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org/wiki/Microscope A microscope (from the Greek: , mikrs, "small" and , skopen, "to look" or "see") is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy. Microscopic means invisible to the eye unless aided by a microscope. There are many types of microscopes, the most common and first to be invented is the optical microscope which uses light to image the sample. Other major types of microscopes are the electron microscope (both the transmission electron microscope and the scanning electron microscope) and the various types of scanning probe microscope.

Contents
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1 History o 1.1 The rise of modern light microscopy o 1.2 Electron microscopy o 1.3 Scanning probe microscopy o 1.4 Fluorescence and light microscopy 2 Types o 2.1 Optical o 2.2 Electron o 2.3 Scanning probe o 2.4 Other 3 See also 4 References 5 External links

History
Main article: Timeline of microscope technology The first microscope to be developed was the optical microscope, although the original inventor is not easy to identify. An early microscope was made in 1590 in Middelburg, Netherlands.[1] Two eyeglass makers are variously given credit: Hans Lippershey (who developed an early telescope) and Zacharias Janssen. Giovanni

Faber coined the name microscope for Galileo Galilei's compound microscope in 1625 [2] (Galileo had called it the "occhiolino" or "little eye").

The rise of modern light microscopy

See also: Optical microscope The first detailed account of the interior construction of living tissue based on the use of a microscope did not appear until 1644, in Giambattista Odierna's L'occhio della mosca, or The Fly's Eye.[3] It was not until the 1660s and 1670s that the microscope was used extensively for research in Italy, Holland and England. Marcelo Malpighi in Italy began the analysis of biological structures beginning with the lungs. Robert Hooke's Micrographia had a huge impact, largely because of its impressive illustrations. The greatest contribution came from Antoni van Leeuwenhoek who discovered red blood cells and spermatozoa and helped popularise microscopy as a technique. On 9 October 1676, Leeuwenhoek reported the discovery of micro-organisms.[3] In 1893 August Khler developed a key technique for sample illumination, Khler illumination, which is central to modern light microscopy. This method of sample illumination gives rise to extremely even lighting and overcomes many limitations of older techniques of sample illumination. Further developments in sample illumination came from Fritz Zernike in 1953 and George Nomarski 1955 for their development of phase contrast and differential interference contrast illumination which allow imaging of transparent samples.

Electron microscopy

An ant as imaged using a scanning electron microscope (SEM) See also: electron microscope In the early 1900s a significant alternative to light microscopy was developed, using electrons rather than light to generate the image. Ernst Ruska started development of the first electron microscope in 1931 which was the transmission electron microscope (TEM). The transmission electron microscope works on the same principle as an optical microscope but uses electrons in the place of light and electromagnets in the place of glass lenses. Use of electrons instead of light allows a much higher resolution. Development of the transmission electron microscope was quickly followed in 1935 by the development of the scanning electron microscope by Max Knoll.[4] Electron microscopes quickly became popular following the Second World War. Ernst Ruska, working at Siemens developed the first commercial transmission electron microscope and major scientific conferences on electron microscopy started being held in the 1950s. In 1965 the first commercial scanning electron microscope was developed by Professor Sir Charles Oatley and his postgraduate student Gary Stewart and marketed by the Cambridge Instrument Company as the "Stereoscan".

Scanning probe microscopy

See also: scanning probe microscope

The 1980s saw the development of the first scanning probe microscopes. The first was the scanning tunneling microscope in 1981, developed by Gerd Binnig and Heinrich Rohrer. This was closely followed in 1986 with Gerd Binnig, Quate, and Gerber's invention of the atomic force microscope.

Fluorescence and light microscopy

See also: fluorescence microscope, immunofluorescence, and confocal microscope The most recent developments in light microscope largely centre on the rise of fluorescence microscopy in biology. During the last decades of the 20th century, particularly in the post-genomic era, many techniques for fluorescent labeling of cellular structures were developed. The main groups of techniques are small chemical staining of cellular structures, for example DAPI to label DNA, use of antibodies conjugated to fluorescent reporters, see immunofluorescence, and fluorescent proteins, such as green fluorescent protein. These techniques use these different fluorophores for analysis of cell structure at a molecular level in both live and fixed samples. The rise of fluorescence microscopy drove the development of a major modern microscope design, the confocal microscope. The principle was patented in 1957 by Marvin Minsky, although laser technology limited practical application of the technique. It was not until 1978 when Thomas and Christoph Cremer developed the first practical confocal laser scanning microscope and the technique rapidly gained popularity through the 1980s. Main article: Microscopy#Sub-diffraction techniques Much current research (in the early 21st century) on optical microscope techniques is focused on development of superresolution analysis of fluorescently labeled samples. Structured illumination can improve resolution by around two to four times and techniques like stimulated Emission Depletion microscopy are approaching the resolution of electron microscopes.

Types

Types of microscopes Microscopes can be separated into several different classes. One grouping is based on what interacts with the sample to generate the image, i.e., light (optical microscopes), electrons (electron microscopes) or a probe (scanning probe microscopes). Alternatively microscopes can be classed on whether they analyse the sample via a scanning point (confocal optical microscopes, scanning electron microscopes and scanning probe microscopes) or analyze the sample all at once (wide field optical microscope and transmission electron microscopes). The wide field optical microscope and transmission electron microscope use the theory of lenses (optics for light microscopes and electromagnet lenses for electron microscopes) in order to magnify the image generated by the passage of a wave through the sample, or reflected by the sample. The waves used are electromagnetic (in optical microscopes) or electron beams (in electron microscopes). Resolution in these microscopes is limited by the wavelength of the radiation used to image the sample, shorter wavelengths allow a higher resolution. Scanning optical and electron microscopes, like the confocal microscope and scanning electron microscope, use lenses to focus a spot of light/electrons onto the sample then analyze the reflected and/or transmitted waves. The point is then scanned over the sample to analyze a rectangular region. Magnification of the image is achieved by displaying the data from scanning a small sample area on a large screen. These microscopes have the same resolution limit as wide field optical and electron microscopes. Scanning probe microscopes also analyze a single point in the sample and then scan the probe over a rectangular sample region to build up an image. As these microscopes do not use electromagnetic or electron radiation for imaging they are not

subject to the same resolution limit as the optical and electron microscopes described above.

Optical
Main article: Optical microscope The most common type of microscopeand the first inventedis the optical microscope. This is an optical instrument containing one or more lenses producing an enlarged image of a sample placed in the focal plane. Optical microscopes have refractive glass and occasionally of plastic or quartz, to focus light into the eye or another light detector. Mirror-based optical microscopes operate in the same manner. Typical magnification of a light microscope, assuming visible range light, is up to 1500x with a theoretical resolution limit of around 0.2 micrometres or 200 nanometres. Specialized techniques (e.g., scanning confocal microscopy, Vertico SMI) may exceed this magnification but the resolution is diffraction limited. The use of shorter wavelengths of light, such as the ultraviolet, is one way to improve the spatial resolution of the optical microscope, as are devices such as the near-field scanning optical microscope. Sarfus, a recent optical technique increases the sensitivity of standard optical microscope to a point it becomes possible to directly visualize nanometric films (down to 0.3 nanometre) and isolated nano-objects (down to 2 nm-diameter). The technique is based on the use of non-reflecting substrates for cross-polarized reflected light microscopy.

CBP Office of Field Operations agent checking the authenticity of a travel document at an international airport using a stereo microscope Ultraviolet light enables the resolution of microscopic features, as well as to image samples that are transparent to the eye. Near infrared light images circuitry embedded in bonded silicon devices, as silicon is transparent in this region. Many wavelengths of light, ranging from the ultraviolet to the visible are used to excite fluorescence emission from objects for viewing by eye or with sensitive cameras.

Phase contrast microscopy is an optical microscopy illumination technique in which small phase shifts in the light passing through a transparent specimen are converted into amplitude or contrast changes in the image. A phase contrast microscope does not require staining to view the slide. This microscope made it possible to study the cell cycle. The traditional optical microscope has recently been modified into a digital microscope, where, instead of directly viewing the object, a charge-coupled device (CCD) is used to record the image, which is then displayed on a computer monitor.

Electron
Main article: Electron microscope Three major variants of electron microscopes exist:

Scanning electron microscope (SEM): looks at the surface of bulk objects by scanning the surface with a fine electron beam. See also environmental scanning electron microscope (ESEM). Transmission electron microscope (TEM): passes electrons through the sample, analogous to basic optical microscopy. This requires careful sample preparation, since electrons are scattered so strongly by most materials.This is a scientific device that allows people to see objects that could normally not be seen by the naked or unaided eye.

An instrument used to obtain an enlarged image of a small object. The image may be seen, photographed, or sensed by photocells or other receivers, depending upon the nature of the image and the use to be made of the information of the image. A simple microscope, hand lens, or magnifier usually is a round piece of transparent material, ground thinner at the edge than at the center, which can form an enlarged image of a small object. Commonly, simple microscopes are double convex or planoconvex lenses, or systems of lenses acting together to form the image. The compound microscope utilizes two lenses or lens systems. One lens system forms an enlarged image of the object and the second magnifies the image formed by the first. The total magnification is then the product of the magnifications of both lens systems (see illustration).

Compound microscope diagram. (After F. A. Jenkins and H. E. White, Fundamentals of Optics, 4th ed., McGraw-Hill, 1976) The typical compound microscope consists of a stand, a stage to hold the specimen, a movable body-tube containing the two lens systems, and mechanical controls for easy movement of the body and the specimen. The lens system nearest the specimen is called the objective; the one nearest the eye is called the eyepiece or ocular. A mirror is placed under the stage to reflect light into the instrument when the illumination is not built into the stand. For objectives of higher numerical aperture than 0.4, a condenser is provided under the stage to increase the illumination of the specimen. Various optical and mechanical attachments may be added to facilitate the analysis of the information in the doubly enlarged image. See also Electron microscope; Fluorescence microscope; Interference microscope; Lens (optics); Optical microscope; Phase-contrast microscope; Reflecting microscope; X-ray microscope.

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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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he 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

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