3 Light microscopy
Visible light occupies a very narrow portion of the electromagnetic spectrum. The electromagnetic spectrum extends from radio and microwaves all the way to gamma rays. Electromagnetic energy is complex, having properties that are both wave-like and particle-like. A discussion of these topics is well beyond the scope of this chapter. Suffice to say, visible light is that portion of the electromagnetic spectrum that can be detected by the human eye. In physics texts, this range is generally defined as wavelengths of light ranging from approximately 400 nm (deep violet) to 800 nm (far red). Most humans cannot see light of wavelengths much beyond 700 nm (deep red).
It is common practice to illustrate the electromagnetic spectrum as a sine wave. This is a convenient representation as the distance from one sine peak to another represents the wavelength of light (Fig. 3.1). Light that has a single wavelength is monochromatic: that is, a single color. The majority of sources of light provide a complex mixture of light of different wavelengths, and when this mixture approximates the mixture of light that derives from the sun, we perceive this as ‘white’ light. By definition, white light is a mixture of light that contains some percentage of wavelengths from all of the visible portions of the electromagnetic spectrum. It should be understood that almost all light sources provide a mixture of wavelengths of light (exceptions being devices such as lasers, which generate monochromatic, coherent light). One measure of the mixture of light given off by a light source is color temperature. In practical terms, the higher the color temperature, the closer the light is to natural daylight derived from the sun. Natural daylight from the sun is generally stated to have a color temperature of approximately 5200 kelvin (K). Incandescent light, from tungsten bulbs, has a color temperature of approximately 3200K. These values will be familiar to those using color film for photography, as film type must be chosen to suit the illumination source. As a general rule, the higher the color temperature, the more ‘blue’ or white the light appears to the eye. Lower color temperatures appear more red to yellow, and are regarded as being ‘warmer’ in color.
Shorter wavelengths of light (toward the blue to violet end of the spectrum) have a higher energy content for a given brightness of light. As one goes to even shorter wavelengths of the electromagnetic spectrum, the energy content becomes even higher (X-rays and gamma rays). The energy content of light is generally expressed as an energy level, or amplitude based on the electron volts per photon (the particle representation of light). Visible light has an energy level of approximately one electron volt per photon, and the energy level increases as one moves toward the violet and ultraviolet range of the spectrum. Approaching the soft X-ray portion of the spectrum, the energy level per photon ranges from 50 to 100 electron volts (eV). It is this higher energy in the shorter wavelengths of light (the ultraviolet and blue end of the spectrum) that is exploited to elicit fluorescence in some materials.
Light sources give off light in all directions, and most light sources consist of a complex mixture of wavelengths. This mixture of wavelengths is what defines the color temperature of the light source. It should also be noted that the mixture of wavelengths is influenced by the type of material making up the source. Since the majority of light sources used in microscopy are either heated filaments or arcs of molten metal, each source will provide a specific set of wavelengths related to the material being heated. This is referred to as the emission spectrum. Some sources provide relatively uniform mixtures of wavelengths, although of different amplitudes or intensities, such as tungsten filament lamps and xenon lamps. Others, such as mercury lamps, provide very discrete wavelengths scattered over a broad range, but with distinct gaps of no emission between these peaks.
Although light sources are inherently non-coherent (with the exception of lasers), standard diagrams of optics always draw light rays as straight lines. This is a simplification, and it should be remembered that the actual light consists of every possible angle of light rays from the source, not just the single ray illustrated in the diagram. Another property of light that is important for an understanding of microscope optics is absorption of some of the light by the medium through which the light passes (Fig. 3.2). This is seen as a reduction in the amplitude, or energy level, of the light. The medium through which the light passes can also have an effect on the actual speed at which the light passes through the material, and this is referred to as retardation.
Media through which light is able to pass will slow down or retard the speed of the light in proportion to the density of the medium. The higher the density, the greater the degree of retardation. Rays of light entering a sheet of glass at right angles are retarded in speed but their direction is unchanged (Fig. 3.3a). If the light enters the glass at any other angle, a deviation of direction will occur in addition to the retardation, and this is called refraction (Fig. 3.3b). A curved lens will exhibit both retardation and refraction (Fig. 3.3c), the extent of which is governed by:
Figure 3.3 (a) Rays passing from one medium to another, perpendicular to the interface, are slowed down at the same moment. (b) Rays passing at any other angle to the interface are slowed down in the order that they cross the interface and are deviated. (c) Rays passing through a curved lens exhibit both retardation and refraction.
The angle by which the rays are deviated within the glass or other transparent medium is called the angle of refraction and the ratio of the sine values of the angles of incidence (i) and refraction (r) gives a figure known as the refractive index (RI) of the medium (Fig. 3.4a). The greater the RI, the higher the density of the medium. The RI of most transparent substances is known and is of great value in the computation and design of lenses, microscope slides and coverslips, and mounting media. Air has a refractive index of 1.00, water 1.30, and glass a range of values depending on type but averaging 1.50.
Figure 3.4 (a) Angle of incidence (i) and refraction (r). (b) Ray C–D is lost through the edge of the lens. Ray E–F shows total internal reflection. (c) Parallel rays entering a curved lens are brought to a common focus.
As a general rule, light passing from one medium into another of higher density is refracted towards the normal, and when passing into a less dense medium it is refracted away from the normal. The angle of incidence may increase to the point where the light emerges parallel to the surface of the lens. Beyond this angle of incidence, total internal reflection will occur, and no light will pass through (Fig. 3.4b).
Parallel rays of light entering a simple lens are brought together by refraction to a single point, the ‘principal focus’ or focal point, where a clear image will be formed of an object (Fig. 3.4c). The distance between the optical center of the lens and the principal focus is the focal length. In addition to the principal focus, a lens also has other pairs of points, one either side of the lens, called conjugate foci, such that an object placed at one will form a clear image on a screen placed at the other. The conjugate foci vary in position, and as the object is moved nearer the lens the image will be formed further away, at a greater magnification, and inverted. This is the ‘real image’ and is that formed by the objective lens of the microscope (Fig. 3.5).
If the object is placed yet nearer the lens, within the principal focus, the image is formed on the same side as the object, is enlarged, the right way up, and cannot be projected onto a screen. This is the ‘virtual image’ (Fig. 3.6), and is that formed by the eyepiece of the microscope of the real image projected from the objective. This appears to be at a distance of approximately 25 cm from the eye – around the object stage level. Figure 3.7 illustrates the formation of both images in the upright compound microscope, as is commonly used in histopathology.
White light is composed of all spectral colors and, on passing through a simple lens, each wavelength will be refracted to a different extent, with blue being brought to a shorter focus than red. This lens defect is chromatic aberration (Fig. 3.8a) and results in an unsharp image with colored fringes. It is possible to construct compound lenses of different glass elements to correct this fault. An achromat is corrected for two colors, blue and red, producing a secondary spectrum of yellow/green, which in turn can be corrected by adding more lens components – the more expensive apochromat.
Microscope objectives of both achromatic and apochromatic types (see Fig. 3.11) are usually overcorrected for longitudinal chromatic aberration and must be combined with matched compensating eyepieces to form a good-quality image. This restriction on changing lens combinations is overcome by using chromatic, aberration-free (CF) optics, which correct for both longitudinal and lateral chromatic aberrations and remove all color fringes, being particularly useful for fluorescence and interference microscopes.
Other distortions in the image may be due to coma, astigmatism, curvature of field, and spherical aberration, and are due to lens shape and quality. Spherical aberration is caused when light rays entering a curved lens at its periphery are refracted more than those rays entering the center of the lens and are thus not brought to a common focus (Fig. 3.8b).
Light, of course, is an essential part of the system; at one time sunlight was the usual source. A progression of light sources has developed, from oil lamps to the low-voltage electric lamps of today. These operate via a transformer and can be adjusted to the intensity required. The larger instruments have their light sources built into them. Dispersal of heat, collection of the greatest amount of light, and direction and distance are all carefully calculated by the designer for greatest efficiency. To obtain a more balanced white light approximation, these light sources must often be operated at excessive brightness levels. The excess brightness is reduced to comfortable viewing levels through the use of neutral density filters.
Light from the lamp is directed into the first major optical component, the substage condenser, either directly or by a mirror or prism. The main purpose of the condenser is to focus or concentrate the available light into the plane of the object (Fig. 3.9). Within comfortable limits, the more light at the specimen, the better is the resolution of the image.
Many microscopes have condensers capable of vertical adjustment, in order to allow for varying heights or thickness of slides. Once the correct position of the condenser has been established, there is no reason to move it, as any alteration will change the light intensity and impair the resolution. In most cases condensers are provided with adjustment screws for centering the light path. Checking and, if necessary, adjusting the centration before using the instrument should be a routine procedure for every microscopist. All condensers have an aperture diaphragm with which the diameter of the light beam can be controlled.
Adjustment of this iris diaphragm will alter the size and volume of the cone of light focused on the object. If the diaphragm is closed too much, the image becomes too contrasty and refractile, whereas if the diaphragm is left wide open, the image will suffer from glare due to extraneous light interference. In both cases the resolution of the image is poor. The correct setting for the diaphragm is when the numerical aperture of the condenser is matched to the numerical aperture of the objective in use (Fig. 3.10) and the necessary adjustment should be made when changing from one objective to another. This is achieved, approximately, by removing the eyepiece, viewing the substage iris diaphragm in the back focal plane of the objective, and closing it down to two-thirds of the field of view.
With experience the correct setting can be estimated from the image quality. Under no circumstances should the iris diaphragm be closed to reduce the intensity of the light; use filters or the rheostat of the lamp transformer. Many condensers are fitted with a swing-out top lens. This is turned into the light path when the higher-power objectives are in use. It focuses the light into a field more suited to the smaller diameter of the objective front lens. Swing it out of the path with the lower power objectives, or the field of view will only be illuminated at the center. When using apochromatic or fluorite objectives the substage condenser should also be of a suitable quality, such as an aplanatic or a highly corrected achromatic condenser.
Above the condenser is the object stage, which is a rigid platform with an aperture through which the light may pass. The stage supports the glass slide bearing the specimen, and should therefore be sturdy and perpendicular to the optical path. In order to hold the slide firmly, and to allow the operator to move it easily and smoothly, a mechanical stage is either attached or built in. This allows controlled movement in two directions, and in most cases Vernier scales are incorporated to enable the operator to return to an exact location in the specimen at a later occasion.
Within the objective there may be from 5 to 15 lenses and elements, depending on image ratio, type and quality (Fig. 3.11). The main task of the objective is to collect the maximum amount of light possible from the object, unite it, and form a high-quality magnified real image, some distance above. Older microscopes used objectives computed for an optical tube length of 160 mm (DIN standard), or 170 mm (Leitz only), but these fixed tube length systems have now been largely replaced by infinity corrected objectives that can greatly extend this tube length and permit the addition of other devices into the optical path.
The ability of an objective to resolve detail is indicated by its numerical aperture and not by its magnifying power. The numerical aperture or NA is expressed as a value, and will be found engraved on the body of the objective. The value expresses the product of two factors and can be calculated from the formula:
where n is the refractive index of the medium between the coverglass over the object and the front lens of the objective, for example air, water, or immersion oil, and u is the angle included between the optical axis of the lens and the outermost ray that can enter the front lens (Fig. 3.12).
Figure 3.12 The refractive index of the medium between the coverglass and the surface of the objective’s front lens (in this case air, RI = 1.00), and the sine of the angle (u) between the optical axis and the outermost accepted ray (r), gives the numerical aperture (see text).
In Figure 3.12 the point where the axis meets the specimen is regarded as a light source; rays radiate from this point in all directions. Some will escape to the outside, and some will be reflected back from the surface of the coverglass. Ray r is the outermost ray that can enter the front lens; the angle u between ray r and the axis gives us the sin value required. In theory the greatest possible angle would be if the surface of the front lens coincided with the specimen, giving a value for u of 908. In the above formula, with air (RI = 1.00) as the medium, and a value for u of 908 (sin u × 1), the resulting NA = 1.00. Of course this is impossible as there must always be some space between the surfaces, so a value of 908 for u is unobtainable. In practice the maximum NA attainable with a dry objective is 0.95. Similar limitations apply to water and oil immersion objectives; theoretical maximum values for NA are 1.30 and 1.50, respectively. In practice values of 1.20 and 1.40 are the highest obtainable.
The resolving power of the objective is its ability to resolve the detail that can be measured. In summary, as the NA of an objective increases, the resolving power increases but working distance, flatness of field, and focal length decrease.
Objectives are available in varying quality and types (Fig. 3.11). The achromatic is the most widely used for routine purposes; the more highly corrected apochromats, often incorporating fluorite glass, are used for more critical work, while plan-apochromats (which have a field of view that is almost perfectly flat) are recommended for photomicrography. For cytology screening, flat-field objectives – often plan-achromats – are particularly useful. On modern microscopes, up to six objectives are mounted onto a revolving nosepiece to enable rapid change from one to another and, ideally, the focus and field location should require the minimum of adjustment. Such lenses are said to be par-focal and par-central.
Most objectives are designed for use with a coverglass protecting the object. If so, a value giving the correct coverglass thickness should be found engraved on the objective. Usually this is 0.17 mm. Some objectives, notably apochromats between 40 : 1 and 63 : 1, require coverslip thickness to be precise. Some are mounted in a correction mount and can be adjusted to suit the actual thickness of the coverglass used.
Above the nosepiece is the body tube. Three main forms are available: monocular, binocular, and the combined photo-binocular. The last sometimes has a prism system allowing 100% of the light to go either to the observation eyepieces, or to the camera located on the vertical part, and sometimes has a beam-splitting prism dividing the light, 20% to the eyes and 80% to the camera. This facilitates continuous observation during photography. Provision is made in binocular tubes for the adjustment of the interpupillary distance, enabling each observer to adjust for the individual facial proportions. Alteration of this interpupillary distance may alter the mechanical tube length, and thus the length of the optical path. This can be corrected either by adjusting the individual eyepiece tubes, or by a compensating mechanism built into the body tube.
Modern design tends towards shortening the physical lengths of the components, and in consequence, the intermediate optics are sometimes included in the optical path to compensate. These lenses are mounted on a rotating turret and are designated by their magnification factor. Additionally, a tube lens may be incorporated for objectives that are infinity corrected, as these objectives form only a virtual image of the object, which must be converted to a real image focused at the lower focal plane of the eyepiece.
Eyepieces are the final stage in the optical path of the microscope. Their function is to magnify the image formed by the objective within the body tube, and present the eye with a virtual image, apparently in the plane of the object being observed; usually this is an optical distance of 250 mm from the eye.
Early types of eyepiece, like objectives, were subject to aberrations, especially of color. Compensating eyepieces were designed to overcome these problems and can be used with all modern objectives. The eyepiece designed by Huyghens in 1690 is still available, together with periplanatic (flat-field) and wide-field types, and eyepieces for holding measuring graticules and photographic formats. High focal point eyepieces are designed for spectacle wearers. For older fixed tube length microscopes, manufacturers often placed different amounts of the various corrections in the optical train in either the objective or the eyepiece. Therefore it is important to use eyepieces from the same manufacturer with objectives from that manufacturer. Eyepieces designed for infinity objectives must be used with the newer infinity-corrected systems.