microscopy

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

History

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

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

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

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

Microscope design and image formation

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

The characteristics of microscope imaging systems

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

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

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

Three-dimensional imaging in microscopy

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

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

David M. Shotton

Bibliography

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

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