applied to materials that are not electrically conducting, such as quartz.

Microscopy is a fascinating subject that has quite literally given scientists and educators a whole new view of the world. In the over 400 years since the field began, massive advances have been made, both in terms of what is technically possible and also in what can be discovered using a microscope. During the last 50 years of the twentieth century, the reliance on light and light microscopes has been reduced in favor of ones that are more powerful. The twenty-first century has opened up even more ways of looking more closely at the world. Current advances in microscopy include new techniques such as multi-photon fluorescence and computerized wide-field deconvolution microscopy. A major advance has come to scientists at the University of St. Andrews, in Scotland, who are developing what they call a super-microscope for use with leading-edge research in such diseases as cancer and Alzheimer diesase. The biophotonics microscope will allow one instrument to perform multiple functions in cellular research; such activities as imaging, organizing, separating, and making holes into cells as small as one-hundredth of a millimeter.

One of the most amazing recent developments in microscopy involves the manipulation of individual atoms. Through a novel application of the STM, scientists at International Business Machines Corporation (IBM) were able to arrange individual atoms on

KEY TERMS

Compound microscope A light microscope that uses two or more glass lenses to produce an image.

Electron A negatively charged particle, ordinarily occurring as part of an atom. The atoms electrons form a sort of cloud about the nucleus.

Glass converging lens A circular disk with one or two convex curved surfaces used in focusing (converging) light or producing magnified images.

Magnetic lens A magnet used to focus an electron beam for producing magnified images in an electron microscope.

Micrometer One millionth of a meter, or 0.001 millimeter (one thousandth of millimeter).

Refraction The bending of light that occurs when traveling from one medium to another, such as air to glass or air to water.

Wavelength The distance between two consecutive crests or troughs in a wave.

X ray Short wavelength light between wavelengths of 0.01 and 0.00001 micrometer.

a surface and spell out the letters IBM. This has opened up new directions in microscopy, where the microscope is both an instrument with which to observe and to interact with microscopic objects. Future trends in microscopy will most likely probe features within the atom.

Resources

BOOKS

Becker, Wayne M. Guide to Microscopy. San Francisco, CA: Benjamin Cummings Publishing Company, 2003.

Murphy, Douglas B. Fundamentals of Light Microscopy and Electronic Imaging. New York: Wiley-Liss, 2001.

Sluder, Greenfield, and David E. Wolf, eds. Digital Microscopy: A Second Edition of Video Microscopy. Amsterdam, Netherlands, and Boston, MA: Elsevier Academic Press, 2003.

Spector, David L., and Robert D. Goldman, eds. Basic Methods in Microscopy: Protocols and Concepts from Cells (A Laboratory Manual). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2006.

Torok, Peter, and Fu-Jen Keo, eds. Optical Imaging and Microscopy: Techniques and Advanced Systems. Berlin, Germany, and New York: Springer-Verlag, 2003.

Kurt Vandervoort

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Microscopy

Microscopy

Microscopy is the science of producing and observing images of objects that cannot be seen by the unaided eye . A microscope is an instrument which produces the image. The primary function of a microscope is to resolve, that is distinguish, two closely spaced objects as separate. The secondary function of a microscope is to magnify. Microscopy has developed into an exciting field with numerous applications in biology , geology , chemistry , physics , and technology.


The light microscope

The most common, inexpensive, and easy to use microscope is the light microscope, which produces a magnified image of the object by bending and focusing light rays. The light microscope uses a variety of glass lenses to produce a magnified image that is focused before the eye. The magnifying properties of a converging lens , like that which is used in a typical magnifying glass or camera. Light from the object is bent, or refracted, as it passes through the lens producing an image which is inverted and magnified. In the simplest compound microscope, two converging lenses are used. The image from the first lens (objective) becomes the object for the second lens (eyepiece). The final image is much larger than either lens could produce independently. With a little effort, you can reproduce this effect yourself by using two magnifying glasses.

The wavelength of visible light ultimately limits the resolving power of the light microscope. Therefore, two objects separated by distances significantly less than about 0.4 micrometers (the smallest wavelength of visible light) cannot be distinguished as separate. This is because the light microscope produces its images by reflecting from or transmitting visible light through a specimen. An analogy can be made to ocean waves at the beach, with wavelengths of a few meters. If two people were wading into the surf only a few inches apart (a separation much less than the wavelength of the ocean waves), it would be impossible to distinguish them as separate by analyzing the ocean waves that reflected from them. Despite these limitations, the resolution of the light microscope is sufficient to produce excellent images of many of the important cell structures and organelles, and consequently still has many applications, chiefly in biology.


History of light microscopy

Since the time of the Romans, it was realized that certain shapes of glass had properties that could magnify objects. By the year 1300, these early crude lenses were being used as corrective eyeglasses. It wasn't until the late 1500s, however, that the first compound microscopes were developed.

Robert Hooke (1635-1703) was the first to publish results on the microscopy of plants and animals. Using a simple two lens compound microscope, he was able to discern the cells in a thin section of cork . The most famous microbiologist was 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.

By the mid-nineteenth century, significant improvements had been made in the light microscope design, mainly due to refinements in lens grinding techniques. However, most of these lens refinements were the result of trial and error rather than inspired through principles of physics. Ernst Abbé (1840-1905) was the first to apply physical principles to lens design. Combining glasses with different refracting powers into a single lens, he was able to reduce image distortion significantly. Despite these improvements, the ultimate resolution of the light microscope was still limited by the wavelength of light. To resolve finer detail, something with a smaller wavelength than light would have to be used.


Electron microscopy

In the mid-1920s, Louis de Broglie (1892-1966) suggested that electrons, as well as other particles, should exhibit wave like properties similar to light. Experiments on electron beams a few years later confirmed de Broglie's hypothesis. Electrons behave like waves. Of importance to microscopy was the fact that the wavelength of electrons is typically much smaller than the wavelength of light. Therefore, the limitation imposed on the light microscope of 0.4 micrometers could be significantly reduced by using a beam of electrons to illuminate the specimen. This fact was exploited in the 1930s in the development of the electron microscope.

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 .

For the light microscope, light can be focused and bent using the refractive properties of glass lenses. To bend and focus beams of electrons, however, it is necessary to use magnetic fields. The magnetic lens which focuses the electrons works through the physical principle that a charged particle, such as an electron which has a negative charge, will experience a force when it is moving in a magnetic field. By positioning magnets properly along the electron beam, it is possible to bend the electrons in such a way as to produce a magnified image on a photographic film or a fluorescent screen. This same principle is used in a television set to focus electrons onto the television screen to give the appropriate images.

Electron microscopes are complex and expensive. To use them effectively requires extensive training. They are rarely found outside the research laboratory. Sample preparation can be extremely time consuming. For the TEM, the sample must be ground extremely thin, less than 0.1 micrometer, so that the electrons will make it through the sample. For the SEM, the sample is usually coated with a thin layer of gold to increase its ability to reflect electrons. Therefore, in electron microscopy, the specimen can't be living. Today, the best TEMs can produce images of the atoms in the interior of a sample. This is a factor of a 1,000 better than the best light microscopes. The SEM, on the other hand, can typically distinguish objects about 100 atoms in size.

Scanning tunneling microscopy

In the early 1980s, a new technique in microscopy was developed which did not involve beams of electrons or light to produce an image. Instead, a small metal tip is scanned very close to the surface of a sample and a tiny electric current is measured as the tip passes over the atoms on the surface. The microscope that works in this manner is the scanning tunneling microscope (STM). When a metal tip is brought close to the sample surface, the electrons that surround the atoms on the surface can actually "tunnel through" the air gap and produce a current through the tip. This physical phenomenon is called tunneling and is one of the amazing results of quantum physics. If such phenomenon could occur with large objects, it would be possible for a baseball to tunnel through a brick wall with no damage to either. The current of electrons that tunnel through the air gap is very much dependent on the width of the gap and therefore the current will rise and fall in succession with the atoms on the surface. This current is then amplified and fed into a computer to produce a three dimensional image of the atoms on the surface.

Without the need for complicated magnetic lenses and electron beams, the STM is far less complex than the electron microscope. The tiny tunneling current can be simply amplified through electronic circuitry similar to that which is used in other electronic equipment, such as a stereo. In addition, the sample preparation is usually less tedious. Many samples can be imaged in air with essentially no preparation. For more sensitive samples which react with air, imaging is done in vacuum . A requirement for the STM is that the samples be electrically conducting, such as a metal.


Recent developments in microscopy

There have been numerous variations on the types of microscopy outlined so far. A sampling of these is: acoustic microscopy, which involves the reflection of sound waves off a specimen; x-ray microscopy, which involves the transmission of x rays through the specimen; near field optical microscopy, which involves shining light through a small opening smaller than the wavelength of light; and atomic force microscopy, which is similar to scanning tunneling microscopy but can be applied to materials that are not electrically conducting, such as quartz.

One of the most amazing recent developments in microscopy involves the manipulation of individual atoms. Through a novel application of the STM, scientists at IBM were able to arrange individual atoms on a surface and spell out the letters "IBM." This has opened up new directions in microscopy, where the microscope is both an instrument with which to observe and to interact with microscopic objects. Future trends in microscopy will most likely probe features within the atom.


Resources

books

Burgess, Jeremy, Michael Marten, and Rosemary Taylor. Microcosmos. Cambridge: Cambridge University Press, 1987.

Giancoli, Douglas. Physics. Englewood Cliffs, NJ: Prentice Hall, 1995.

Slayter, Elizabeth and Henry. Light and Electron Microscopy. Cambridge: Cambridge University Press, 1992.

periodicals

Eigler, D.M., and E.K. Schweizer. "Positioning Single Atoms with a Scanning Tunneling Microscope." Nature 344 (1990): 524-526.

Taylor, D., Michel Nederlof, Frederick Lanni, and Alan Waggoner. "The New Vision of Light Microscopy." American Scientist 80 (1992): 322-335.


Kurt Vandervoort

KEY TERMS

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Compound microscope

—A light microscope which uses two or more glass lenses to produce an image.

Electron

—A negatively charged particle, ordinarily occurring as part of an atom. The atom's electrons form a sort of cloud about the nucleus.

Glass converging lens

—A circular disk with one or two convex curved surfaces used in focusing (converging) light or producing magnified images.

Magnetic lens

—A magnet used to focus an electron beam for producing magnified images in an electron microscope.

Micrometer

—0.000001 meter (one millionth of a meter) or 0.001 millimeter (one thousandth of millimeter).

Refraction

—The bending of light that occurs when traveling from one medium to another, such as air to glass or air to water.

Wavelength

—The distance between two consecutive crests or troughs in a wave.

X ray

—Short wavelength light between wavelengths of 0.01 and 0.00001 micrometer.

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"Microscopy." The Gale Encyclopedia of Science. . Encyclopedia.com. (November 14, 2018). https://www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-maps/microscopy

"Microscopy." The Gale Encyclopedia of Science. . Retrieved November 14, 2018 from Encyclopedia.com: https://www.encyclopedia.com/science/encyclopedias-almanacs-transcripts-and-maps/microscopy

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Within the “Cite this article” tool, pick a style to see how all available information looks when formatted according to that style. Then, copy and paste the text into your bibliography or works cited list.

Because each style has its own formatting nuances that evolve over time and not all information is available for every reference entry or article, Encyclopedia.com cannot guarantee each citation it generates. Therefore, it’s best to use Encyclopedia.com citations as a starting point before checking the style against your school or publication’s requirements and the most-recent information available at these sites:

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Notes:
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