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

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.

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

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 (1632–1723). 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.

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).

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

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.

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.

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

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.

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

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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