Perhaps the most critical aspect of observation, which applies to all forms of optical microscopy, is the method of specimen illumination and its effectiveness in revealing the features of interest. Stereomicroscopes are often utilized to examine specimens under both reflected (episcopic) and transmitted (diascopic) illumination schemes, employing a variety of light sources and configurations, which are strategically positioned in the appropriate locations.
In many circumstances, reflected and transmitted light sources are combined to take advantage of particular specimen characteristics in a manner that most effectively reveals the features of interest. This review focuses on the wide variety of techniques and equipment currently in use to illuminate a multitude of specimens observed with reflected light techniques. Many of the specimens examined with stereomicroscopes are three-dimensional, and require a significant degree of creativity on the part of the microscopist to most effectively illuminate the specific details of interest. Presented in Figure 1 is a modern stereomicroscope (Nikon SMZ1500) equipped with several of the common reflected light illuminators that are available for these instruments. Included in the configuration are a ring light, coaxial illuminator, and bifurcated fiber optics light pipe, which represent three of the most useful and versatile reflected light illumination sources for stereomicroscopy.
A number of similarities exist between the challenges faced in illumination for stereomicroscopy and those encountered in close-up or macro photography using conventional camera and lens combinations. The lower magnifications utilized with stereomicroscopy overlaps the reproduction ratios possible using traditional camera lenses coupled to extension devices, or specialized macro lenses, and many objects can be effectively imaged with either type of equipment. Many of the illumination techniques that have proven useful in photomacrography can be applied with the stereomicroscope, and vice versa.
Stereomicroscopy techniques commonly vary a great deal from those developed for "standard" compound microscopes employed in conventional optical microscopy. This is particularly true with regard to many of the illumination strategies. For a majority of the image contrast-enhancing methods utilized with the compound microscope, the primary optical configuration and illumination strategy, based on Köhler principles, remains the same. This fundamental illumination scheme is modified for the various contrast-enhancing methods by the addition of auxiliary filters and other optical components, such as Nomarski or Wollaston prisms for differential interference contrast (DIC), a polarizer and analyzer (often with a quarter-wavelength or full-wave retardation plate) for polarized light techniques, phase plates for phase contrast, and interference filters for fluorescence excitation and emission. In the stereomicroscope, with its much longer working distance, smaller numerical apertures, and lower magnifications, many of these techniques are not applicable.
There exists no single optimum illumination strategy that is the correct choice for the wide variety of specimens that the stereomicroscope is designed to accommodate. Each specimen under examination can be illuminated by a variety of different mechanisms, and employing a nearly infinite number of variations or combinations of techniques. For a given specimen or object, although there may be several possible illumination schemes that produce acceptable results, a single approach may be discovered that, after careful refinement, produces exceptional results.
Choice of Illumination Strategy
Specimen characteristics should be carefully considered in selecting an illumination strategy to suit the needs of visual observation, photomicrography, or digital imaging. The opacity of the specimen is, in general, the most important characteristic, and will determine the basic type of illuminator that should be employed-reflected (episcopic), transmitted (diascopic), or in some cases, a combination of both. Opaque specimens are typically illuminated from above (with reflected light), using orientations ranging from on-axis (parallel to the microscope optics) to highly oblique (up to a 90-degree incident angle from the optical axis), as required to reveal the features or characteristics of interest.
Once it has been determined that the specimen's opacity suggests the use of a particular general category of illuminator, then a number of other factors should be considered to further refine particular variations on the basic illumination scenario that will likely produce the desired results. Figure 2 illustrates a variety of potential pathways for specimen illumination using reflected light. A simple tungsten (or tungsten-halogen) illuminator, shown oriented at different angles to the specimen surface, and a ring light mounted on the objective lens body, provide lighting that is independent of the microscope optical path. The illumination path for a coaxial illuminator, which functions within the microscope optical train, is illustrated in the cutaway section of the instrument.
Opaque specimens most commonly benefit from reflected illumination, while translucent and transparent objects usually produce the best results with some variation of transmitted illumination (brightfield, polarized, oblique, or darkfield). This is not invariably true, however, and translucent objects may benefit from having at least a portion of their illumination directed from a source placed above them. Aside from opacity, a number of other factors should be considered in planning a lighting strategy. These include the basic physical characteristics of the specimen, the type of information that is required from the examination, digital or photographic imaging requirements, and how the information will be utilized.
The geometrical profile, topography, and morphology of the specimen are all important factors in choosing and configuring illumination in a manner that will reveal the desired information. Specimens that are highly three-dimensional (have high relief) should be illuminated differently than those that are flat, smooth, or even highly polished. For example, highly angular lighting can produce shadows on rough surfaces, obscuring surface detail that may be important. Highly diffuse light originating from directly above a rough-surfaced specimen may uniformly illuminate "peaks" and "valleys", but if textural information is required to characterize the object as to smoothness, flatness, or other topographic variables, this type of lighting may not be optimum. A large spectrum of other factors influence how the illumination interacts with the specimen, and a number of these are discussed in more detail in the following sections.
Among other specimen characteristics that influence the choice of an appropriate lighting scheme, the composition is crucial, and directly affects both the surface and internal reflectivity. Metals, plastics, ceramics, glasses, and natural materials, such as minerals or gems, all behave differently with regard to their appearance under different lighting conditions. Some specimens may have specific environmental requirements that affect their suitability for illumination with various source types. For example, living aquatic organisms may require immersion in water during observation. Metallic objects often are best studied while covered with oil or another protective coating, or may be highly polished. Such specimens may produce artifacts by reflecting images of the light source (or sources) into the microscope objective. These reflections usually produce glare and obscure important detail, or distract attention from the important elements that are being observed and imaged. The most difficult specimens may even require a special lighting technique just to be rendered visible.
Another important factor that must be considered, in many cases, is whether the specimen material is sensitive to heat or ultraviolet light, both of which are significant emission components of some illumination sources. Light sensitivity may require limiting the amount of time that the specimen is illuminated. When the time available for observation is limited, the choices of possible illumination techniques become far more restricted. A similar problem occurs if a specimen is being studied for observation or recording of a transient, or short-lived, phenomenon or property, in which case the intensity of the illumination may become the primary factor in choosing a lighting strategy.
The purpose of the microscope examination, or the specific kind of information that is required from the specimen being studied, will often strongly influence the strategy that is chosen for illumination. It may be necessary to enlist a variety of schemes to reveal minute details, larger features, or gross characteristics. Depending upon the information that must be obtained from a given specimen, the illumination technology employed can be extremely simple, or much more elaborate, and may require a combination of techniques. For example, if the only important property of a specimen is its color, then the lighting employed can be very simple physically, and is only required to provide accurate color rendition. If both color determination and fracture pattern analysis are important, then more attention must be paid to lighting geometry so that all features of interest are revealed.
Photographic or digital imaging requirements are another important factor that must be taken into consideration in choosing specimen illumination. If a traditional film camera is employed to record images, the color temperature (and possibly other spectral characteristics) of the light source must be appropriate for the film used, in order for the specimen to be accurately represented. The intensity of the lighting must also be adequate to ensure exposures that are of reasonable duration for the camera/film combination employed. This is especially important in manufacturing, industrial, or clinical laboratory settings. Digital image capture systems require many of the same considerations as film systems, although white balance adjustment on the imaging device (digital camera) allows considerable latitude in matching camera response to the color characteristics of various light sources. If video recording is to be conducted, illumination intensity may be an even greater issue.
Effective matching of the microscope and specimen with an illumination system often depends to a large degree upon the skill and training of the operators that will use the system, and the setting or type of environment in which it will be employed. Many illumination systems that have the flexibility to be adapted to a wide range of applications require skilled operators with considerable training and expertise. In a manufacturing or industrial setting, where relatively unskilled operators may utilize microscopes for assembly or production inspection during long work shifts, the simpler lighting systems that are preset to a fixed configuration are preferable. This simplicity will present fewer variables in operation, and more consistency from operator to operator, and from shift to shift. This strategy is only possible, however, in situations where there is considerable uniformity in the objects or specimens that are being examined. Any unique or unusual lighting situation will probably require a more flexible lighting system, and a more skilled technician.
Another demand on lighting systems designed to be utilized in any setting where repetitive operations must be efficiently conducted, is the ergonomic characteristics of the combined microscope and illumination system. Comfort and ease of use is undoubtedly important in any serious application of microscopy, although the work environment that perhaps best validates this concern is the clinical laboratory. In clinical laboratory situations, a fatiguing or difficult-to-use lighting configuration can reduce the accuracy of critical specimen analyses, even when conducted by a skilled microscopist.
General Factors Influencing Illumination Characteristics
The lighting angle (or angles) from which episcopic illumination is directed dramatically influences the appearance of the specimen being studied. No single angle is "correct" for all objects being illuminated, and the best light source positions are usually determined empirically by experimentation. Varying the angle at which the light strikes the specimen, with respect to the direction of observation (or optical axis), will produce marked differences in the features or characteristics that are emphasized.
The nature of the specimen being studied will determine the lighting angle that best reveals the desired properties. In lighting a specimen whose surface is roughly textured, a small adjustment of the lighting angle from on-axis (vertical) to slightly oblique can produce strong emphasis of the surface texture. In contrast, a surface that is nearly flat, with fine detail such as small scratches, may not display a significant effect from illumination angle variations until the light source is highly oblique. Moving a light almost 90 degrees off-axis, so that the wavefronts are just glancing the specimen surface, is sometimes beneficial in revealing fine surface detail or features that are not visible when the light strikes the specimen from a more direct angle. If more than one light source is employed, they may be positioned at different angles in order to combine the effects of oblique and direct illumination. There are no rules that can predict the effects of lighting angle for all specimens, and controlled experimentation is probably the best approach to develop a lighting scheme for a given requirement.
The size of the light source, in comparison to the field area being illuminated, strongly influences the overall lighting effect. In a general sense, a light source that is small can be considered more directional, with greater coherency, and can produce higher contrast images with bright highlights, dark shadows, and sharp, well-defined edges. A larger light source will generally provide lighting that is less directional, resulting in images having lower contrast between light and dark areas. In addition, these images will have shadowed regions that are not very dark, with softer edges delineating areas of unequal brightness.
Light sources can, by their design, be of a specular or diffuse nature, although this characteristic is interconnected with the illuminator size and its distance from the specimen. Illuminators that include lenses can be focused into more coherent, tighter beams, which produce specular (or harder) illumination. Other sources (for example, fluorescent ring lights) produce an even, more diffuse, softer illumination, partially because of the nature of the lamp itself, and partially due to the ring light position on the microscope objective. Figure 3 presents examples of the contrast in specimen appearance resulting from illumination by a small specular source (fiber optic light pipe) and by a relatively large fluorescent ring light. Diffusion accessories are available to modify the output of specular light sources, but they are unlikely to have the desired effect on a small source unless the diffuser is large, relative to the specimen that is being illuminated. A considerable amount of confusion exists regarding these variables in light source design and implementation, but the most important factor to consider is the directionality of illumination with respect to the specimen. Directionality depends not only on the light source design, but also on its size and distance from the specimen.
As previously discussed, the utilization of multiple light sources provides additional flexibility in achieving the desired illumination effect for a wide variety of specimens. One lighting arrangement that exemplifies the multiple-source strategy is to position one light source at a low angle to the specimen surface (highly oblique lighting) to emphasize topography and surface texture, and another light closer to the optical axis to partially illuminate the shadows and reveal some of the detail in those areas. In the terminology of general photography, these illumination sources would be referred to as the main (oblique) and fill (on-axis) lights. Balancing the relative intensity of the two lights (or the lighting ratio) will usually require some experimentation to achieve the optimum effect.
Another important consideration, when developing a reflected lighting strategy for stereomicroscopy, is the working distance of the microscope objective, which can seriously restrict the flexibility in positioning reflected illuminators. This distance is measured between the objective lens and the specimen, and covers a range of several centimeters (for lower aperture and magnification objectives) to only a few millimeters for the highest numerical aperture objectives. In the familiar studio setting in general photography, the photographer has a significant amount of latitude in placing lights in nearly any arrangement that is necessary to achieve the desired lighting effect. In contrast, the size of the "studio" under a stereomicroscope objective may measure only a few centimeters or millimeters, and impose severe limitations on the choices in lighting scheme.
A small working space not only restricts the type of illuminator that may be used, but also the range of angles from which light can "reach" the specimen field. The limited area between the objective front lens and specimen may force placement of illuminators farther off-axis than desired, and often prevents the elimination of shadows on rough-surfaced specimens. Figure 4 illustrates a situation in which the short objective working distance restricts illumination to a highly oblique angle, and prevents even illumination from being achieved. An effective method of providing more effective illumination, in this circumstance, is to place small mirrors or other reflective surfaces on the side of the specimen opposite the light source. The simple illuminator type presented in Figure 4 is capable of providing adequate illumination at a longer working distance than shown in the illustration. However, at shorter working distances, the microscope objective physically obstructs the full illumination of the specimen when the light source is positioned at a smaller angle, closer to the microscope optical axis. Depending upon the working distance available with the instrument configuration, multiple lights and reflectors can be employed, and their relative distances and angular positions varied to achieve the required proportions of direct and indirect (reflected) illumination.
In situations where on-axis lighting is necessary, ring lights or coaxial illuminators may be a possible solution, but these sources, too, have optimum working distances and angles. At extremely long microscope working distances, ring-style lighting may become too dispersed and provide insufficient intensity. In contrast, at very short working distances, the specimen will lie in the darker central region of the light cone, and will be unevenly illuminated. The optimum working range for a ring light illuminator is presented in Figure 5. Note that the cone of illumination is well defined with this type of illumination source.
In stereomicroscopy, the angle of view for the two eyes is slightly different, each being oriented at an angle of 5 to 7 degrees with respect to the microscope optical axis. The difference in viewing angle for the two eyes is the primary factor that enables the brain to create the perception of a three-dimensional image. Because the light reflection angle from the specimen surface equals the incident angle for illuminating light rays, reflections observed with one eye may appear differently to the other eye. Furthermore, it is important to keep in mind that when images are being recorded, light passing to the camera system travels only through a single channel in the microscope, producing a slightly off-axis view of the specimen. This factor may affect illumination effects and must be evaluated and compared with respect to the appearance of the specimen through the eyepieces.
Another factor that may govern illuminator placement, and which consequently influences the strategy chosen to meet lighting requirements, is that the tungsten or tungsten-halogen lamps provided in many microscope illuminators produce a considerable amount of infrared radiation. This invisible radiation can result in considerable heat gain at the specimen plane that may not be tolerated by living organisms, and which can possibly deform, or even melt, some materials. When heat sensitive specimens are being studied, positioning the lamps farther away is one strategy to reduce the heat input. If repositioning the lamps is not an adequate solution, or is not an option, utilization of lighting components that are designed to minimize infrared radiation should be considered.
Specimen heating is reduced by the nature of some illuminator designs, such as fiber optic devices, by virtue of the physical placement of the lamp itself at some distance from the point at which the light is output. Considerable heat may still be delivered at the luminous fiber end, however. As a further measure to reduce the problem, many illuminators have infrared-cut filters (also called heat filters or hot mirrors) to attenuate infrared transmission. Alternatively, light sources may have projector type lamps incorporating dichromatic reflectors (termed cold mirrors) that reflect visible light for illumination, while allowing infrared to pass through the reflector and away from the light path.
Lighting Components for Stereomicroscopes
Ambient light conditions in the laboratory may be sufficient for observation when very low magnifications are employed (1-3x) in the stereomicroscope, and may be considered as the most basic lighting system. The primary drawback in using room lighting for microscope illumination is the lack of control over the intensity, position, and color temperature of the light, and it is probably unrealistic to rely on this light source for any serious application.
A majority of stereomicroscope manufacturers offer at least one basic incandescent (tungsten or tungsten-halogen) illuminator that can be mounted directly on the focusing stand, or that is secured by a flexible arm enabling convenient attachment to the stand. Several varieties of these simple illuminators are illustrated in Figure 6. Typically, small incandescent illuminators employ 10 or 20-watt tungsten or quartz-halogen lamps that provide an adequate amount of light for viewing a wide variety of specimens. More advanced stereomicroscope stands are equipped with a housing for a built-in reflected light source, which provides similar illumination with improved convenience.
Incandescent illuminators are usually inexpensive, require little space, and are very easy to configure. Their main disadvantage is the limited amount of light that is available from the low power lamps, which often is insufficient to properly illuminate all necessary areas of the specimen, especially when photomicrography, digital, or video imaging is required. A secondary problem is the highly directional, and somewhat specular, nature of the light produced by these illuminators, which can result in undesirable shadows. Incandescent illuminators can be used in conjunction with mirrors or diffusers to modify the beam spreading characteristics to some degree, although intensity limitations and small area covered cannot be completely overcome. When this type of light source is placed in close proximity to the specimen, the thermal energy delivered to the illuminated area can be too great for some heat-sensitive materials. In general, however, simple incandescent light sources are durable, practical, and are ideal for student microscopes, for transportation to and use in the field, or for simple industrial inspection or assembly.
Of all the illumination sources available for stereomicroscopy, fiber optic illuminators are probably the most versatile and popular. Many different light source designs, fiber types and configurations, and accessory attachments are available. A fiber optic light system can be configured to meet the stringent requirements of almost any application. Generally powered by high-intensity tungsten-halogen lamps, fiber optic illuminators are relatively bright sources, and by utilization of appropriate filters, can be color-balanced for video or still image recording. Configured as cold light sources (through the addition of infrared filters), fiber optic systems are much more suitable for investigations of heat-sensitive specimens than are basic incandescent illuminators.
The fiber optic ring light is one of the most widely used configurations among the fiber optic-based illuminators. A fixed means of attachment, surrounding the microscope objective, eliminates any variables in adjustment, and ensures that the illumination is of consistent quality, and highly reproducible, from specimen to specimen. Because the illumination path is nearly coincident with the optical axis of the microscope, the viewing area is evenly illuminated and nearly shadowless. These characteristics can be beneficial, but are not well suited to textural investigation, where more directional lighting is advantageous. However, ring lights are very commonly employed for electronic assembly and quality control applications, including solder joint inspection on printed circuit boards having attached components, which can cast shadows in other types of illumination. The diffuse illumination provided by ring lights, directed nearly on-axis, eliminates the shadows, while still providing adequate contrast for visual inspection.
Other common applications for ring light sources include animal surgery and the study of anatomical specimens. The illumination provided by ring lights is adequate for most opaque objects, but is not the preferred technique for observing many specimens, especially for image recording purposes. The fiber bundle ring units are available in different sizes, and with a variety of accessory attachments, such as diffusers, polarizers, and toric lenses, which serve to modify the light distribution. A fiber optic ring light (with a partial cutaway revealing a portion of the assembly construction detail) is illustrated in Figure 7, mounted on the objective lens of a common main objective (CMO) stereomicroscope.
If a particular specimen requires greater flexibility in varying the angle and direction of illumination, or more control of image contrast than a fixed ring light provides, a possible solution is the use of flexible light guides coupled with a tungsten-halogen light source. These guides are available as either a single light pipe or in double or triple units, such as the bifurcated light pipe (one light input into two outputs; Figure 8). A variety of light guides and attachments are illustrated in Figure 8, including a fiber optic ring light. Several light pipe designs offer significant flexibility, enhancing their utility for illuminating difficult-to-reach areas, such as those occurring in some machine installations. These light pipes must be clamped or loosely attached to remain in place, however, and they are not as popular for microscope use as are the semi-rigid designs.
A semi-rigid light guide maintains its shape and positioning without clamping, and can be employed in conjunction with the light source base as a stand-alone unit. In general, light pipes provide simple control of illumination, since they are easily positioned, and filters can be added to the light source for color balancing, heat reduction, polarization, and other purposes. Focusing lenses for light pipes are available that concentrate the illumination into a smaller beam, increasing intensity and resulting in shorter exposure times during image recording, or less noise in video recordings.
Fiber optic light pipe sources are specular (especially with focusing lenses) and directional, and may produce uneven illumination, which requires them to be carefully positioned to avoid undesirable shadowing effects in the illuminated area. By adding one or more additional light pipes, such as with a dual (bifurcated) pipe system, two fiber optic sources can be employed as main and fill lights to eliminate shadows and generally provide more even illumination. Alternatively, light pipes can be directed independently to selectively illuminate different areas for the purpose of emphasizing desired features. Using multiple light pipes provides one technique for achieving more even illumination, while retaining the specular, higher contrast appearance that is sometimes desirable, and which could not be obtained with sources that are more diffuse. Light pipes are very popular illumination sources for many stereomicroscopy applications, including integrated circuit and other electronic parts inspection operations, dissection tasks in biology, jewelry assembly and repair, and materials failure analysis.
In order to provide diffuse shadow-less illumination, the fluorescent ring light is probably without equal. Similar in many characteristics to fiber optic ring lights, these sources incorporate a ring-shaped fluorescent tube as a large, diffuse, nearly-on-axis light source that produces relatively low contrast images. The primary applications for fluorescent ring lights are electronics assembly and industrial inspection tasks, where the ease of use, low heat output, even illumination, and consistent color temperature are ideal. Fluorescent lamp tube life is very long, and may extend for years before replacement is required. There are several disadvantages to these lamps, however, that make fluorescent illuminators more suitable for visual inspection than for image recording. Some models exhibit a high frequency flicker that, while unnoticeable to the eye, can produce artifacts in video images through the rapid intensity fluctuations. In addition, the light emission spectrum produced by fluorescent lamps exhibits a sharp peak in the green wavelength region, and in some cases they exhibit spectral discontinuities, factors that complicate matching these light sources to the response of color film.
Light sources designed to place the incident light path as close as possible to the optical axis, but not on-axis, are classified as near-vertical illuminators. In stereomicroscopes of the Greenough design, a mirror is located directly between the two eye paths at the base of the microscope body, and directs light from the source downward, nearly vertically, to the specimen surface.
In the common main objective (CMO) design, a mirror is placed between the objective and the zoom body (the same distance off-center as the two eye paths), so that the three optical paths are coincident at the plane of the specimen. In this design, the objective assists in concentrating the light, in addition to its image-forming function. Figure 9 illustrates illumination and imaging optical pathways for the two (Greenough and CMO) stereomicroscope designs.
Vertical illuminators provide true on-axis illumination by the addition of a half-reflecting surface, which is placed beneath the microscope objective at a 45-degree angle to the optical axis. The reflector directs light, from an illuminator placed at right angles to the optical axis, downward toward the specimen, while allowing light reflected from the specimen to pass back through the microscope optical system. In the stereomicroscope, half-reflecting mirrors are commonly employed to perform the beam-splitting function. Illuminators made for Greenough microscopes must be designed to accommodate each eye path (at specific angles to each other), and may incorporate angled optical elements to satisfy this requirement. For a single light path, such as that utilized in photomacrography, the reflector can be simply a thin piece of glass.
Vertical illuminators may incorporate either condensing lenses or diffusers between the light source and the half-reflecting mirror. In a condenser system, the rays from the light source are focused in a fashion similar to reflected light Köhler illumination. The illuminating rays converge, after being reflected from the beam-splitting mirror, at the exit pupil (rear aperture) of the objective lens. This type of system maximizes the effective numerical aperture of the illumination path, producing images with relatively high contrast, superior resolution, and good rendition of minute surface detail.
Systems designed for vertical illumination that place a diffusing element (rather than a condenser) in the light path before the mirror, generally feature a lower illumination numerical aperture. These designs are easier to align, however, and produce lower contrast images, with fewer shadows. Small surface details are not as well resolved as with the condenser systems, although this type of diffuse axial illumination is ideal for many tasks that require specular surfaces to be evaluated. Among these applications are inspection of CD-ROM surfaces and silicon wafers, character reading on small parts, and solder pad imaging and component inspection on printed circuit boards, in addition to the study of biological and medical specimens. Vertical illuminators can be configured so that simple illuminators or fiber optic systems can be employed as the light source. Custom illuminators are often coupled to fiber optic guides of specific dimensions, or to multi-branch fiber guides designed as an accessory for a particular illuminator.
Coaxial illuminators are similar (conceptually) to on-axis vertical illuminators, and produce comparable results in specimen lighting characteristics. A major difference, however, is that the illumination path for coaxial illumination lies within the optical system of the microscope, instead of between the microscope and specimen. The technique can be described as through the lens illumination, as the primary image forming optical train of the stereomicroscope acts as its own condenser, in a manner similar to the function of classical metallurgical microscopes. The primary advantage of this technology is that the illumination system numerical aperture is altered in concert with that of the objective. As magnification is increased in the zoom body of the stereomicroscope, the numerical aperture also increases, for both the image-forming and illumination pathways. This manifestation offsets the loss in image intensity with increased magnification that is characteristic of other lighting techniques, such as simple vertical illumination. Consequently, the field of view through the eyepieces is equally bright throughout the magnification range of the zoom optical system.
The coaxial illuminator is positioned (as illustrated in Figure 1) in the stereomicroscope above the zoom body, and below the binocular tube and auxiliary beam splitters employed for photographic equipment adapters. Figure 10 presents a cutaway illustration of a typical coaxial illuminator and the microscope zoom body with the other components removed for clarity. Light is directed through two independent pathways and lens systems (for the right and left eyes) in the zoom body by the placement of half-reflecting mirrors. Polarizing components are utilized to eliminate internal reflections from optical elements, and other sources of glare, that would lower the image contrast. The primary polarizers are placed between the light source and mirrors to polarize light entering the zoom body. Analyzers (or secondary polarizers) positioned above the half-reflecting mirrors eliminate undesirable reflections before they reach the eyepieces. In order to allow image-forming light reflected from the specimen to pass through the upper polarizers to the eyepieces or camera attachment, a quarter-wave retardation plate, which functions as a de-polarizer, is mounted over the front lens element of the common main objective. In use, the retardation plate can be rotated to an angular position to ensure the brightness and contrast of the image is optimized for the specimen being studied.
Target applications for microscopes equipped with coaxial illuminators are the same as for the vertical illuminator counterparts, and include inspection of integrated circuits and semiconductor wafers, metals and materials analysis, and any task requiring even illumination of polished surfaces. The on-axis light produced is not ideal for rough surfaces or surfaces that are not positioned at right angles to the optical axis. Surfaces oriented at right angles to the illumination axis appear bright in the image, while other orientations appear dark because light is reflected away from the imaging path. This characteristic of the coaxial illumination technique enables beneficial application to defect analysis of polished or honed surfaces.
A significant limitation of coaxial illuminators is a restriction on the lowest microscope magnification that can be employed. For magnifications in which the view field size approaches the diameter of the effective objective aperture, the illumination reflected from the field edges may not be able to enter the zoom body. As a consequence, illumination will dramatically decrease ("fall off") in intensity at the edges of the image. In addition, other image problems related to this limitation may result, and depending upon the microscope characteristics, body magnifications below about 2x to 3x may not be possible. Another consideration in the utilization of coaxial illuminators is that the illuminator module itself may add a magnification factor (of perhaps 1.5x) that is multiplied by the total basic microscope magnification, further limiting the maximum useable viewfield diameter.
Light Emitting Diode Illuminators
One of the newest technologies for illumination in microscopy, particular with application to stereomicroscopy, is based on the white light emitting diode (LED). A relatively recent technological development, diode sources that emit white light have gained acceptance in machine vision applications, and are being increasingly applied in microscopy. Several companies now market ring lights incorporating white LEDs in different variations, including spot-focused, diffuse, and versions optimized for shorter or longer than normal working distances. Other LED-based illuminator configurations are available, and include spotlights, backlight panels, linear arrays, and diffuse axial illuminators. Figure 11 illustrates the construction details of a ring illuminator incorporating an array of light emitting diodes.
Light emitting diodes have the advantage of being a cold light source, and most designs have a constant spectral output over their extremely long life spans. Suppliers of the illuminators rate the LEDs for 40,000 hours or more, with possible lifetimes of over 100,000 hours (compared to a halogen lamp's typical life of approximately 1000 hours). Because of the long life exhibited by these sources, they essentially never require replacement, and manufacturers have the option of sealing the light source and associated optics. This can be a significant advantage in many applications, because of time saved in disassembling illuminators for lamp replacement, and in the often-tedious realignment of microscope components following maintenance.
A significant problem with the current white light emitting diodes is their relatively low intensity, and this may limit their application to lower magnifications in the stereomicroscope if direct visual examination is necessary. For documentation on photographic film or by digital capture, the low intensity can be compensated, to some degree, by increased exposure times. Another disadvantage of currently available white diode light sources is that the output color temperature cannot be easily filtered to modify spectral characteristics. This effect occurs because many LEDs do not produce a true red-green-blue output that can be shaped in a straightforward manner by filtration.
Light emitting diodes are inherently monochromatic devices, with the color being determined by the band gap of the semiconductor material utilized in their construction. Following the early red-emitting devices, materials were developed that enabled the production of orange, yellow, and green LEDs. However, it was not until the recent development of semiconductor materials producing high-brightness blue and ultraviolet wavelengths that it became technologically feasible to produce solid-state white light. Most white light LEDs are fabricated from gallium nitride blue-emitting semiconductor dies surrounded by a phosphorescent material, which emits a range of longer visible wavelengths when excited by the blue light. The phosphor emission is dominated by yellow light, which combines with the complementary blue color through additive mixing to produce the appearance of white. Other techniques employed to produce the apparent white output include the mixing of colors from two sharply monochromatic complementary sources (dichromatic LEDs), or three monochromatic sources (trichromatic LEDs) in the proper ratio to achieve the perception of white. The combination of wavelengths can produce "white" light having a relatively high color temperature, which is in the range suitable for optical microscopy applications.
Another methodology for achieving white emission, which is similar to the mechanism of fluorescent light tubes, employs a phosphor that emits over a wide range of visible wavelengths to produce the broad spectral output of white light. This type of LED usually relies upon a semiconductor material that emits in the ultraviolet to excite the phosphor, and the entire visible output from the device is the result of the secondary phosphor emission. LED illuminators are available that are reported to exhibit daylight color temperature (approximately 5,500 K), but several of their other spectral characteristics may not be easily matched to the response of photographic film. As a result, these LEDs may be more appropriate for use with digital camera systems. This is especially true for the dichromatic and trichromatic devices, which produce light that appears to be white, but has spectral characteristics that are not suitable for all applications.
Among the numerous advantages of solid-state illumination sources is relatively low power consumption requirements, enabling these devices to be operated on battery power for reasonable periods. This benefit dramatically enhances the utility of LED-powered microscopes in field applications. Typically, LED illuminators are operated with 1 to 3-volt power supplies at 10 to 100 milliamperes. Ring light illuminators utilizing LEDs should exhibit the same general behavior as fiber optic and other ring lights, and their many advantages would seem to give them great potential in microscopy applications, especially as they are improved over time. Alternative illuminator configurations utilizing LEDs, as they evolve in development, should have nearly unlimited potential in their flexibility for use with the stereomicroscope.
Paul E. Nothnagle - Avimo Precision Instruments, 78 Schuyler Baldwin Drive, Fairport, New York, 14450.
William Chambers - Microscopy Consultant, Nikon Instruments Inc., Melville, New York 11747.
Thomas J. Fellers and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.