MICROSCOPE

instrument for producing enlarged images of extremely small objects. With the aid of a scanning tunneling microscope, objects as small as atoms may be seen and studied. Microscopes are indispensable in many scientific fields. The simplest microscope comprises a single lens. Such magnifiers were in use by the mid-15th century, and by 1674 the Dutch naturalist Antonie van Leeuwenhoek had produced lenses powerful enough to enable him to observe bacteria 23 micrometres in diameter. In the compound microscope an additional lens is employed to enlarge the image produced by a primary lens. This instrument was invented in the Netherlands about 1600, but it did not come into universal use until after 1830, when the English microscopist Joseph Lister established the theoretical principles that made possible the construction of lenses free of chromatic and spherical aberration. By the end of the 19th century there were available microscopes that achieved resolution equal to the theoretical limit set by numerical aperture and the wavelengths of visible light and ultraviolet radiation. In 1924 the French physicist Louis de Broglie showed that a beam of electrons may be considered as a form of wave motion having a much shorter wavelength than that of light. Based on that idea, the electron microscope, in which specimens are illuminated by an electron beam that is focused by an electrostatic or electromagnetic field, was developed in the 1930s by the German electrical engineer Ernst Ruska. Modern electron microscopes provide detailed images at magnifications of more than 250,000. The field lenses of electron microscopes, however, suffer the same defects as do the glass lenses of optical microscopes, including chromatic and spherical aberrations. High-frequency sound waves (above the range of human hearing) have wavelengths comparable to those of visible light. This understanding led to use of sound rather than optical waves in microscopy. Although the idea dates to the 1940s, the first working acoustic microscopes were not developed until the 1970s. Because sound waves can penetrate opaque materials, acoustic microscopes are capable of providing images of the internal structures, as well as the surfaces, of many objects that cannot be viewed with an optical microscope. The scanning tunneling microscope, invented in the 1980s by Gerd Binnig and Heinrich Rohrer, measures variations in an electric current that is generated between the microscope probe and the surface of a specimen. The image of the surface that is produced is of such high resolution that individual atoms can be seen. instrument that produces enlarged images of small objects, allowing them to be viewed at a scale convenient for examination and analysis. The image may be formed by optical, acoustic, or electronic means, and it is received by direct imaging, electronic processing, or a combination of these methods. The microscope may be static, in which the object is viewed directly, or dynamic, in which the image is built up by successive scans of the object. The magnifying power of a microscope is an expression of the number of times the object being examined appears to be enlarged. Magnifying power is a dimensionless ratio. The resolution of a microscope is a measure of the size of the smallest detail on the object that can be observed. Resolution is expressed in linear units, usually millimetres. The most familiar type of microscope is the optical, or light, microscope, in which lenses are used to form the image. Optical microscopes can be simple, consisting of a single lens, or compound, consisting of several optical components in tandem. Simple optical magnifiers may have magnifying powers of from 1 to 10 magnitudes, with resolutions to about 0.01 millimetre (mm) possible. Compound optical microscopes have magnifying powers ranging from about 2.5 to 1,000 magnitudes and provide resolutions of 0.01 to 0.0002 mm. Other types of microscopes utilize the wave nature of various physical processes, the most important being the electron microscope, which uses a beam of electrons in its image formation. Electron microscopes have magnifying powers of 1,000 to 1,000,000 magnitudes possible, with resolutions ranging from 0.001 to 0.000 000 01 mm. Special microscopes, such as acoustic microscopes or scanning tunneling microscopes, use other physical effects, further extending the range of objects that can be viewed. Indeed, with a scanning tunneling microscope, even individual molecules and atoms can be seen. Some of these specialized microscopes, as well as the more common optical and electron microscopes, are described in this article. A brief history of microscopy is also included. Acoustic microscopes General considerations The optical microscope reveals much of the microscopic world, but its domain is restricted; it does not include opaque materials, opalescent liquids, nor media that absorb light completely. Sergei Y. Sokolov at the V.I. Ulyanov (Lenin) Electrotechnical Institute, Leningrad, suggested that these restrictions could be lifted if sound waves were used in a new form of microscope. Sokolov recognized that a microscope using sound waves with a frequency of 3,000 megahertz (MHz) would have a resolution equal to that of the optical microscope. He advanced his idea in the early 1940s, at a time when the required technology did not exist. Since then, however, the technology has been developed, and the high frequencies required for Sokolov's microscope are found in the microwave systems used for radar and for satellite communications. During the 1970s, several groups of researchers in the United States employed these frequencies to build sound systems. The microscope that evolved from this effort is known as the scanning acoustic microscope. Sound is a term commonly associated with the waves in air that can be detected by the human ear. The phenomenon is more general, however; sound waves travel through liquids and solids as well. The descriptive term acoustics is used for these waves. Acoustic waves easily propagate through materials impervious to optical waves. The energy is carried by the periodic motion of particles, where alternate regions of compression and rarefaction move through the media at the velocity of sound. The differences between the optical and acoustic instruments can be clarified with a short description of three parameters used to characterize waveswavelength, wave velocity, and frequency. These parameters are easily visualized by observing ocean waves. The wavelength is the distance between the crests, the wave velocity is the speed of the moving crests, and the frequency is the frequency of impact of the crests on the shoreline. If the spacing between crests is 10 metres and if they move with a velocity of 10 metres per second, the crests will hit the shoreline with a frequency of 1 per second. If the velocity is reduced to 0.1 metre per second and a distance of 10 metres is maintained between crests, the frequency of impact will be reduced to 0.1 per second. Similarly, in sound systems, where the velocity is reduced by a factor of 100,000 below the velocity of optical waves, the acoustic wavelength is comparable to the optical wavelength if the sound frequency is reduced by the same factor of 100,000 below the frequency of optical waves. The resolution of an acoustic microscope operating at 3,000 MHz is equal to the resolution of the optical microscope. Instrumentation The instrumentation of an acoustic microscope is completely different from that of a traditional optical microscope. The central component of the acoustic system is a small sapphire rod. A thin film of piezoelectric material is deposited on the flat end of the sapphire crystal, and a spherical depression on the opposite end of the crystal rod acts as the lens. The endface with the cavity is immersed in water, along with the specimen. The thin film of piezoelectric material, usually zinc oxide (ZnO), expands and contracts when a periodic electric field is applied across it. The film acts as a transducer to convert electromagnetic energy into acoustic energy. The periodic expansions then travel through the crystal as acoustic waves. The acoustic velocity in water is less than it is in sapphire, and the waves that cross the crystal-water interface are refracted through a large angle. The refraction is so large that the plane waves in the sapphire rod are converted in the liquid into spherical waves that converge to a narrow waist, or focus, near the centre of curvature of the spherical lens. In a scanning system, the minimum resolvable feature is determined by the waist diameter. The diameter of the acoustic beam at the waist is less than one wavelength. The elastic properties of the sample are examined with the acoustic beam that emerges from the lens. The examination is carried out point by point by mechanically scanning the lens over the area under investigation. The waves reflected from the sample travel back through the liquid, through the lens, and through the crystal to the ZnO transducer. There they are reconverted into electrical signals. In this way, the energy is quickly transferred into and out of the acoustic system. The images are displayed on the screen of a monitor and recorded by photographing the display on the screen. Mechanical scanning, together with the reconversion to electronic signals, forms the analog to the photographic film of an optical microscope. In another configuration, a cylindrical lens is used to focus the acoustic energy into a narrow line. It is used to measure features on the surface along a given direction and characterize the anisotropic properties of surfaces. The acoustic microscope utilizes three types of acoustic waves in its various operating modes; these are (1) longitudinal waves, in which the particle motion is in the direction of wave motion, (2) shear waves, in which the particle motion is transverse to the direction of wave motion, and (3) surface waves, in which the energy clings to the interface between the sample and the liquid. (The surface wave, first described by the 19th-century scientist Lord Rayleigh, now bears his name.) Longitudinal waves are employed in the most common mode, where the acoustic pulse reflected from the sample surface is recorded for each position of the probing beam. Changes in the elastic properties of the sample show up as differences in both amplitude and phase of the reflected signal. Rayleigh waves are extraordinarily effective in enhancing the contrast for obscure surface structure. When the incident beam is defocused by moving the lens toward the sample, the longitudinal waves in the liquid are coupled to the surface waves on the sample interface. In this circumstance, the interchange of energy between the longitudinal and surface modes combine in such a way as to create an interference pattern at the transducer. These patterns are sensitive to small changes in the elastic properties of the surface. Minute cracks or scratches show up with excellent contrast. The electronic components in the acoustic microscope are adapted from the technology used to generate the short pulses of radio-frequency energy in radar systems. The system operates with coherent signals over a wide range of frequencies. In water, the frequency range extends from 5 to 5,000 MHz; the wavelength ranges from 300 to 0.3 micrometres. Cryogenic instruments with liquid helium as the working fluid are used to extend the system to even shorter wavelengths. The velocity of sound in helium is about one-sixth that of water, which means that the wavelength is reduced by the same factor. Furthermore, at very low temperatures (near 0.1 K, or -459 F), the sound absorption in helium is less than it is in water. These two factors have been exploited to build an instrument operating at 16,000 MHz with a resolution of 15 nanometres. Additional reading General surveys of the subject, with details on features and applications, are found in H.N. Southworth, Introduction to Modern Microscopy (1975); L.C. Martin, The Theory of the Microscope (1966); Theodore George Rochow and Eugene George Rochow, An Introduction to Microscopy by Means of Light, Electrons, X-Rays, or Ultrasound (1978); Eric A. Ash, Scanned Image Microscopy (1980); W.G. Hartley, Microscopy (1962, reissued 1979 as Hartley's Microscopy; also published as How to Use a Microscope, 1964); and Jeremy Burgess, Michael Marten, and Rosemary Taylor, Microcosmos (1987).A wide-ranging and well-illustrated introduction to electron microscopy, suitable for beginners as well as for more advanced users, is offered in Ian M. Watt, The Principles and Practice of Electron Microscopy (1985). Joseph I. Goldstein et al., Scanning Electron Microscopy and X-Ray Microanalysis (1981), studies in detail all facets of the scanning electron microscope and electron-probe microanalyzer; it is a technical book but not unduly mathematical. P.B. Hirsch et al., Electron Microscopy of Thin Crystals (1965, reissued 1977), is a classic treatise on transmission electron microscopes and microscopy, fairly difficult in places but an invaluable reference.Discussions of acoustic microscopy are concentrated mostly in periodical literature. V. Jipson and C.F. Quate, Acoustic Microscopy at Optical Wavelengths, Applied Physics Letters 32(12):789791 (June 15, 1978), surveys experimental evidence of the equivalence of the resolution of the acoustic microscope operating at room temperature to that of the optical microscope. Foundations of our understanding of the use of acoustic waves in microscopy are presented in the following review papers: R.A. Lemons and C.F. Quate, Acoustic Microscope, Physical Acoustics: Principles and Methods 14:292 (1979); C.F. Quate, A. Atalar, and H.K. Wickramasinghe, Acoustic Microscopy with Mechanical Scanning: A Review, Proceedings of the IEEE 67(8):10921114 (August 1979); C.F. Quate, Acoustic Microscope, Scientific American 241:6270 (October 1979), and Acoustic Microscopy, Physics Today 38(8):3442 (August 1985); and J.E. Heiserman and C.F. Quate, The Scanning Acoustic Microscope, pp. 343394 in D. Sette (ed.), Frontiers in Physical Acoustics (1986). The entire collection of papers in the special March 1985 issue of the IEEE Transactions on Sonics and Ultrasonics SU-32(2) is devoted to the subject of acoustic microscopy. Andrew Briggs, An Introduction to Scanning Acoustic Microscopy (1985), is a concise illustrated handbook.An informative introduction to the scanning tunneling microscopes, without the heavy technical details, is provided in the following review articles: G. Binnig and H. Rohrer, The Scanning Tunneling Microscope, Scientific American 253(2):5056 (August 1985); J.A. Golovchenko, The Tunneling Microscope: A New Look at the Atomic World, Science 232:4853 (April 4, 1986); and C.F. Quate, Vacuum Tunneling: A New Technique for Microscopy, Physics Today 39(8):2633 (August 1986). R.J. Behm, N. Garcia, and H. Rohrer (eds.), Scanning Tunneling Microscopy and Related Methods (1990), is an advanced series of lectures relating to the new microscopy.For history, see S. Bradbury, The Evolution of the Microscope (1967); and E. Ruska, The Development of the Electron Microscope and of Electron Microscopy, Reviews of Modern Physics 59(3/1):627638 (July 1987), a fascinating look at the birth and growth of electron microscopy, by its founding father. Robert R. Shannon David C. Joy Calvin F. Quate Development of the microscope The simple microscope The earliest simple microscopes used drops of water captured in a small hole to function as a magnifying lens. It is not clear when crude glass lenses first began to be employed, but by the 17th century Antonie van Leeuwenhoek, a Dutch scientist, had developed techniques for making good-quality ground lenses for simple microscopes. These were limited in magnifying power and were used primarily for botanical studies. The compound microscope The compound microscope, using an objective and an eyepiece, was first described in the 16th century, but all drawings of the period indicate very impractical arrangements of lenses. The first useful compound microscope was constructed in the Netherlands sometime between 1590 and 1608. Three spectacle makersHans Jansen, his son Zacharias, and Hans Lippersheyhave each received credit for the invention. This early microscope was limited by the use of very simple lenses, and the resolution was limited by blurring due to spherical aberration and chromatic aberration. Further development of the compound microscope in the 17th and 18th centuries occurred primarily in England and Italy, albeit along slightly different paths. Many Italian instruments were designed for the examination of opaque objects by use of reflected light. The Italian microscope designer Eustachio Divini introduced the idea of using two plano-convex lenses to form an eyepiece that permitted some aberration correction. Many mechanical innovations, including equipping body tubes with screw-thread focusing mechanisms and using spring stages to hold specimen slides, were developed by the Italian microscopists. In England the 17th-century scientist Robert Hooke introduced several techniques for improving the quality of microscopes. His instruments began a trend in England toward the development of elegant devices, incorporating many mechanical advances. English optics, however, continued to be constructed of simple lenses that suffered from spherical aberration and chromatic aberration. The spherical aberration could be limited somewhat by placing a stop (a disk with a small central aperture) behind the lens; but the chromatic aberration, which was believed to be intrinsic to refracting lenses, remained uncorrected until 1733, when Chester Moor Hall, an amateur optician, found by trial and error that combining a convex crown glass lens with a concave lens made of new lead-containing flint glass corrected the problem. While the original invention was intended for making achromatic telescope lenses, several workers recognized the application to microscopes, and in 1774 Benjamin Martin used a set of achromats for a microscope objective. This led to major improvements in the quality of microscope images and to many new designs for microscope objectives. As a result, by 1820 there were many types of new achromatic microscopes on the market. The appearance of new varieties of optical glasses encouraged continued development of the microscope in the 19th century, and considerable improvements were made in understanding the geometric optics of image formation. The English scientist Joseph Jackson Lister in 1830 published a work describing a theoretical approach to the complete design of microscope objectives. In 1834 George Biddell Airy, an English astronomer, determined that the resolving power in telescopes was limited by the finite wavelength of light due to diffraction. In 1873 Ernst Abbe related this concept to the ability of the microscope to resolve fine periodic structure in microscopic objects. Thus was born the modern theory of the microscope, which included the effects of light interference in determining the limit of resolution of the microscope. Abbe correctly related the limit of optical resolution to the wavelength of light and the numerical aperture of the objective, and he explained the relation between the type of light illuminating the object in the microscope and the appearance of the image. Abbe also invented the oil-immersion objective, in which the resolution was improved by immersion of the object in a medium with a high refractive index. Electron microscopes General considerations Fundamental research by many physicists in the first quarter of the 20th century suggested that cathode rays (i.e., electrons) might be used in some way to increase microscope resolution. Louis de Broglie, a French physicist, in 1924 opened the way with the suggestion that electron beams might be regarded as a form of wave motion. De Broglie derived the formula for their wavelength, which showed, for example, that, for electrons accelerated by 60,000 volts, the effective wavelength would be 0.05 angstromi.e., 1/100,000 that of green light. If such waves could be used in a microscope, then a valuable increase in resolution would result. In 1926 it was demonstrated that magnetic or electrostatic fields could serve as lenses for electrons or other charged particles, and this discovery initiated the study of electron optics. In 1931 a two-lens electron microscope was devised. It produced enlarged images of the electron source. Later, in 1933, a true electron microscope was built. A year or so later, the resolution of the optical microscope was surpassed, but the specimen was severely damaged by the heating effects of the electron beam, a problem that was not solved for some years. In 1935 the construction of the first commercially built electron microscope was begun in England, and it was soon followed by the production of microscopes in Germany and the United States. Progress in the construction of electron microscopes was rapid, with a great impetus to the achievement of high resolution being the introduction, in 1946, of a device called a stigmator, which compensates for astigmatism of the objective lens. Electron microscopes are now available that are easy to operate and reliable and that give point-to-point resolutions of less than 0.2 nanometre. Such high resolutions, coupled with suitable advances in the methods of specimen preparation, permit the direct visualization of many molecules and some atoms. There are two basic types of electron microscope: the transmission electron microscope (TEM), which can only image specimens a fraction of a micrometre or less in thickness, and the scanning electron microscope (SEM), in which a beam of electrons is scanned over the surface of a solid object and used to build up an image of the details of the surface structure. There are also several special types of electron microscope. Among the most valuable is the electron-probe microanalyzer, which allows a chemical analysis of the composition of materials to be made by using the incident electron beam to excite the emission of characteristic X radiation by the various elements composing the specimen. These X rays are detected and analyzed by spectrometers built into the instrument. Such probe microanalyzers are able to produce an electron scanning image so that structure and composition may be easily correlated. Differences of types There are many differences between optical and electron microscopes. The electron microscope requires that the electron beam be in a vacuum, because electrons cannot travel any appreciable distance in air at atmospheric pressure. The column of the electron microscope is evacuated by pumps, and the specimen, photographic plates, and any other necessary apparatus are introduced into the vacuum by means of air locks. Unlike the optical microscope, in which the lenses are of fixed focus and the distance between specimen and objective lens is varied, the electron microscope has variable-focus lenses, and the distance between specimen and objective lens and separation of the lenses remain constant. The magnification is determined mainly by the value of the current (for magnetic lenses) through the intermediate and projector lens coils. The image is focused by changing the current through the objective lens coil. Another difference is that the optical microscope is usually operated so that the image is a virtual one, while in the electron microscope the final image is invariably real and is visualized on a fluorescent screen or recorded for study on a photographic plate. In the optical microscope the image is formed by absorption of light in the specimen; in the electron microscope the image results from a scattering of electrons by atoms in the specimen. A heavy atom is more effective in scattering than one of low atomic number, and the presence of heavy atoms will increase the image contrast. The electron microscopist may incorporate more heavy atoms into the specimen for this purpose.