Introduction to Stereomicroscopy
The first stereoscopic-style microscope having twin eyepieces and matching objectives was designed and built by Cherubin d'Orleans in 1671, but the instrument was actually a pseudostereoscopic system that achieved image erection only by the application of supplemental lenses.
A major drawback of the d'Orleans design was that the left-side image was projected to the right eyepiece and the right-side image project to the left eyepiece. It wasn't until over 150 years later when Sir Charles Wheatstone wrote a treatise on binocular vision that enough interest was stimulated in stereomicroscopy to provide the impetus for further work.
During the mid-nineteenth century, Francis Herbert Wenham of London designed the first truly successful stereomicroscope. Wenham incorporated a novel approach by utilizing an achromatic prism to split the light beam at the rear of a single objective. A few years later, John Ware Stephenson produced a similar instrument (see Figure 1). The Wenham binocular, as the microscope design became known, suffered from artifacts brought about by the single lens and did not actually produce a true stereoscopic effect.
In the early 1890's, Horatio S. Greenough, an American instrument designer, introduced a novel design that was to become the forefather of modern stereomicroscopes. Greenough convinced the Carl Zeiss Company of Jena to produce the microscope, but instead of incorporating Greenough's lens erecting system, Zeiss engineers designed inverting prisms to produce an erect image. This design has withstood the test of time (and a large number of microscopists), and was a workhorse in medical and biological dissection throughout the twentieth century. The microscope is still a favorite for many specific applications.
Stereomicroscopes manufactured during the first half of the twentieth century, or dissection microscopes as they were called, were much like traditional compound microscopes of the era. They were heavy, constructed mainly from brass, utilized prisms for image erection, and had simple lens systems consisting of one or two doublets. The working distance was inversely proportional to the magnification, and was quite short at the highest available magnifications. These microscopes were employed primarily for dissection, because there were very few industrial applications involving small assemblies that required a microscope for examination. Even watchmakers used monocular loupes!
The first modern stereomicroscope was introduced in the United States by the American Optical Company in 1957. Named the Cycloptic®, this breakthrough design featured a die-cast aluminum housing, a constant working distance (that, at four inches, was the one of the longest produced), and an internal magnification changer, which allowed the observer to increase the objective magnification from 0.7x to 2.5x in five steps. In addition, the microscope utilized one-piece glass erecting prisms, was equipped with a variety of accessories including stands, arms, and illuminators, and conformed to 1950's styling with a two-tone gray paint scheme (see Figure 2). The microscope's name was derived from a single large central objective at the bottom of the body through which both the left and right channel accumulated light from the specimen.
In later microscopes, the Cycloptic feature was renamed Common Main Objective (CMO). This design uses a single large objective lens which, when focused on the specimen, forms an image at infinity. The Cycloptic, unlike most of the early stereomicroscope designs, had a threaded mount in the lower microscope body to secure the objective into position just beneath a rotatable drum containing two pairs of afocal Galilean-style telescopes. As the drum rotated, the telescope lenses were used in both forward and reversed orientations (magnifying and minifying), to yield four different magnifications. The fifth magnification resulted from an open channel with no glass. Galilean lens systems have the advantage of a small focal length, a very small field diameter, and seldom have magnifications exceeding 2x or 3x. A 2x Galilean lens will provide either 2x or 1/2x magnification, depending upon orientation, and matched pairs can be arranged to produce many variations. The Cycloptic's head contained what is now known as tube lenses, erecting prisms, and a pair of eyepieces. This microscope quickly became popular with early semiconductor manufacturers, most notably Western Electric.
Two years later (in 1959), Bausch & Lomb introduced a stereomicroscope to compete with the Cycloptic, but with a cutting-edge advance: continuously variable, or zoom, magnification. Named the StereoZoom®, this microscope was the first stereomicroscope without erecting prisms and was fashioned around the basic Greenough design, which will be discussed in detail below. It was generally the same size and shape as the Cycloptic (Figure 3), and had a comparable magnification range (0.7x to 3.0x) with similar working distances. The microscope also featured a new Bausch & Lomb invention: four first-surface mirrors with enhanced aluminum coatings, which were strategically positioned to perform the function of both inclination prisms and Porro erecting prisms. In stereomicroscopy erect images are useful because microscopists often must perform interactive manipulations on the specimen while under observation. Tasks such as dissection, micro-welding, industrial assembly, or microinjection of oocytes are more conveniently conducted when the specimen has the same physical orientation on the microscope stage as it does when viewed through the eyepieces. Also, the study of true spatial relationships between specimen features is aided by a natural, erect image.
In addition to having a reduced cost when compared to prism-equipped microscopes, the StereoZoom was also lighter in weight. The basic microscope system or "Power Pod", as it was called, was complemented by an enormous selection of auxiliary lenses, eyepieces, illuminators, arms and stands, all produced with a trend-setting style that endured for over 40 years. Acceptance of the StereoZoom by a rapidly emerging semiconductor industry was immediate and long-lived. This novel design dominated the stereomicroscope market for many years until production was halted in 2000 by Leica, which in the 1980's had combined the microscope resources of American Optical, Bausch & Lomb, Leitz, Reichert, and Wild.
During the early 1960's, zooming stereomicroscopes were introduced by Nikon, Olympus, Unitron, and other (not so well known) Japanese companies that were beginning to make their presence known in the United States. Collectively, the Japanese, American, and European microscope manufacturers continued advancing the development of "bigger and better" stereomicroscopes having a host of new features. These advances were accelerated by the invention of high-speed computers, which made it feasible for optical designers to tackle the complex problem of creating an effective variable magnification zoom lens system with well-corrected optical aberrations.
Today's stereomicroscope designs feature high numerical aperture objectives that produce high contrast images, which have a minimum amount of flare and geometrical distortion. The observation tubes will accommodate high-eyepoint eyepieces having a field of view up to 26 millimeters, with a diopter adjustment that allows the image and reticle to be merged into focus simultaneously. In addition, many models sport high zoom ratios (up to 12x-15x) that provide a wide magnification range (between 2x and 540x) and reduce the necessity to change objectives. Ergonomic features incorporated into the microscope designs help to reduce fatigue during long hours of operation, and new accessories enable modern stereomicroscopes to image specimens that were impractical just a few years ago.
The human eyes and brain function together to produce what is referred to as stereoscopic vision, which provides spatial, three-dimensional images of the objects surrounding us. This is because of the brain's interpretation of the two slightly different images received from each of the retinas. The average human eyes are separated by a distance of approximately 64-65 millimeters, and each eye perceives an object from a somewhat different viewpoint that differs by a few degrees from the other. When transmitted to the brain, the images are fused together, but still retain a high degree of depth perception, which is truly remarkable. The stereomicroscope takes advantage of this ability to perceive depth by transmitting twin images that are inclined by a small angle (usually between 10 and 12 degrees) to yield a true stereoscopic effect.
In some stereomicroscope systems, specimens are imaged utilizing two separate compound microscope optical trains, each consisting of an eyepiece, an objective, and intermediate lens elements. Other designs employ a common objective shared between two individual optical channels. Two distinct images, originating from slightly different viewing angles, are projected onto the microscopist's retinas, where they stimulate nerve endings to transfer the information to the brain for processing. The result is a single three-dimensional image of the specimen whose resolution is limited by the microscope optical system parameters and the frequency of nerve endings in the retina, much like the limiting grain size in photographic film or the pixel density in a charged coupled device (CCD) digital camera.
Stereomicroscopes can be roughly divided into two basic families, each of which has both positive and negative characteristics. The oldest stereomicroscopic system, named after the inventor Greenough, utilizes twin body tubes that are inclined to produce the stereo effect. A newer system, termed the common main objective (introduced above), utilizes a single large objective that is shared between a pair of eyepiece tubes and lens systems. Either type of microscope can be equipped with step-type individual lenses to change magnification, or a continuously variable zoom-type magnification system. The following discussion addresses the advantages and disadvantages of both the Greenough and common main objective stereomicroscope designs.
The Greenough design, introduced by Zeiss at the turn of the twentieth century, consists of two identical (and symmetrical) optical systems each containing a separate eyepiece and objective arranged in accurate alignment within a single housing (Figure 4). A major advantage of this design is the high numerical apertures that can be obtained because the objectives are very similar in design to those utilized in classical compound microscopes. In general, the lower portions of the body tubes, containing the slender objectives, are tapered and converge at the best focus of the object plane. The upper end of the body tubes project a pair of images into the observer's eyes, normally with a pair of standard eyepieces. The size, focus, rotation, and centering of the two images must be held constant within very tight tolerances, so that the eyes view essentially the same scene. The one departure from sameness is the slightly different viewing angle at which each image is projected onto the retina. Because of the convergence angle, typically ranging from 10 to 12 degrees in modern designs, the left eye views the object from the left side while the right eye views the same object from a slightly different perspective on the right side.
A pair of erecting prisms or mirror system is utilized to de-rotate and invert the magnified image received from the objectives and present it to the observer as it would appear without a microscope. The body tubes are built to provide a straight line-of-sight in some designs, while others enlist the aid of additional prisms to allow inclination of the tubes and a more natural viewing position for the microscopist. Because the image-forming light rays pass through the complex lens system on center, the quality of the image is symmetrical about its center, as is the case with most compound microscopes. In addition, correction for optical aberrations in Greenough-type microscopes is less difficult than with common main objective designs, because the lenses are smaller, axially symmetrical, and do not rely heavily on light rays passing through the objective periphery.
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A distortion artifact arises in the Greenough microscope design due to the oblique separation of each body tube from a common axis. Termed the Keystone effect, this distortion causes the area on the left side of the right eye to appear slightly smaller than that on the right-hand side of the same image, and of course the reverse is true for the left eye's image (see Figure 5). Keystone distortion arises from the fact that the intermediate images produced by each body tube are inclined with respect to the specimen plane, and tilted relative to each other, so that only the central regions are in simultaneous focus at identical magnifications. The result is that peripheral portions of the viewing field are focused either slightly above or below the actual specimen plane and have very small differences in magnification, although the eyes usually compensate for this effect and it is often not noticeable to the microscopist. During prolonged observation periods, however, fatigue and eyestrain can be accelerated by the Keystone effect.
The small change in magnification and focus across the field of view in Greenough stereomicroscopes might be noticed in a photograph or video image produced through one side of the instrument, especially if the object is primarily flat and rectilinear. In photomicrography, focus discontinuities brought on by the inclination angle are easily compensated by tilting either the specimen or one of the beam paths so that the microscope optical axis is perpendicular to the lateral specimen plane. When undertaking measurements with a reticle, the linear eyepiece grid should be positioned in a vertical direction to minimize the Keystone effect. Another solution is to tip the specimen or the microscope five or six degrees and negate the convergence.
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Common main objective stereomicroscope designs center on the refracting action of a single, large diameter objective lens, through which both the left and right channels view the object . Each channel operates as an independent optical train parallel to the other (this is the reason they are also known as parallel microscopes; Figure 4), and there is collimated light between the individual channels and the objective (the image is projected to infinity). This arrangement guarantees that convergence of the left and right optical axes coincide with the focal point in the specimen plane. Because this parallel axis arrangement is usually extended to include the eyepieces, the left and right images are viewed by the microscopist's eyes with little or no convergence. A major advantage of the common main objective system is that the optical axis of the objective is normal to the specimen plane, and there is no inherent tilt of the image at the eyepiece focal plane.
Although in most situations there are the usual 10 to 12 degrees of convergence at the specimen, the brain is not used to interpreting three-dimensional images without convergence, leading to a unique anomaly that is specific to CMO stereomicroscopes. When viewing specimens through this type of microscope, the center portions of the specimen appear to be slightly elevated, so that a flat specimen now appears to have a convex shape. For example, a coin will have the appearance of being thicker in the center, so it would rock from side to side when inverted on a flat surface. This artifact is referred to as a perspective distortion, but should not cause concern unless the microscope is utilized to judge flatness or height (see Figure 5). Specimens with complex or rounded shapes, while displaying a certain amount of perspective distortion, often do not appear to be distorted when viewed through the stereomicroscope.
Perspective distortion is sometimes referred to as doming or the globular effect, and results from a combination of keystone and pincushion distortion. As an example, presented in Figure 5 is a slightly exaggerated illustration of how a United States Lincoln penny, a disc-shaped flat coin, would appear in a stereomicroscope with severe perspective distortion. The original penny is shown at the top of the illustration to have a flat surface. Just beneath are the images projected simultaneously by the microscope to both the left and right eyes, which demonstrate an asymmetrical pincushion distortion directed toward the central axis of the microscope. The final result is perception of a dome- or globe-shaped object when the images from both eyepieces are projected onto the retinas and fused together in the brain. Most high-end research grade common main objective stereomicroscopes produced by the major manufacturers have virtually eliminated this artifact, but it still occurs in some less expensive microscopes.
Another artifact often encountered with common main objective stereomicroscopes is that small amounts of off-axis aberrations such as astigmatism, coma, and lateral chromatic aberration appear in the center of each image. This occurs because each optical channel is receiving light rays from an off-center region of the large objective instead of directly from the center, where aberrations (especially those occurring off-axis) are at a minimum or practically non-existent in lenses with the best optical corrections. The effect is generally not noticed when both eyes are employed to view the specimen, but a photomicrograph or digital image may have asymmetric geometry across the field.
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In general, the chromatic aberrations are difficult and expensive to correct, especially considering the large size and volumes of glass used in manufacture of the objectives. Some CMO stereomicroscope designs have made this a non-issue by providing the facility to offset the large central objective, positioning it on the axis of either the left or right side channel. Other microscope designs even provide a means for replacing the large objective with a conventional infinity-corrected objective that can be utilized to view and photograph specimens at high magnifications (and numerical apertures).
The greatest design feature and practical advantage of a common main objective stereomicroscope, as with most modern microscopes, is the infinity optical system. A collimated light pathway, with two parallel axes for the channels, exists between the objective and removable head/observation tube assembly (labeled infinity space in Figure 6). This allows the effortless introduction of accessories, such as beamsplitters, coaxial episcopic illuminators, photo or digital video intermediate tubes, drawing tubes, eyelevel risers, and image transfer tubes into the space between the microscope body and head. It is also possible to place these accessories in the space between the objective and zoom body, although this is rarely done in practice. Because the optical system produces a parallel bundle of light rays between the body and microscope head, the added accessories do not introduce significant aberrations or shift the position of images observed in the microscope. Such versatility is not available in stereomicroscopes designed around the Greenough principles.
It is a difficult task to determine which of the two designs (CMO or Greenough) is superior, because there are no universally accepted criteria for comparing performance between the stereomicroscope systems. Common main objective microscopes, in general, have a greater light-gathering power than the Greenough-design and are often more highly corrected for optical aberration. Some observations and photomicrography might best be conducted utilizing a CMO microscope, while other situations may call for features exclusive to the Greenough design. As a consequence, each microscopist must make the determination whether one design will be more appropriate for the task at hand and use this information to develop a strategy for stereomicroscopy investigations.
In most circumstances, the choice between Greenough or common main objective stereomicroscopes is usually based on the application, and not whether one design is superior to the other. Greenough microscopes are typically employed for "workhorse" applications, such as soldering miniature electronic components, dissecting biological specimens, and similar routine tasks. These microscopes are relatively small, inexpensive, very rugged, simple to use, and easy to maintain. Common main objective microscopes are generally utilized for more complex applications requiring high resolution with advanced optical and illumination accessories. The wide spectrum of accessories available for these microscopes lends to their strength in the research arena. In many industrial situations, Greenough microscopes are likely to be found in production lines, while common main objective microscopes are limited to the research and development laboratories. Another consideration is the economics of microscope purchase, especially on a large scale. Common main objective stereomicroscopes can cost several times more than a Greenough microscope, which is a chief consideration for manufacturers who may require tens to hundreds of microscopes. However, there are exceptions. If a common main objective microscope is the better tool for a job, the true cost of ownership may be lower in the end.
Magnification in Stereomicroscopy: Objectives and Eyepieces
The total magnification achieved in a stereomicroscope is the product of the objective and eyepiece magnifications, plus that contributed by any intermediate or external auxiliary magnifying lens systems. Over the years, a number of independent methods have been developed to change (increase or decrease) the magnification factor of stereomicroscopes. In the simplest microscopes, the objectives (or single objective in a CMO design) are permanently mounted in the lower body housing, and magnification can only be altered by introducing eyepieces of varying power. Slightly more complex microscopes have interchangeable objectives that allow total magnification factors to be adjusted either by using a higher or lower power objective or by substituting eyepieces of differing magnification. Objectives in these models are mounted by screw threads or clamps, which enable relatively quick changeover to a new magnification.
Mid-level stereomicroscopes are equipped with either a sliding objective housing or a rotating turret containing several matched sets of objectives to produce varying magnification factors. In order to adjust the microscope magnification, the operator simply twists the turret to position a new auxiliary paired set of objectives beneath the channel tubes. Microscopes having this design were once very popular, but are rarely manufactured today.
The highest quality stereomicroscopes are equipped with a zoom lens system or a rotating drum containing Galilean telescopes that are utilized to increase and decrease overall magnification. The rotating drum system functions as an integral intermediate tube (or piece) containing paired sets of lenses that can be installed into the optical pathway by rotating the drum. In most models, positive détentes are employed to act as "click stops" to secure the lens mounts into correct alignment, and are marked to notify the operator of the new magnification factor. The drum usually has a pair of empty lens mounts that are devoid of auxiliary lenses and can be positioned into the optical path to allow use of the objective and eyepiece combination without additional magnification.
Zoom systems (illustrated in Figure 7) provide a continuously variable magnification range that can be adjusted by turning a knob located either on the periphery of the microscope body or integrated within the body itself. This design eliminates the blank-out that occurs with possible visual loss of spatial relationships between specimen features when magnification is changed in discrete, stepped settings. In some of the older literature, zoom systems are often referred to as pancratic systems after the Greek words pan for "each" and kratos for "power". Zoom ratios vary between 4:1 and 15:1, depending upon the microscope age, manufacturer, and model. In general, a zoom lens system contains a minimum of three lens groups, enlisting two or more elements for each group, which are strategically positioned with respect to each other. One element is fixed within the channel tube, while the other two are smoothly translated up and down within the channel by precision cams. The system is designed to allow rapid and continuous changes in magnification while simultaneously keeping the microscope in focus. Following the zoom system, additional lens elements are utilized to relay and/or erect the image before projecting it into the eyepieces. Several of the newer stereomicroscope models employ a positive click-stop that alerts the microscopist at selected magnification positions in the zoom range. This distinction is essential for calibration of the magnification level at a given power step, a feature often found useful when performing linear measurements.
Early stereomicroscope zoom lens systems had a magnification range of approximately 7x to 30x. The magnification factors slowly grew as optical performance improved in this class of microscopes, and more recent student microscopes now feature zoom ranges between 2x and 70x. Mid-level stereomicroscopes have zoom magnification factors with an upper magnification limit between 250x and 400x, while high-end research microscopes sport zoom systems that can reach over 500x in magnification. This wide magnification range is complemented by a depth of field and working distances that are much larger than are found in compound microscopes having equivalent magnifications. The working distance on modern stereomicroscopes varies between 20 and 140 millimeters, depending upon the objective magnification and zoom ratio. With the addition of specialized auxiliary attachment lenses, working distances of 300 millimeters or more can be achieved. Field diameters are also much wider than those attainable with compound microscopes.
Auxiliary attachment lenses can be fitted to the objective barrel on specially designed stereomicroscopes (Figure 8). In general, the attachment lenses are threaded to rotate into a matching thread set on the front of the objective barrel. Other versions attach to the barrel with a clamping device. These lenses enable the microscopist to either increase or decrease the magnification of the primary objective.
Attachment lenses are useful when image quality is not the overriding factor, because optical corrections cannot be as accurately performed due to the fact that the lens is not mounted in the identical position each time it is attached. In addition, attachment lenses modify the objective working distance (the distance between the specimen and the objective front lens element). A lens that increases the microscope magnification will also simultaneously render a short working distance, while an attachment lens that serves to decrease magnification produces a corresponding increase in working distance.
Modern stereomicroscopes are equipped with standardized widefield high-eyepoint eyepieces that are available in magnifications ranging from 5x to 30x in approximately 5x increments. Most of these eyepieces can be utilized with or without eyeglasses, and protective rubber cups are available to avoid contact between a microscopist's eyeglasses and the eyepiece eyelens.
Eyepieces generally are equipped with a diopter adjustment to allow simultaneous focusing of the specimen and measuring reticles, and binocular microscope observation tube mounts (heads) now have moveable tubes that enable the operator to vary the interpupillary distance between eyepieces over a range of 55 to 75 millimeters. The interpupillary adjustment is often accomplished by rotating the prism bodies with respect to their optical axes. Because the objectives are fixed in their relationship to the prisms, the adjustment does not alter the stereoscopic effect. This convenience reduces fatigue during extended observation periods, but requires re-adjustment when the instrument is used by more than one operator. Note that microscopists who wear eyeglasses to correct for shortsightedness and differences in vision between eyes should also wear their glasses for microscopy. Eyeglasses worn only for close-up work should be removed during observation because the microscope produces the image at some distance.
The field of view (sometimes abbreviated FOV), which is visible and in focus when observing specimens in a microscope, is determined by the objective magnification and the size of the fixed field diaphragm in the eyepiece. When the magnification is increased in either a conventional or stereomicroscope, the field of view size is decreased if the eyepiece diaphragm diameter is held constant. Conversely, when magnification is decreased, the field of view is increased at fixed eyepiece diaphragm diameters. Changing the size of the eyepiece diaphragm opening (this must be done during manufacture) will either increase the field of view at fixed magnification (for a larger diaphragm size), or decrease the field of view (smaller diaphragm size).
In most compound and stereomicroscope eyepieces, the physical diameter of the field diaphragm (located either in front or behind the eyepiece field lens) is measured in millimeters and called the field number, which is often abbreviated and referred to simply as FN. The actual physical size of the field diaphragm and apparent optical field size can vary in eyepiece designs having a field lens below the diaphragm. Measuring and photomicrography reticles are placed in the plane of the eyepiece field diaphragm, so as to appear in the same optically conjugate plane as the specimen.
The field number of the eyepiece, usually inscribed on the housing exterior, is divided by the magnification power of the objective to quantitatively determine the field of view size. Included in the calculation should also be the zoom setting and any additional accessories inserted into the optical path that may have a magnification factor. However, the eyepiece magnification is not included, which is a relatively common mistake made by novices in microscopy. When a wider field of view is desired, the microscopist should choose eyepieces with a higher field number. In the lower magnification ranges, stereomicroscopes have substantially larger fields of view than classical laboratory compound microscopes. The typical field size with a 10x eyepiece and a low power objective (0.5x) is around 65 to 80 millimeters (depending upon the zoom factor), which greatly exceeds the size observed (about 40 millimeters) with a compound microscope at comparable magnification. These large field sizes require a high degree of illumination, and it is often difficult to provide a continuous level of illumination across the entire viewfield.
Resolution and Depth of Field in Stereomicroscopy
Resolution in stereomicroscopy is determined by the wavelength of illumination and the numerical aperture of the objective, just as it is with any other form of optical microscopy. The numerical aperture is a measure of the resolving power of the objective and is defined as one-half the angular aperture of the objective multiplied by the refractive index of the imaging medium, which is usually air in stereomicroscopy. By dividing the illumination wavelength (in microns) by the numerical aperture, the smallest distance discernible between two specimen points is given by the equation (the Raleigh Criterion):
Resolution (d) = 0.61 × λ / (n × sin(θ))
where d is the smallest resolvable distance, λ is the illuminating wavelength (usually a mixture centered around 550 nanometers in stereomicroscopy), n is the refractive index of the medium between the objective and specimen, and θ is the objective one-half angular aperture. As an example, a Nikon SMZ1500 stereomicroscope equipped with a 1.6x apochromatic objective having a numerical aperture of 0.21, will have a maximum resolution of approximately 1.6 micrometers when the specimen is illuminated with white light having an average wavelength of 550 nanometers. Note that the resolution calculated for the 1.6x objective assumes the imaging medium between the specimen and the objective is air. Objective lenses manufactured for common main objective stereomicroscopes typically vary in magnification from 0.5x to 2.0x, with three or four intermediate values.
The magnification, working distance, and numerical aperture of typical stereomicroscope objectives at varying magnification are presented in Table 1. In the past, several manufacturers have assigned color codes to their stereomicroscope objective magnification values. Table 1 also lists the color code assignment for a series of Nikon stereomicroscope objectives having this identifying information. Note that many manufacturers do not assign a specific color code to stereomicroscope objectives, and the codes listed in Table 1 are intended only to alert readers that some objectives may display this and other specialized proprietary nomenclature.
Stereomicroscope Objective Specifications
The resolving power of stereomicroscope objectives is determined solely by the objective numerical aperture and is not influenced by optical parameters of the eyepiece. Overall resolution will not be affected when exchanging 10x eyepieces for 20x or higher magnification eyepieces, although specimen detail that is not visible at the lower magnification will often be revealed when the eyepiece magnification is increased. The highest power eyepieces (30x or higher) may approach empty magnification, especially when the total microscope magnification exceeds that available from the objective numerical aperture. In order to gauge and compare the performance of one microscope to another, the resolution value is often expressed in terms of line pairs per millimeter (lp/mm). In the case of the Nikon 1.6x objective discussed above, the resolution approaches 630 line pairs per millimeter under optimum conditions.
Auxiliary attachment lenses, which range in power from 0.3x to 2.0x, can alter the working distance and resolving power of a stereomicroscope optical system. In general, the resolving power influence is proportional to the magnification factor of the attachment lens. The field diameter is inversely proportional to the magnification factor, while the depth of field is inversely proportional to the magnification factor squared. Changes in working distance are also inversely proportional to the magnification factor, but are difficult to compute because the function is not linear. In addition, use of these auxiliary lenses will not have significant impact on image brightness in most cases.
Numerical Aperture and Equivalent f-Number Values
Lenses designed for general photography are rated with a system that is based on f-numbers (abbreviated f), rather than numerical aperture (Table 2). In fact, these two values appear different, but actually express the same quantity: the light gathering ability of a photography lens or microscope objective. F-numbers can be easily converted to numerical aperture (and vice versa) by taking the reciprocal of twice the other's value:
f-Number (f) = 1 / (2 x NA) and NA = 1 / (2 x f)
Numerical aperture (in microscopy) is equal to the refractive index of the imaging medium multiplied by the angular aperture of the objective. The f-number is calculated by dividing the focal length of the lens system by the aperture diameter. If a 50-millimeter focal length lens has the same aperture diameter as a 100-millimeter lens, the shorter lens has twice the f-number as the longer. In cases where the maximum diameter is the same in both lenses, the size is f/2 for the 50-millimeter lens and f/4 for the 100 millimeter lens.
The aperture diameter is fixed in a stereomicroscope objective, similar to the situation with conventional compound microscope objectives. As the microscope magnification is increased or decreased by changing the zoom factor, the focal length is also altered accordingly. At higher magnifications, the ratio of the aperture diameter to focal length increases, and the opposite is true as magnification is decreased.
The focal length of a 2.0x stereomicroscope objective is half that of a 1.0x objective, which in turn, is half that of a 0.5x objective. In some of the Nikon SMZ series stereomicroscopes (U, 10a, 800, and 1000), the 0.5x objective has a focal length of 200 millimeters, while the 1.0x is 100 millimeters, and the 2.0x objective focal length is 50 millimeters. The relative size of the zoom system aperture (as compared to that of the objective) functions to control the f-number (and numerical aperture) of the entire microscope system. In late model microscopes, such as the SMZ1500, objective focal lengths have been reduced in order to increase the total system numerical aperture. Thus, a 0.5x objective designed for the SMZ1500 has a 160-millimeter focal length, with the 1.0x and 2.0x objectives having focal lengths equal to one-half and one-quarter that of the 0.5x lens, respectively.
Some manufacturers supply adapter rings that allow objectives designed for a specific microscope to be used on other (usually earlier model) stereomicroscopes. In several cases, two objectives having the same magnification can have different focal lengths due to variations in tube lens and zoom channel aperture specifications. As an example, the Nikon SMZ-U stereomicroscope 1.0x objective has a focal length of 100 millimeters, while the later model SMZ1500 microscope employs a focal length of 80 millimeters for an objective having similar magnification and optical corrections. The difference between the two microscope designs is the size of the zoom system aperture, which results in shorter focal lengths for the SMZ1500 series objectives. When interchanging objectives having the same magnification but different focal lengths, an additional factor must be introduced into total magnification calculations to correct for the focal length differences.
Depth of Field in Stereomicroscope Objectives
Depth of field is an important concept in stereomicroscopy (perhaps even more so than with other common forms of optical microscopy), and is strongly influenced by the total magnification of the instrument, including the contribution from both the objective and auxiliary attachment lenses. At a magnification of 50x, using a 1x objective (numerical aperture 0.10), 10x eyepieces, and a zoom factor of 5, the depth of field exhibited by a typical stereomicroscope is approximately 55 micrometers. If a 2x attachment lens is added to the microscope when it is configured for operation at 50x, the new magnification will be 100x, but the depth of field drops to about 14 micrometers, a substantial decrease from the value (55 micrometers) without the auxiliary lens. In this situation, it is wiser to change the eyepiece magnification from 10x to 20x to achieve the added magnification so as to retain the larger depth of field value (see Table 3). Increasing the objective numerical aperture through enhanced optical correction (for instance, from achromat to apochromat) will also produce a modest decrease in field depth.
Depth of field values for a Nikon plan apochromatic 1x objective are presented in Table 3, where they are listed as a function of zoom magnification factor and eyepiece magnification. It is clear from the data in the table that numerical aperture increases with increasing zoom magnification, while the depth of field decreases with increasing eyepiece and zoom magnification factors.
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Reducing the size of the double iris diaphragm positioned between the objective and the eyepieces can enhance depth of field. This diaphragm is opened and closed using a wheel or lever in the microscope body housing. There are actually two diaphragms, one for each of the channels, in the common main objective stereomicroscope design. The role of these diaphragms is to produce an increase in field depth while simultaneously improving specimen contrast observed in the eyepieces. Depth of field and numerical aperture variations, as a function of diaphragm opening size, are presented in Table 4 for the Nikon plan apochromatic 1x objective at the highest zoom magnification factor (11.25). As the diaphragm size is ramped down, the depth of field utilizing a 10x eyepiece increases from 26 to 89 millimeters, approximately a 200 percent increase. Simultaneously, the numerical aperture drops from a value of 0.131 to 0.063, or almost 100 percent. Similar effects are observed at higher eyepiece magnifications.
Depth of Field and Numerical Aperture
Iris Diaphragm Opening Size
Closing the iris diaphragms will also produce a decrease in overall light intensity, increasing exposure times for both digital and film camera systems. In most cases, the optimum setting for the diaphragms is determined by experimentation. As the diaphragms are slowly closed, the image begins to display more contrast as illumination intensity slowly fades. At some point, depending upon the optical configuration of the microscope, the image begins to degrade and specimen details exhibit diffraction phenomena while minute structural details disappear. The best setting is a balance between maximum specimen detail and maximum contrast as seen in the eyepieces, on film, or in digital images.
Photomicrography and Digital Imaging
Both Greenough and common main objective stereomicroscopes are readily adaptable to image capture utilizing traditional photomicrography techniques (film) or through advanced digital imaging. Often photomicrography is employed as a tool for recording the spatial distribution of specimen details prior to observation and imaging with a higher-power compound microscope. This technique is often necessary for biological specimens, where dissecting, staining, and selective mounts are performed.
The principal concern with digital imaging and photomicrography in stereomicroscopy is the low numerical aperture of the objectives, and the inability to capture on film (or in a digital image) the tremendous depth of field observed through the eyepieces. There are also several limiting factors that should be considered when photographing specimens through a single body tube utilizing a Greenough-style stereomicroscope. Because the microscope objective is positioned at a slight angle to the specimen, depth and resolution seen in the microscope eyepieces is not recorded on film. Some manufacturers once provided accessories that help to alleviate these problems, but many of the older microscopes have spare and accessory parts inventories that are exhausted, limiting the choices for photomicrographers.
Older stereomicroscopes can be equipped with a digital or film camera using attachments that are available over the Internet or through optics and science supply houses. These attachments exist for almost every conceivable camera system, and many will fit the camera directly onto an observation tube with the eyepiece left in place. Newer stereomicroscopes have trinocular heads or photographic intermediate tubes (sometimes requiring a projection eyepiece) as an option, but these are often limited in use to the camera systems specified by the microscope manufacturer.
The microscope presented in Figure 9 is a state-of-the-art Nikon research-level stereomicroscope equipped for both traditional imaging with Polaroid film and with a digital video camera. The camera systems are coupled to the microscope through a beamsplitter attachment that is attached as an intermediate piece between the microscope body and the binocular head. Both single and double-port beamsplitters are available from Nikon for use with either one or two camera systems. The optical path is directed into the camera ports with a selection lever located on the front portion of the intermediate piece. Standard c-mount, f-mount, and proprietary coupling systems are available to support a wide variety of camera systems. In addition, Nikon offers projection lenses of varying magnification that can be utilized to vary the image size on film or in digital images.
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A photo reticle can be inserted into one of the eyepieces for composing images for capture, or the focus finder in the exposure monitoring system can be utilized for the same purpose. Magnification in photomicrographs or digital images is calculated by the product of the projection lens magnification (if used) times the zoom magnification and the objective magnification. Some beamsplitter ports also introduce a fourth magnification factor, usually 0.5x to 2.5x that must be included in the calculation. Other microscope manufacturers offer similar camera systems designed exclusively for their stereomicroscope product line-ups.
A unique aspect of photomicrography in stereomicroscopy is the ability to compose images that are stereo pairs, by employing specimens having significant three-dimensional spatial relationships among structural details. The first step is to photograph the specimen using the left eyepiece, followed by another photograph through the right eyepiece. An alternative procedure that can also be utilized with common main objective stereomicroscopes involves tilting the specimen on the horizontal (stage) axis by an angle of seven to eight degrees to the left of the microscope optical axis. After capturing a photomicrograph or digital image, the specimen is tilted an identical amount to the right of the optical axis and another photomicrograph (digital image) is recorded. This maneuver produces the same effect as taking two sequential photographs with a Greenough-style stereomicroscope.
After printing (or digital image processing) the photomicrographs, they can be mounted (or displayed on a computer monitor) side-by-side and viewed with a stereo viewer, rendering specimen details in striking three-dimensional displays. It is important that the orientation and alignment of the stereo pairs coincides with the requirements of the stereo viewer.
Magnification is often thought of as the most important criteria for judging the performance of an optical microscope. This is far from true, because the correct magnification is the one sufficient for the task at hand and should not be unnecessarily exceeded. Many classical investigations into the basis of cellular structure and function, and the minute details of semiconductor anatomy, are best conducted with classical transmitted and reflected compound optical microscopes. Magnifications in the 400x to 1000x range are required for these studies, which usually do not rely heavily on large depths of field for successful observation. On the other hand, a wide variety of specimens must be examined at smaller magnifications, but require a larger depth of field with a high degree of contrast.
Stereomicroscopes have characteristics that are valuable in situations where three-dimensional observation and perception of depth and contrast is critical to the interpretation of specimen structure. These instruments are also essential when micromanipulation of the specimen is required in a large and comfortable working space. The wide field of view and variable magnification displayed by stereomicroscopes is also useful for construction of miniature industrial assemblies, or for biological research that requires careful manipulation of delicate and sensitive living organisms.
Considering the wide range of accessories currently available for stereomicroscope systems, this class of microscopes is extremely useful in a multitude of applications. Stands and illuminating bases are available from all of the manufacturers, and can be adapted to virtually any working situation. There are a wide choice of objectives and eyepieces, enhanced with attachment lenses and coaxial illuminators that are fitted to the microscope as an intermediate tube. Working distances can range from 3-5 centimeters to as much as 20 centimeters in some models, allowing for a considerable amount of working room between the objective and specimen.
Modern stereomicroscopes are designed with ergonomic issues in mind, and most of the optical assemblies are sealed pods that are protected against dust and tampering, and contain lens shields to protect the optical elements from environmental hazards. Antireflective coatings vaporized onto the surface of large objective front lenses serve to protect these delicate parts from attack by corrosive liquids or gasses, or from abrasive particles that might cause chips and scratches.
The utility of stereomicroscopes is limited only by their resolving power. These microscopes are enjoying widespread use in a variety of disciplines that have tasks requiring the features found in modern instruments of this class. Among them are education (biology, chemistry, botany, geology, and zoology), medicine and pathology, the semiconductor industry, metallurgy, textiles, and other industries that require assembly and inspection of miniature components.
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.
Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.