Basic Microscope Ergonomics

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Walk into a busy laboratory and it isn't unusual to see microscopes sitting on books, tilted at odd angles, and otherwise propped in a variety of positions to accommodate their users. Microscopes have undergone a remarkable evolution since their invention in the early 1600s, but most of the new developments and improvements have been in the area of contrast enhancement accessories and the microscope optical train.

While issues of usability have taken a back seat to optical performance for the past 400 years, they haven't been completely disregarded by microscopists. As early as the 1830s, Sir David Brewster noted in his Treatise on Optics, "The best position for microscopical observations is when the observer is lying horizontally on his back . . . . The worst of all positions is that in which we look downwards vertically." Unfortunately, Sir Brewster's suggestions were never utilized in microscope design and sitting or standing at the instrument became the status quo. While conventional microscope design has not necessarily been a problem for short-term use, long-term sessions have historically created problems for scientists and technicians who used the instruments, making them, quite literally a pain in the neck. Microscopists were expected to suffer for the greater good of science and many have paid the price over the years with physical complaints and sometimes even permanent injuries. The Occupational Safety and Health Administration (OSHA), in the United States Department of Labor, finds that "Microscope work is straining both to the visual system and the musculoskeletal system. Operators are forced into an unusual exacting position,

with little possibility to move the head or the body. They are often forced to assume an awkward work posture such as the head bent over the eye tubes, the upper part of the body bent forward, the hand reaching high up for a focusing control, or with the wrists bent in an unnatural position." Once an exotic and esoteric piece of scientific equipment, during the 20th century microscopes became commonplace in geological, biological, and medical laboratories and in factories manufacturing electronic components and integrated circuits for computers and the consumer electronics industry. As microscope use grew, so did concerns about usability. During the 1980s and 90s, microscope manufacturers began introducing ergonomic features into their instruments to make them safer and more comfortable to use for extended periods of time, up to six or eight hours a day. Illustrated in Figure 1 is a 1980s era Nikon SMZ10 stereomicroscope equipped with several aftermarket ergonomic accessories. To ease operator posture, the microscope is fitted with an extended set of eye tubes (also see Figures 3 and 4), an optical wedge that places the observation tubes closer to a horizontal angle, and a flexible adapter that allows the individual user to adjust the microscope's height. Adjacent to the microscope is a pair of sloping armrests that eliminate the necessity to remove arms from the laboratory bench in order to adjust the microscope. The SMZ10 was designed during a period when stereomicroscope focus controls were mounted high on the rack, and required considerable forearm motion to continually adjust focus. In addition, magnification is altered by rotating a large knurled ring in the central portion of the microscope body. This ring contains several pairs of Galilean telescopes that increase or decrease magnification. Operation of the microscope for extended periods of time requires constant changing of both focus levels and magnification factors, both of which have controls located a significant distance from the desktop. A digital video camera system is attached to the microscope through an aftermarket adapter. Operating the microscope with the camera enables the operator to compose and focus images on a computer monitor instead of through the microscope eyepieces, thus reducing eyestrain. Basic Ergonomics Ergonomics is about finding a better fit between people and the things they do, the objects they use, and the environments in which they live, work, travel, and play. The science of ergonomics is the study and application of human anatomy, biomechanics, and biology to the design of objects, systems, and environments. Also called human engineering, or human factors, it is a relatively new branch of science that was founded in 1949, spurned by the development of new technologies during World War II. Throughout this period, it had become clear that, in order to be used safely and effectively, new technologies and products needed to account for human and environmental factors. Over the past 50 years, ergonomics has become widely applied, from factory work and information systems to the home, sports, and leisure -- just about every aspect of life.

In the workplace, the goal of ergonomics is to improve efficiency, quality, and job satisfaction by making routine and repetitive tasks more comfortable and easier to do. This reduces stress, both physical and psychological, by lowering the fatigue factor and human error. In some jobs, particularly in the nuclear and chemical industries and transportation (e.g. air traffic control), the cost of human error can be catastrophic, injuring or killing hundreds of people or resulting in widespread environmental disasters. Percentage of Medical Problems Reported by Microscope Operators Anatomical Location Employee Percentage Neck Shoulders Back (Total) Lower Back Lower Arms Wrists Hands and Fingers Legs and Feet Eyestrain Headaches Table 1 For the vast majority of jobs, however, it is the individual workers who are primarily affected, suffering discomfort, injuries, or outright disabilities, classified as work-related musculoskeletal disorders (MSDs or WMSDs). MSDs are medical conditions affecting the muscles, nerves, tendons, ligaments, joints, cartilage, and/or spinal discs. MSDs are referred to by a number of names (and acronyms). The terminology includes Repetitive Strain Injuries and Repetitive Stress Injuries (RSIs), Cumulative Trauma Disorders (CTDs), and Overuse Syndrome, although these are umbrella terms and don't refer to any MSD in particular. Some examples of specific MSDs are carpal tunnel syndrome, tendonitis, ganglion cysts, and lower back pain. General warning signs of MSDs are fatigue, stiffness, persistent burning or aching, reduced coordination, and a loss of grip strength in the hands. Numerous studies have established the following ergonomic risk factors as most likely to cause or contribute to an MSD: force, repetition, awkward postures, static postures, vibration, contact stress, and cold temperatures. Of these risk factors, force (i.e., forceful exertions), repetition, and awkward postures are most often associated with the occurrence of serious MSDs.

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Exposure to one ergonomic risk factor may be enough to cause or contribute to an MSD. Most often, ergonomic risk factors act in combination to create a hazard. Jobs that have multiple risk factors have a greater likelihood of causing an MSD, depending on the duration, frequency, and/or magnitude of exposure to each factor. It is important to consider ergonomic risk factors in light of their combined effect in causing or contributing to an MSD, as well as their individual effects. Ergonomics has become a topic of increasing importance in the workplace throughout the past several decades. A mismatch between the physical capacity of workers and the physical demands of their jobs can result in MSDs. In the USA, 1.8 million workers report work-related MSDs such as carpal tunnel syndrome, tendonitis, and back injuries, every year. Approximately 600,000 MSDs are serious enough to warrant taking time off from work to recover and sometimes even require surgical intervention. Evidence suggests that another 1.8 million MSDs go unreported every year. MSDs are estimated to cost up to $50 billion a year. Employers pay between $15-$18 billion in workers' compensation costs alone; $1 out of every $3 spent on workers' compensation goes for MSD-related claims. This does not include billions of dollars spent on medical treatment and hidden costs associated with work-related injuries. Recent increases in reported MSDs suggest that employers should be vigilant in creating work environments that are conducive to both good health and high productivity. Microscope Ergonomics The human body is a wonder of biomechanics, accommodating and adapting to a wide variety of postures and activities. The keyword for a healthy, well-maintained body is "activity." The human body works best when it is constantly moving or changing positions. Sitting or standing for hours on end, bent over a microscope eyepiece is not an activity for which the body is well adapted. Microscope work requires the head and arms to be held in a forward position and inclined toward the microscope with rounded shoulders, a posture that can irritate soft tissues, such as muscles, ligaments, and disks. If the feet are placed on the ring-style footrests that are common to many lab stools, the position is further exaggerated.

Poor posture and awkward positioning are the primary risk factors for MSDs that can affect full-time microscopists, who often experience pain or injury to the neck, wrists, back, shoulders, and arms. Eyestrain, leg, and foot discomfort have also been documented with long-term microscope use. In the semiconductor industry, the second leading cause of work-related medical problems is found in microscope technicians, trailing only maintenance workers who traditionally have high injury rates. A regional survey of cytotechnologists, heavy users of microscopes, found that slightly over 70 percent reported having neck, shoulder, or upper back symptoms, while 56 percent had an increased incidence of hand and wrist symptoms. Other studies have indicated that around 80 percent of microscopists in all fields have experienced job-related musculoskeletal pain and that 20 percent have missed work because of medical problems related to microscope use. The rather high 5- to 10-year drop out rate for cytotechnologists is attributed, in part, to physical discomfort associated with long hours examining specimens through the microscope. Table 1 lists the range of percentages reported in the literature for medical complaints associated with long-term microscope use. A majority of reported problems occur with the neck, back, shoulders, and arms, with a smaller percentage of microscopists reporting discomfort or injury to the wrists, hands, legs, feet, and eyes. Many of these conditions can be avoided or at least mitigated. Two studies at Duke University Medical Center during the 1990s suggested that people suffered fewer discomforts when using new ergonomically designed microscopes or even conventional microscopes modified to better accommodate the user. In either case, adaptability was the key. Microscopes that could be adapted to an individual user, rather than forcing the user to adapt to the microscope, were more comfortable and caused fewer problems. Factors believed to be causing these problems are head inclinations up to 45 degrees and upper back inclination at angles up to 30 degrees, awkward positioning of the arms and hands, and repetitive motions. An unaccommodating workstation that requires a microscopist to sit in awkward positions for long periods can also cause fatigue and MSDs.
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Proper Microscope Posture

Explore how new ergonomic microscope designs aid the operator in assuming a more correct posture as a function of height for seated observations. The major factor with using conventional microscopes is that viewing specimens requires users to maintain a flexed neck posture while the hands are in a relatively fixed position. From the viewpoint of biomechanics, having to maintain even a slight incline of 30 degrees from the vertical can produce significant muscle contractions, muscle fatigue,

and pain. In fact, it has been documented that nerves can often be pinched when the neck is overextended by this amount. Repetitive motions of the hands and the contact stress of arms resting on a hard surface can cause pain and nerve injury, leading to repetitive stress injuries and/or carpal tunnel syndrome. Later studies have suggested that to permit a more neutral erect working posture, the optical path (distance from the ocular lenses to the specimen being viewed) should range between 45 and 55 centimeters (18 to 21.5 inches). The eyepieces should be no more than 30 degrees above the horizontal plane of the desktop (Figure 2). A majority of older microscopes, however, have much shorter optical path dimensions (25 to 30 centimeters or 10 to 12 inches) with the eyepieces angled at 60 degrees above horizontal. This creates a dilemma for the user. If the microscope is raised high enough to prevent neck flexion, then the user is forced to bend the wrists into an unnatural position. If the microscope is lowered to bring the stage to a more neutral position, with the forearm parallel to the floor, then the neck is forced to bend. Most workers compensate for this by finding some "happy medium" between the two extreme postures, resulting in discomfort for the neck, shoulders, forearms, wrists, and hands. Eye fatigue can also be a major problem for operators, especially if they have poor vision resulting from near and far sightedness or astigmatism. The diopter adjustment provided on most microscope eyepieces can be employed to compensate for minor focus problems (near and far sightedness), but microscopists who have moderate to severe astigmatism should wear glasses even when viewing specimens through the eyepieces. In order to accommodate the longer eyepoints necessary for observation with eyeglasses, manufacturers offer specialized high eyepoint eyepieces. Many problems associated with eyestrain during extended periods of microscope use can be alleviated by employing video camera systems that display the specimen on a computer monitor or television screen. In fact, many future microscope designs may be capable of eliminating the eyepieces altogether, substituting instead a CCD or CMOS image sensor for the classical observation tubes. The digital imaging chip will be coupled to a sophisticated software analysis package that controls image capture and storage, digital processing, and other features such as time-lapse cinemicrography and real-time video movies. Ensuring that the microscope images are as bright, sharp, and crisp as possible will also help to reduce eye fatigue and associated headaches. It is important to train operators in correct alignment of the microscope lamp and optical pathway to optimize image quality. This is true regardless of whether the image is observed through the eyepieces or on a computer monitor. Many of the newer microscopes have expanded viewfields through the use of eyepieces with larger field diaphragms. Coupled to objectives with higher numerical aperture values, better aberration correction, and longer working distances, the images produced show a tremendous amount of specimen detail in exquisite clarity with flat fields from edge-to-edge. These factors ease the burden of visually searching for tiny specimen details, and reduce associated eye stress and fatigue during extended periods of observation.

Some companies now produce adapters that allow conventional microscopes to be modified to better fit individual users (Figures 1 through 4). Body tube extensions can increase the distance between the eyepieces and stage adjustments, and optical wedges can provide a greater angle of adjustability, between 30 and 80 degrees. Aftermarket microscope stands allow an instrument to be elevated and rotated for increased comfort. A recent solution to the problem of microscope usability has been the incorporation of ergonomic features into modern designs by the microscope manufacturers. Although these models were prohibitively expensive for the vast majority of labs when they were first developed, ergonomic features are increasingly becoming standard on new microscope models. Another study at Duke examined the new ergonomic designs with a group of cytotechnologists who used traditional microscopes in their work, and who had a variety of complaints associated with using that equipment. The study replaced conventional Zeiss model microscopes, used by the workers, with a Nikon Eclipse E400 having a tilting and telescopic head, optional eye-level riser tubes, single-hand focus controls, and in-line focusing. Users were significantly more comfortable in the neck and shoulder regions after switching to the ergonomic design, indicating that redesigning microscopes with posture and ease of manipulation in mind will help to reduce discomfort associated with long hours of use. The study also showed that symptoms of eye fatigue and midback discomfort decreased, though not to a statistically significant degree. Eliminating or reducing eye fatigue is most easily accomplished by equipping the microscope with a digital video camera that displays specimen images on a television screen or computer monitor. As mentioned above, this allows operators who have eye problems, such as myopia and astigmatism, to comfortably wear their glasses during inspection of specimens. New Microscope Designs

The microscope manufacturers have been instrumental in stepping up the pace of ergonomic development with new designs featuring the latest technology to ease operator fatigue and reduce stress levels and associated health problems. Microscopes can be broadly divided into four categories: stereomicroscopes, upright compound microscopes, reflected light microscopes, and inverted microscopes. Each class is designed for a specific type of observation, and each has its own unique ergonomic requirements, although there are many shared attributes. The most important features are operator control, posture boundaries, eye-level adjustment, stage placement, instrument body and stand rigidity, and specimen handling. All of these specifications are discussed in detail for each microscope class in the following sections. Stereomicroscopes Stereomicroscopes, the largest segment of the microscope market with a share of about 50 percent of total microscope sales, are the focus of many new ergonomic features. Each year over 20,000 of these ubiquitous and popular instruments are sold to educators, researchers, and industrial manufacturers. Traditionally, stereomicroscopes are constructed with the body and eye tubes mounted on a long pillar that allow specimens having a wide spectrum of sizes to be examined and manipulated. The eye tubes in older stereomicroscopes are fixed into position, usually at a 45 to 60 degree angle from the horizontal bench, and the focus knob is mounted high on the body near the pillar rack. This design has a number of significant drawbacks in terms of ergonomics and has taken a heavy toll in operator trauma over the years.

Recent advances in stereomicroscope design have addressed the many ergonomic issues facing manufacturers who employ large numbers of technicians that spend long periods examining and manipulating specimens with stereomicroscopes. The primary concentration has been on eyepiece tube inclination angles and the relative height of the tubes in relation to the laboratory bench top. Inclination angles have been modified in current microscope designs to include low eye-level and tilting tubes that offer a wide range of adjustments to fit the needs of operators who span a large range of body sizes and heights (Figures 2 to 4). These re-designed eye tubes allow comfortable observation regardless of whether the microscopist is sitting or standing, and enable the utilization of

eye-level risers, intermediate tubes, and photography ports without compromising operator comfort. Coupled to advanced eyepieces with a high eyepoints, diopter adjustments, and large field numbers, modern stereomicroscope observation ports have made significant strides in ergonomic design and have led to improved efficiency with fewer medical incidents. Another ergonomic feature incorporated into recent stereomicroscope designs involves lowering the position of focus knobs to ensure quick, accurate, and effortless focusing of specimens. Located within easy reach of the operator, the lowered focus knobs eliminate the need for an operator to twist shoulders while adjusting the microscope focus. This feature, alone, has significantly improved stereomicroscope operation, especially for technicians who must examine large complex objects that require constant focus adjustments. Presented in Figure 5 is an illustration of a late model state-of-the-art stereomicroscope designed by Nikon, the SMZ1500. This instrument features lowered focus knobs, an eyelevel riser intermediate piece, and ergonomic tilting eyepiece tubes to increase user comfort and reduce the amount of fatigue and stress associated with operation of the microscope. Also available for this microscope are a number of accessories including an ergonomic 1x objective, which allows the operator to control eye-level position, and extended eyepiece tubes that further modify the viewing angle. The eyepieces have an adjustable diopter range and are made with high eyepoints to ease the burden of wearing glasses while observing specimens in the microscope.

New ergonomic auxiliary objectives, designed specifically for stereomicroscopes, aid in establishing the correct eye-level position by allowing the operator to freely adjust focal lengths to match seating requirements. Streamlined microscope bases with a variety of illumination capabilities also enable operators to manipulate specimens while resting

their arms in a natural position. Longer working distance objectives that offer excellent aberration correction, high numerical apertures, and improved light transmission add further to operator comfort and reduction of fatigue. Traditional Upright Compound Microscopes Next to stereomicroscopes, upright compound microscopes command the most attention and are sold by the thousands every year. The design of these microscopes has also received considerable attention from the manufacturers with respect to ergonomic factors. Included in the new list of features are one-handed stage and focus control, optimum eyelevel positioning (Figure 2), low-profile stages, rigid instrument body standards, and strain-free posture for operators while examining specimens. Perhaps the most significant feature of new compound microscope designs is positioning of the stage handle and focus control knobs in an equidistant configuration from the operator (Figure 6). This allows a more relaxed posture with the hands resting comfortably on the desktop. In addition, operators no longer need to twist their shoulders to simultaneously manipulate the stage and focus controls, considerably reducing the amount of strain associated with long-term observations. Other important microscope controls, such as the field diaphragm, light intensity potentiometer, and auto-illumination photomicrography preset switch, are located in the front of the microscope base at desktop height and within easy reach of the operator (see Figure 6 for details). Pushbutton filter engagement levers are also positioned near the main controls to further enhance operational ease. Many microscopes are equipped with a refocusing stopper that allows quick change of specimens with an instant return to focus. In addition, coarse focus tension controls allow the operator to customize the action of z-direction stage translation to suit individual tastes. A few microscopes also offer detachable fine focusing knobs that can be fitted to either side of the instrument to suit the operator's preferences, and some knobs are coated to enhance traction and allow easy operation with only a single finger.

Lowered stage assemblies, in recent instrument designs, have heights ranging from approximately five to eight inches, considerably shorter than those found on previous microscopes. Many also feature space-saving dual pulley or pulley and guide rod systems that replace older rack and pinion mechanisms to govern stage movement. Some models feature focusing adjustments that translate the nosepiece, rather than the entire stage, to maintain a constant stage height, reducing the amount of forearm movement necessary to move the stage or change the specimen. More advanced designs eliminate protrusion of the x-directional guide from the side of the stage, thus reducing interference with focusing action. Rotational movement of modern stages often exceeds 200 degrees and some can even achieve over 250 degrees of rotation about the microscope optical axis. Any microscopist who has spent time framing images for capture on film or by digital imaging will appreciate this advanced feature, which not only saves time, but a considerable amount of frustration. Eyepiece tube angles are almost infinitely adjustable on some of the latest microscope designs (Figures 2 and 4). In addition to a tilt range between 25 and 40 degrees, some binocular tubes have a telescoping adjustment that enables the eyepieces to be moved backward and forward over a span of 30-50 millimeters to accommodate the operator. Microscopes can be fitted with eye-level riser blocks to increase the height of eyepiece tubes and some have eyepoint adjusters, which are mounted on swivel joints to increase or decrease the eyepoint position (Figure 4). The result is that almost every microscopist, regardless of size and height, can peer straight into the eyepieces without tilting the head, a considerable improvement in design that ensures a comfortable working position. Even when the posture inevitably changes after several hours of observation, the eyepoint can be adjusted to match the new position to reduce fatigue. Easy diopter and interpupillary diameter adjustments in modern eyepieces also serve to reduce operator discomfort, and reduced diameter eyepiece lenses in some models aid observation by operators with deep eye sockets. A majority of new microscope designs incorporate pre-centered illuminators allowing rapid bulb replacement, and some even feature pre-centered condensers. These elements allow the operator to concentrate on specimen observation with a minimum of disruption to reconfigure the microscope after lamp failure. In addition, some objectives are intensity equalized to reduce the necessity to adjust illumination intensity when changing objectives, thus eliminating the discomfort of exposure to sudden changes in light intensity when rotating objectives. Designs incorporating lowered nosepieces also reduce operator fatigue by allowing the arms to rest on the desktop while changing objectives. Modern microscopes are designed with the help of computer-aided engineering (CAE) to achieve both high vibration resistance and structural rigidity to ensure that these instruments operate at peak optical levels. Vibrations and base deformations lead to image deterioration, which is manifested in bad photomicrographs and/or digital images. This is also a leading cause of fatigue and frustration by the microscopist intent on capturing the best images possible. Collectively, the new ergonomic features on today's upright compound microscopes enable observations over extended periods of time with a

minimum of stress and fatigue, enhancing both the performance and efficiency of the microscopist. Reflected Light Microscopes Industrial reflected light widefield and confocal microscopes are becoming increasingly popular as process monitoring and quality control tools in the booming semiconductor industry. Operators attending workstation monitors in wafer assembly plants often spend up to eight hours a day examining integrated circuits for defects, improper mask alignment, and process errors. Paramount in the development of these microscopes is an advanced optical system providing superior illumination, greater depth of focus, highly corrected objectives, and contrast enhancing light conditioners. The instruments are often equipped with specialized stages designed specifically for rapid manipulation of wafers having varying dimensions (Figure 7). To increase stability, semiconductor inspection microscopes are built with rigid and sturdy bases having sufficient weight to resist blur and image shifts induced by floor vibrations.

Several standards have been adapted by the semiconductor industry (SEMI S2-93A and S8-95) to provide safety and ergonomic features for microscopes designed to be operated in wafer fabrication plants. Among the requirements for these instruments are controls and knobs that are positioned low and close to the operator while the eyepoint is set at the proper height for comfortable operation. The manufacturers have designed microscopes that have focus, illumination, and objective controls positioned in the base, beneath the

stage and directly in front of the operator or in a separate keypad that can be conveniently located. These features minimize hand movement and enable "blind" operation, which reduces the distraction of a search for adjustment knobs or manual rotation of the objectives. In addition, most models have eyepieces moved to a forward position on the microscope where they are located closer to the operator, allowing a more erect seated posture. The latest microscopes are equipped with tilting eyepiece tubes that provide continuous adjustment of the tilt angle through a range of zero (horizontal) to 45 degrees for viewing at the optimum eyepoint level. Some models also have telescoping tubes that enable the operator to shift the viewing position closer or farther away from the microscope body and some even sport image-erecting prisms. These features are important in situations where operators must inspect wafers in a standing position, and are thus subject to a more rapid onset of fatigue. Stage evolution has also progressed in the design of modern industrial microscopes. Many feature custom wafer and mask holders that attach directly to the manually operated or motorized stages or allow the microscope to be interfaced with wafer loaders. Stage control has also been the focus of ergonomic considerations, and the x-y fine movement translators in many semiconductor microscopes do not move with the stage. Instead, they are placed close to the front of the microscope in a lowered position, which allows the operator to control movement without lifting the arms. These controls are also located near focus, illumination, and objective rotation buttons so the microscope can be operated with one hand. In addition, many stages feature a coarse/fine selectable movement control having an electric clutch handle that allows the operator to quickly navigate to a selected area, then zero in on fine details with a minimum of effort.

Other features, such as motorized nosepieces, tilting breath shields, remote focus handles and jog dials simplify operation of industrial microscopes. High-end models feature illumination blanking when objectives are rotated to protect the operator's eyes from bright light flashes. Remote keypads on reflected light confocal and widefield microscopes now control epi-polarizer rotation, aperture diaphragm opening size, epi/diascopic illumination selection, focus, stage position, and observation mode selection. Together, these ergonomic considerations have dramatically improved the efficiency of semiconductor inspection operations, while simultaneously reducing the health risks associated with long hours spent at the microscope. Inverted Microscopes A dramatic transition of fluorescence microscopy and electrophysiology to the forefront of biomedical research has occurred over the past ten years. In this regard, inverted microscopes designed to examine and manipulate cells and tissues in culture have simultaneously acquired ergonomic features to increase operator comfort and reduce fatigue. Frequently utilized control elements, such as focus, illumination intensity, optical path direction, and stage translation, have been moved to the front of these instruments to increase operation efficiency and minimize operator stress and strain. Like other modern designs, inverted microscopes are equipped with adjustable eyepiece tubes that allow the operator to alter the eyepoint height over a wide range. On many microscopes, the tubes can also be rotated over a 90+ degree range to increase flexibility in specimen examination and manipulation. Additional observation tube accessories include auxiliary magnification and Bertrand lenses (for phase ring alignment), and some models are equipped with a light-limiting shutter and built-in photo reticle. Stages on inverted microscopes have been redesigned to afford lower heights with ample room for incubators, large culture vessels, in vitro fertilization micromanipulator systems, electrophysiology attachments, and patch clamp accessories. Translation handles are often mounted with universal joints to allow simultaneous focus operations while the specimen is being scanned. Beneath the stage, larger nosepiece clearance and revolution orientation enables operators to identify and rotate objectives more quickly and efficiently. The combination of these features helps to reduce operator MSD complaints resulting from extended observation periods. Other features common on high-end research inverted microscopes include tilting illumination pillars, threaded installation holes for accessories, and condenser/stage refocusing stopper combinations. On inverted microscopes, the pillar supports a long working distance condenser and usually a tungsten halogen lamp house for brightfield, darkfield, phase contrast, and differential interference illumination. Some microscopes are equipped with a detachable pillar that can also be tilted backwards up to 45 degrees to allow micromanipulation equipment to be installed or to change specimens without raising the condenser. These features ease the burden of complex and repetitive microscope reconfiguration, and are a much-improved ergonomic design over previous models. Refocusing stoppers allow the operator to quickly rack down the coarse motion

to change specimens and return to the exact focus point. Likewise, condenser-refocusing stoppers eliminate the need for the operator to manually replace the condenser into the correct focal position. Once set, the stopper enables a quick changeover of specimens with a rapid return to the preset condenser focal point. Current inverted microscope bodies are now much sturdier and heavier than their former counterparts. This dramatically reduces vibration and provides long-term stability for examination and manipulation of specimens and the utilization of time-lapse cinemicrography digital imaging and photomicrography techniques. Collectively, the new ergonomic features on inverted microscopes have come a long way towards easing the fatigue associated with long periods on the microscope, while at the same time, increasing the efficiency and health of microscopists who spend countless hours investigating the mysteries of life. Aftermarket Ergonomic Products for Older Microscopes A number of aftermarket products designed to enhance the usability of microscopes are now available from a variety of manufacturers. Included in this new array of ergonomically designed add-ons are extended eye tubes for binocular observation heads (Figure 3), armrests, eye-level height extenders, lenticular microarrays to ease viewing specimens (Figures 9 and 10), digital viewing screens, bellows-type body extensions (Figure 8), optical wedges, and ocular-less digital imaging video systems. Extended eye tubes (Figures 1 and 3) are considerably longer than conventional models and enable the operator to move away from the bench while maintaining a more supported neutral posture during extended observation periods. Observation tube lengths are available in sizes up to 90 millimeters, which is ideal for microscopes placed near machines, heating stages, solder stations, or in fume hoods. In addition, the extended tubes provide an additional magnification factor of almost 2x, which compensates for the decrease in magnifying power accompanying long working distance objectives. Several models also offer larger interpupillary adjustments (up to 90 millimeters to accommodate all users) and are designed to maintain parfocality and true optical alignment throughout the working-angle range of the observation tubes. A useful and easily adapted product is the optical wedge, which adds flexibility in configuration of the eye level height. This attachment is positioned between the binocular head and the body of the microscope to provide additional viewing angles and added operator comfort. The optical wedge can be coupled to flexible body-extending adapters (Figures 1 and 3) to provide a wider range of eyepiece height adjustment, thus allowing the microscopist to assume a more neutral posture. A locking knob is utilized to modulate the adapter height, and most units of this type allow rotation of the observation tubes from side to side to increase flexibility in microscope configuration. Microscope positioning plates are also available to raise, lower, or tilt the microscope into a position that meets the ergonomic requirements of the operator's body dimensions. These devices consist of an adjustable base plate that enables the microscopist to raise the

instrument height between 1.5 and 4 inches, and stacker plates can be added to attain even greater heights. Individual legs on the positioning plates are adjustable to allow tilting of the microscope, and to provide a high degree of precision with respect to instrument height and viewing angle.

Increasing the field of view and the distance from the observer's eyes to the microscope has been the target of a growing number of aftermarket products. Utilizing lenticular array technology, Vision Engineering has introduced a new ergonomic accessory termed Isis, which can be retrofitted to existing microscopes by insertion into standard observation tubes (Figure 9). This product increases the effective distance of the eyes to about 38-40 millimeters from the eyepieces, expanding the pupil image and providing the operator greater freedom of head movement with better posture. The manufacturer also claims that Isis reduces the distraction of eye floaters, which move across the field of vision and are accentuated by viewing specimens in bright illumination. Lenticular array technology is based on a rotating optical disk that contains several million tiny individual microlenses (termed lenticules) that act in concert to expand the depth of focus and field of view when the disk is spun at high rotational speeds (Figure 10). The transparent disks are about 15 centimeters in diameter and are utilized in either a transmitted or reflected mode to enhance observation. Each of the individual lenticules is about 70 microns in size, but they are merged to deliver a smooth image when the disk is spun at speeds up to 3,500 rpm.

More advanced applications of lenticular array technology are manifested in microscope designs that contain viewing heads devoid of eyepieces (Figure 11). First applied to stereomicroscopes, this approach substitutes a lenticular array-driven screen that is positioned on top of the microscope body to allow the operator wide latitude in viewing angle and distance. Operators who wear eyeglasses can easily view specimens for long periods of time without the discomfort associated with repeated re-focusing when the glasses are removed. These microscopes are also designed with ergonomic standards in mind, and have focusing, zoom, and illumination adjustments that are positioned low on the base for easier operation. Another new technology roaming the horizon involves a microscope design devoid of eyepieces and lenticular arrays, which substitutes a CCD or CMOS image sensor to project the image on a computer monitor, as discussed above. Although this configuration is easily attainable with current instruments by simple addition of a digital camera system, the eyepiece-less microscopes are accompanied with software packages that aid in image capture, and feature a number of applications such as multiple file storage formats, digital image processing software, and time-lapse cinemicrography. The versatility of these systems should ease the fatigue and stress associated with long term microscope use and increase operator efficiency through enhanced software features.

The importance of proper alignment of the microscope optical components cannot be overstressed. Inadequate illumination, image deterioration from lens artifacts, improper use of filters, and other errors contribute not only to inferior images, but also increase the strain of imaging specimens. Each operator should be thoroughly trained in proper use of the microscope, including lamp changeovers and centration, optical alignment, correct filtration techniques, and image capture. Prevention On one account, Sir David Brewster was absolutely correct. Peering down vertically through a microscope is "the worst of all positions" for making observations. His suggestion that microscopists should lie on their backs may not be entirely feasible, but it does capture one essential truth. The body can endure stationary positions for extended periods if it is in a neutral posture, a position that can be maintained without a concerted effort or contortions. A neutral body posture is essential to working efficiently and effectively at the microscope for long hours. Not everyone is in a position to buy a new ergonomically designed microscope or workstation. For conventional microscope workstations, the key is finding ways to modify them to fit the user rather than forcing the user into awkward positions. Following are some basic guidelines for achieving and maintaining neutral body posture while using a microscope:

Eyes - eyepieces should rest just below the eyes with the eyes looking downward at an angle 30 to 45 degrees below the horizontal; interocular distance of

binocular eyepieces should be adjusted to ensure that both eyes are focusing comfortably. Neck - the neck and head should bend as little as possible, preferably no more than 10-15 degrees below the horizontal. Back - the individual should be sitting erect, leaning the entire body slightly forward with the lower back and shoulder blades supported by the chair and/or lumbar support cushion. Sitting for long periods places undue strain on the lower back, which can be alleviated with the proper support. Arms/wrist - the upper arms should be perpendicular to the floor, elbows close to the body (not winged or sticking out), the forearms parallel to the floor; wrists should be straight. Legs - feet should rest firmly on the floor or a footrest, and even pressure should be applied by the chair to the back of the thighs.

To further reduce ergonomic risk factors:

Develop an awareness of posture. Try to maintain the natural curve of the lower back when sitting. Use additional lumbar support if necessary. If the foot ring on a lab stool is too low, raise it to keep the lower back supported by the chair back. Often, laboratory bench leg-wells are utilized as storage facilities for seldom-used equipment and extra supplies. Clear this area so that legs and feet are not impeded while sitting at the bench. Don't lean forward to look through the microscope. Instead, adjust the position of the chair, workstation or microscope to keep the back straight and the head upright. The eyepieces should be in line with, or even extended over, the edge of the bench. If the microscope is too low, raise it by placing a book underneath it or modify the configuration with OEM or aftermarket accessories to keep the head upright. If microscope eye-level risers are not readily available, use a three-ring binder to tilt the microscope so the eyepieces are placed at a more suitable angle. For a longterm solution, either purchase a suitable OEM or aftermarket stand or have the local physical plant construct one adequate for the purpose. Adjust the height of the microscope, bench, or chair to avoid bending or extending the neck, or jutting the chin forward. If standing, the operator should have anti-fatigue mats installed at the microscope workstation to ease the burden on the feet, legs, and lower back. Check the seat platform for tilt and height to maintain even pressure along the back of the thighs. In situations where it is possible, use an industrial-height footstool for better posture and position. This allows the operator to bend forward at the hips rather than the neck, back, and shoulders. Avoid contact stress from forearms resting on sharp bench or counter edges by adding padded edge protectors. Operating the focus and stage controls with the arms separated from the bench (lifted) for extended periods can induce static loading fatigue, which can be reduced with the proper support, such as padded and tilted armrests. Also, if laboratory geometry permits, utilization of cut-out work tables or laboratory benches with recessed tops allow the operator to spread

out and more efficiently employ auxiliary equipment necessary for microscopy observations and manipulations. Ensure the microscope optical train is configured properly and the illumination source is aligned and performing at capacity. Adjust eyepiece interpupillary distance, diopter settings, and check the parfocality. The eyepieces should be approximately the same distance from the observer's eyes, rather than one being closer than the other. Eyepoints should be high enough that the field of vision is completely filled, but far enough away so as to avoid contact of eyepieces with the eyelashes. If the eyepieces are not properly focused, the eyes tend to compensate, which leads to increased headaches and eye fatigue. Buy plancorrected objectives that produce flat viewfields. Microscopes with significant field curvature are difficult to use, especially for extended periods of time in which the operator must be continuously refocusing the specimen to examine the entire field. Excessive microscope illumination can cause an uncomfortably high level of light and contrast, which is easily reduced by proper configuration of the lamp voltage and the condenser aperture. All of these factors are the primary cause of eyestrain. Operators that wear eyeglasses can adjust the eyepieces to accommodate near and far sightedness, but those who have more severe conditions should see an optician to determine whether they are suited for extended observation periods using a microscope. Simple adjustment of the eyepiece diopter cannot correct for astigmatism and some of the other, more serious, visual difficulties. In cases of extreme astigmatism and fusion insufficiency (poor eye coordination), the operator may require assistance of digital video equipment and a computer monitor or television screen to enhance, or replace, the eyepieces. Check the laboratory environment for excessive glare and reflections from overhead fluorescent lighting, and adjust external and internal microscope light to compensate for this artifact. Other environmental factors such as temperature, humidity, air currents, ventilation, excessive noise, and ambient lighting levels will also affect operator comfort and fatigue, especially over extended periods of time. Adjust these variables, whenever possible, to make the laboratory environment as comfortable as possible. The nominal temperature range should lie between 19 and 23 degrees Centigrade (66 to 73 degrees Fahrenheit), with a relative humidity averaging between 40 and 60 percent. Low humidity conditions lead to drying of the eyes, which further aggravates eyestrain. Regular breaks from the microscope, ranging from five to ten minutes per hour, are essential to reduce fatigue, especially for operators who work at microscope workstations for six to eight-hour shifts. Periodic resting of the eyes, neck, and shoulders allows operators to work for extended periods without experiencing stress-related injuries. Bending, flexing, rotation, extension, and stretching exercises during these breaks often helps to alleviate stress and will greatly benefit the operator's health in the long term. In fact, some companies have implemented a routine exercise program during short break periods. Another mechanism employed to relieve fatigue is to intermix other duties on a regular basis to reduce the length of microscopy sessions.

The amount of time a microscopist spends at a workstation should be taken into account when evaluating workstation modifications. The minimum requirement for all seated workstations is good seating, with an adjustable chair and a footrest if necessary; antifatigue mats should be used for workstations that require the microscopist to stand. The chair should have a pneumatic, adjustable seat pan with a sloping "water fall" edge, a backrest that is adjustable for both height and angle, height adjustable armrests, and a five star base with caster wheels. For some seated workstations, a footrest may be appropriate. It should provide stability and firm contact with the floor and a surface texture that keeps the feet from sliding off. It should be readily adjustable to accommodate varying user heights and have an angle of approximately 10 degrees. Toes should be above the heel, allowing the lower leg muscles to stretch. Additional recommendations based on time spent per day at the microscope: 1-2 hours/day

Adequate clearance (a minimum of 2 inches) between the thigh and desk or counter with the leg-well free from obstructions. 2-4 hours/day

Microscope tilted slightly forward or utilization of wedges, extenders, and/or eyelevel adjustments. Proper arm support, keeping the limbs close to the body with the forearm parallel to the floor and resting on the bench top. Use armrests for older microscopes having controls located in high positions. Padded edges for workstations or countertops to avoid contact stress on arms. 4-6 hours/day

Adjustable microscope eyepieces should be installed, if possible. Electrically powered focus adjustments and objective rotation if more than half the total time on the microscope is spent twisting the coarse and fine knobs while transitioning the magnification factor. 6 hours or more/day

Adjustable microscope eyepieces and ergonomically positioned microscope controls. Electrically powered focus and objective rotation. If configurations permit, powered control of the condenser aperture diaphragm, illumination intensity, and beamsplitters. Video monitor or television screen for examination of repetitive specimens (the monitor should be placed in the operator's primary field of view).

Easily adjustable work surface variables, such as bench height, armrest base angle, observation eye-level, and microscope height (essential in a multi-user workstation environment).

Microscopists can also benefit from general workplace ergonomics. Reduce fatigue by reducing or eliminating highly repetitive tasks and take micro-breaks, 20-180 seconds at 10 to 15 minute work intervals. Use this time to stand and/or stretch, and allow the eyes to focus at a distance. Objects that must be accessed frequently should be kept close enough to avoid having to stretch and strain, usually within a distance of 9-19 inches. Less frequently utilized objects can be kept at a distance of 9-25 inches. Conclusion OSHA is continuing to formulate new ergonomics standards that will require employers to assess employee exposure to ergonomic risk factors in general industry jobs. The governmental organization estimates that the new standards, if implemented, will save employers $9.1 billion annually for the next 10 years, and prevent 460,000 reported MSDs a year (perhaps even more, if unreported cases are included). Among the concerns of OSHA officials is that basic information about common MSDs, risk factors, and the importance of reporting symptoms be impressed upon employees who must spend a significant portion of their work day on the microscope. Although many of the ergonomic requirements are now being addressed by microscope manufacturers, there are a considerable number of microscopes "in the field" that are poorly equipped to provide worker comfort and reduce the incidence of injuries. Over time, these microscopes will be replaced with modern, ergonomic-friendly versions, but in the meantime, employers should be concerned about potential medical problems that may arise from extended microscope use. If a worker's job routinely involves exposure to one or more of five known ergonomic risk factors: repetition, force, awkward postures, contact stress and vibration, then some adjustment of the work environment is necessary. Aftermarket accessories, which are available for a wide spectrum of microscopes, may be the answer for a majority of older instruments in the interim. However, the end result should be migration to microscopes designed to optimize both operator safety and comfort, while providing the latest features in regard to optical quality and performance.

Mechanical Tube Length

The mechanical tube length of an optical microscope is defined as the distance from the nosepiece opening, where the objective is mounted, to the top edge of the observation tubes where the eyepieces (oculars) are inserted. The drawing in Figure 1 illustrates the optical path (the red line) defining the mechanical tube length for a typical transmitted light microscope.

For many years, almost all prominent microscope manufacturers designed their objectives for a finite tube length. The designer proceeded under the assumption that the specimen, at focus, was placed at a distance a "little" further than the front focal plane of the objective. The objective then projects a magnified image of the specimen which converges (is brought into focus) at the level of the eyepiece diaphragm, located ten millimeters below the top edge of the openings of the microscope observation tube-where the eyepieces are inserted (see Figures 1 and 2). Tube length has now been standardized to the Royal Microscopical Society (RMS) suggestion of 160 millimeters for finite-corrected transmitted light microscopes. Objectives designed for a 160 millimeter finite tube length microscope bear the inscription "160" (mm) on the barrel as outlined in our discussion on objective specifications and identification. The positioning of the ocular and objectives is reversed in metallographs, which are essentially inverted reflected light microscopes as illustrated in figure 2. Note that in both the examples depicted in Figures 1 and 2, the "tube" is not a straight line and the light waves are transmitted from the objectives to the eyepieces (oculars) with mirrored beamsplitters. This is the case with most modern microscopes, especially those equipped with trinocular heads for photomicrography.

Some older microscopes have mechanical tube lengths that deviate from the 160 millimeter standard. Microscopes produced by Leitz instruments continued to be manufactured with a 170 millimeter tube length long after the RMS standard had been incorporated by other manufacturers. We caution microscopists who attempt to insert objectives designed for one mechanical tube length into a microscope designed for a different tube length. When objectives and tube lengths are mismatched, image quality often suffers due to the introduction of spherical aberrations because the optical tube length is changed. The optical tube length is defined as the distance between the objective rear focal plane and the intermediate or primary image at the fixed diaphragm of the eyepiece. When this tube length is altered to deviate from design specifications, spherical aberrations are introduced into the microscope and images suffer from deterioration in optical quality. Under circumstances where an objective designed for a 170 millimeter tube length is used in a microscope with a 160 millimeter tube length, corrections designed into the objective will cause it to under-compensate for aberrations. The opposite is true when 160 millimeter objectives are used in a 170 millimeter tube length microscope. With a finite tube length microscope system, whenever an accessory such as a polarizing intermediate piece, a DIC Wollaston prism, or a fluorescence illuminator, is placed in the light path between the back of the objective and the eyepiece, the mechanical tube length becomes greater than 160 millimeters. Aberrations may then be introduced when the specimen is refocused. As a result, each such accessory in a finite system must contain optical elements to bring the tube length ostensibly back to 160 millimeters. Often such devices result in a undesirable increase in magnification and lower the overall intensity of

the image. There is also the danger of producing "ghost images"the result of converging rays passing through the beamsplitter of a reflected light accessory.

Specifications and Identification

Identification of the properties of individual objectives is usually very easy because important parameters are often inscribed on the outer housing (or barrel) of the objective itself as illustrated in Figure 1. This figure depicts a typical 60x plan apochromat objective, including common engravings that contain all of the specifications necessary to determine what the objective is designed for and the conditions necessary for proper use.

Microscope manufacturers offer a wide range of objective designs to meet the performance needs of specialized imaging methods, to compensate for cover glass thickness variations, and to increase the effective working distance of the objective. Often, the function of a particular objective is not obvious simply by looking at the construction of the objective. Finite microscope objectives are designed to project a diffraction-limited image at a fixed plane (the intermediate image plane), which is dictated by the microscope tube length and located at a pre-specified distance from the rear focal plane of the objective. Microscope objectives are usually designed to be used with a specific group of oculars and/or tube lenses strategically placed to assist in the removal of residual optical errors. As an example, older Nikon and Olympus compensating eyepieces were used with high numerical aperture fluorite and apochromatic objectives to eliminate lateral chromatic aberration and improve flatness of field. Newer microscopes (from Nikon and Olympus) have objectives that are fully corrected and do not require additional corrections from the eyepieces or tube lenses. Most manufacturers have now transitioned to infinity-corrected objectives that project emerging rays in parallel bundles from every azimuth to infinity. These objectives require a tube lens in the light path to bring the image into focus at the intermediate image plane. Infinity-corrected and finite-tube length microscope objectives are not interchangeable

and must be matched not only to a specific type of microscope, but often to a particular microscope from a single manufacturer. For example, Nikon infinity-corrected objectives are not interchangeable with Olympus infinity-corrected objectives, not only because of tube length differences, but also because the mounting threads are not the same pitch or diameter. Objectives usually contain an inscription denoting the tube focal length as will be discussed. There is a wealth of information inscribed on the barrel of each objective, which can be broken down into several categories. These include the linear magnification, numerical aperture value, optical corrections, microscope body tube length, the type of medium the objective is designed for, and other critical factors in deciding if the objective will perform as needed. A more detailed discussion of these properties is provided below and in links to other pages dealing with specific issues.

Manufacturer - The name of the objective manufacturer is almost always included on the objective. The objective illustrated in Figure 1 was made by a fictitious company named Nippon from Japan, but comparable objectives are manufactured by Nikon, Olympus, Zeiss, and Leica, companies who are some of the most respected manufacturers in the microscope business. Linear Magnification - In the case of the apochromatic objective in Figure 1, the linear magnification is 60x, although the manufacturers produce objectives ranging in linear magnification from 0.5x to 250x with many sizes in between. Optical Corrections - These are usually listed as Achro and Achromat (achromatic), as Fl, Fluar, Fluor, Neofluar, or Fluotar (fluorite) for better spherical and chromatic corrections, and as Apo (apochromatic) for the highest degree of correction for spherical and chromatic aberrations. Field curvature corrections are abbreviated Plan, Pl, EF, Achroplan, Plan Apo, or Plano. Other common abbreviations are ICS (infinity corrected system) and UIS (universal infinity system), N and NPL (normal field of view plan), Ultrafluar (fluorite objective with glass that is transparent down to 250 nanometers), and CF and CFI (chrome-free; chrome-free infinity). The objective in the illustration (Figure 1) is a plan apochromat that enjoys the highest degree of optical correction. See Table 1 for a complete list of abbreviations often found inscribed on objective barrels.
Specialized Objective Designations

ABBREVIATION Achro, Achromat Fluor, Fl, Fluar, Neofluar, Fluotar Apo Plan, Pl, Achroplan, Plano EF, Acroplan N, NPL

TYPE Achromatic aberration correction Fluorite aberration correction Apochromatic aberration correction Flat Field optical correction Extended Field (field of view less than Plan) Normal field of view plan

Plan Apo UPLAN LU L, LL, LD, LWD ELWD SLWD ULWD Corr, W/Corr, CR I, Iris, W/Iris Oil, Oel Water, WI, Wasser HI Gly DIC, NIC CF, CFI ICS RMS M25, M32 Phase, PHACO, PC Ph 1, 2, 3, etc. DL, DLL, DM, BM PL, PLL PM, PH

NL, NM, NH P, Po, Pol, SF U, UV, Universal M NC, NCG EPI TL BBD, HD, B/D

Apochromatic and Flat Field correction Olympus Universal Plan (Brightfield, Darkfield, DIC, and Polarized Light) Nikon Luminous Universal (Brightfield, Darkfield, DIC, and Polarized Light) Long Working Distance Extra-Long Working Distance Super-Long Working Distance Ultra-Long Working Distance Correction Collar Adjustable numerical aperture (with iris diaphragm) Oil Immersion Water Immersion Homogeneous Immersion Glycerin Immersion Differential or Nomarski Interference Contrast Chrome-Free, Chrome-Free Infinity-Corrected (Nikon) Infinity Color-Corrected System (Zeiss) Royal Microscopical Society objective thread size Metric 25-mm objective thread; Metric 32-mm objective thread Phase Contrast Phase Condenser Annulus 1, 2, 3, etc. Phase Contrast: Dark Low, Dark Low Low, Dark medium, Bright Medium Phase Contrast: Positive Low, Positive Low Low Phase Contrast: Positive Medium, Positive High Contrast (Regions with higher refractive index appear darker.) Phase Contrast: Negative Low, Negative Medium, Negative High Contrast (Regions with higher refractive index appear lighter.) Strain-Free, Low Birefringence, for Polarized Light UV transmitting (down to approximately 340 nm) for UV-excited epifluorescence Metallographic (no coverslip) No Coverslip Oblique or Epi illumination Transmitted Light Bright or Dark Field (Hell, Dunkel)

D H U, UT DI, MI, TI

Darkfield For use with a heating stage For use with a universal stage Interferometry, Noncontact, Multiple Beam (Tolanski)
Table 1

Numerical Aperture - This is a critical value that indicates the light acceptance angle, which in turn determines the light gathering power, the resolving power, and depth of field of the objective.

Numerical Aperture

Investigate how the size of the light cone entering the objective front lens changes with the objective numerical aperture value. Start Tutorial Some objectives specifically designed for transmitted light fluorescence and darkfield imaging are equipped with an internal iris diaphragm that allows for adjustment of the effective numerical aperture. Abbreviations inscribed on the barrel for these objectives include I, Iris, and W/Iris. The 60x apochromat objective illustrated above has a numerical aperture of 1.4, one of the highest attainable in modern microscopes using immersion oil as an imaging medium.

Mechanical Tube Length - This is the length of the microscope body tube between the nosepiece opening, where the objective is mounted, and the top edge of the observation tubes where the oculars (eyepieces) are inserted. This aspect of microscope design is discussed in more thoroughly in our mechanical tube length section of the primer. Tube length is usually inscribed on the objective as the size in number of millimeters (160, 170, 210, etc.) for fixed lengths, or the infinity symbol () for infinity-corrected tube lengths. The objective illustrated in Figure 1 is corrected for a tube length of infinity, although many older objectives will be corrected for tube lengths of either 160 (Nikon, Olympus, Zeiss) or 170 (Leica) millimeters. Cover Glass Thickness - Most transmitted light objectives are designed to image specimens that are covered by a cover glass (or cover slip). The thickness of these small glass plates is now standardized at 0.17 mm for most applications, although there is often some variation in thickness within a batch of coverslips. For this reason, some of the more advanced objectives have a correction collar

adjustment of the internal lens elements to compensate for this variation. Abbreviations for the correction collar adjustment include Corr, w/Corr, and CR, although the presence of a movable, knurled collar and graduated scale is also an indicator of this feature. Cover Glass Correction

Discover how internal lens elements in a high numerical aperture dry objective may be adjusted to correct for fluctuations in cover glass thickness. Start Tutorial The interactive Java tutorial linked above allows the visitor to adjust the correction collar on a microscope objective. There are some applications that do not require objectives to be corrected for cover glass thickness. These include objectives designed for reflected light metallurgical specimens, tissue culture, integrated circuit inspection, and many other applications that require observation with no compensation for a cover glass.

Working Distance - This is the distance between the objective front lens and the top of the cover glass when the specimen is in focus. In most instances, the working distance of an objective decreases as magnification increases. Working distance values are not included on all objectives and their presence varies depending upon the manufacturer. Common abbreviations are: L, LL, LD, and LWD (long working distance), ELWD (extra-long working distance), SLWD (super-long working distance), and ULWD (ultra-long working distance). Newer objectives often contain the size of working distance (in millimeters) inscribed on the barrel. The objective illustrated in Figure 1 has a very short working distance of 0.21 millimeters. Specialized Optical Properties - Microscope objectives often have design parameters that optimize performance under certain conditions. For example, there are special objectives designed for polarized illumination signified by the abbreviations P, Po, POL, or SF (strain-free and/or having all barrel engravings painted red), phase contrast (PH, and/or green barrel engravings), differential interference contrast (DIC), and many other abbreviations for additional applications. A list of several abbreviations, often manufacturer specific, is presented in Table 1. The apochromat objective illustrated in Figure 1 is optimized for DIC photomicrography and this is indicated on the barrel. The capital H beside the DIC marking indicates that the objective must be used with a specific DIC Wollaston prism optimized for high-magnification applications.
Objective Numerical Aperture and Working Distance

Optical Correction* Numerical and Aperture Magnification ACH 10x 0.25 ACH 20x 0.40 ACH 40x 0.65 ACH 60x 0.80 ACH 100x (Oil) 1.25 PL 4x 0.10 PL 10x 0.25 PL 20x 0.40 PL 40x 0.65 PL 100x (Oil) 1.25 PL FL 4x 0.13 PL FL 10x 0.30 PL FL 20x 0.50 PL FL 40x 0.75 PL FL 100x (Oil) 1.30 PL APO 1.25x 0.04 PL APO 2x 0.06 PL APO 4x 0.16 PL APO 10x 0.40 PL APO 20x 0.70 PL APO 40x 0.85 PL APO 60x (Oil) 1.40 PL APO 100x (Oil) 1.40 *Abbreviations: ACH, Achromat PL FL, Plan Fluorite PL APO, Plan Apochromat
Table 2

Working Distance (Millimeters) 6.10 3.00 0.45 0.23 0.13 22.0 10.5 1.20 0.56 0.15 17.0 10.00 1.60 0.51 0.10 5.1 6.20 13.00 3.10 0.65 0.20 1.10 0.10

Objective Screw Threads - The mounting threads on almost all objectives are sized to standards of the Royal Microscopical Society (RMS) for universal compatibility. The objective in Figure 1 has mounting threads that are 20.32 mm in diameter with a pitch of 0.706, conforming to the RMS standard. This standard is currently used in the production of infinity-corrected objectives by manufacturers Olympus and Zeiss. Nikon and Leica have broken from the standard with the introduction of new infinity-corrected objectives that have a wider mounting thread size, making Leica and Nikon objectives usable only on their own microscopes. Nikon's reasoning is explained in our section describing

the Nikon CFI60 200/60/25 Specification for biomedical microscopes. Abbreviations commonly used to denote thread size are: RMS (Royal Microscopical Society objective thread), M25 (metric 25-millimeter objective thread), and M32 (metric 32-millimeter objective thread). Immersion Medium - Most objectives are designed to image specimens with air as the medium between the objective and the cover glass.

Immersion Oil and Refractive Index

Explore how variations in the refractive index of the imaging medium effect the ability of an objective to capture light rays emanating from the specimen. Start Tutorial To attain higher working numerical apertures, many objectives are designed to image the specimen through another medium that reduces refractive index differences between glass and the imaging medium. High-resolution plan apochromat objectives can achieve numerical apertures up to 1.40 when the immersion medium is special oil with a refractive index of 1.51. Other common immersion media are water and glycerin. Objectives designed for special immersion media usually have a color-coded ring inscribed around the circumference of the objective barrel as listed in Table 3 and described below. Common abbreviations are: Oil, Oel (oil immersion), HI (homogeneous immersion), W, Water, Wasser (water immersion), and Gly (glycerol immersion).

Color Codes - Microscope manufacturers label their objectives with color codes to help in rapid identification of the magnification and any specialized immersion media requirements. The dark blue color code on the objective illustrated in Figure 1 indicates the linear magnification is 60x. This is very helpful when you have a nosepiece turret containing 5 or 6 objectives and you must quickly select a specific magnification. Some specialized objectives have an additional color code that indicates the type of immersion medium necessary to achieve the optimum numerical aperture. Immersion lenses intended for use with oil have a black color ring, and those intended for use with glycerin have an orange ring, as illustrated with the objective on the left in Figure 2. Objectives designed to image living organisms in aqueous media are designated water immersion objectives with a white ring, and highly specialized objectives for unusual immersion media are often engraved with a red ring. Table 3 lists current magnification and imaging media color codes in use by most manufacturers.
Objective Color Codes

Magnification 1/2x 1x 1.25x 1.5x 2x 2.5x 4x 5x 10x 16x 20x 25x 32x 40x 50x 60x 63x 100x 150x 250x Immersion Media Oil Glycerol Water Special

Color Code No Color Assigned Black Black Black Brown (or Orange) Brown (or Orange) Red Red Yellow Green Green Turquoise Turquoise Light Blue Light Blue Cobalt Blue Cobalt Blue White White White Color Code Black Orange White Red

Table 3

Special Features - Objectives often have additional special features that are specific to a particular manufacturer and type of objective. The plan apochromat objective illustrated in Figure 1 has a spring-loaded front lens to prevent damage when the objective is accidentally driven onto the surface of a microscope slide.

Other features found on specialized objectives are variable working distance (LWD) and numerical aperture settings that are adjustable by turning the correction collar on the body of the objective as illustrated in Figure 2. The plan fluor objective on the left has a variable immersion medium/numerical aperture setting that allows the objective to be used with both air and an alternative liquid immersion medium, glycerin. The plan apo objective on the right has an adjustable working distance control (termed a "correction collar") that allows the objective to image specimens through glass coverslips of variable thickness. This is especially important in dry objectives with high numerical aperture that are particularly susceptible to spherical and other aberrations that can impair resolution and contrast when used with a cover glass whose thickness differs from the specified design value. Although not common today, other types of adjustable objectives have been manufactured in the past. Perhaps the most interesting example is the compound "zoom" objective that has a variable magnification, usually from about 4x to 15x. These objectives have a short barrel with poorly designed optics that have significant aberration problems and are not very practical for photomicrography or serious quantitative microscopy. Parfocal Distance - This is another specification that can often vary by manufacturer. Most companies produce objectives that have a 45 millimeter parfocal distance, which is designed to minimize refocusing when magnifications are changed.

The objective depicted on the left in Figure 3 has a parfocal distance of 45mm and is labeled with an immersion medium color code in addition to the magnification color code. Parfocal distance is measured from the nosepiece objective mounting hole to the point of focus on the specimen as illustrated in the figure. The objective on the right in Figure 3 has a longer parfocal distance of 60 millimeters, which is the result of its being produced to the Nikon CFI60 200/60/25 Specification, again deviating from the practice of other manufacturers such as Olympus and Zeiss, who still produce objectives with a 45mm parfocal distance. Most manufacturers also make their objective nosepieces parcentric, meaning that when a specimen is centered in the field of view for one objective, it remains centered when the nosepiece is rotated to bring another objective into use. Glass Design - The quality of glass formulations has been paramount in the evolution of modern microscope optics, and there are currently several hundred of optical glasses available for the design of microscope objectives. The suitability of glass for the demanding optical performance of a microscope objective is a function of its physical properties such as refractive index, dispersion, light transmission, contaminant concentrations, residual autofluorescence, and overall homogeneity throughout the mixture. Care must be taken by optical designers to ensure that glass utilized in highperformance objectives has a high transmission in the near-ultraviolet region and also produces high extinction factors for applications such as polarized light or differential interference contrast. Cements employed in building multiple lens elements usually have a thickness around 510 microns, which can be a source of artifacts in groups that have three or more lens elements cemented together. Doublets, triplets, and other multiple lens arrangements can display spurious absorption, transmission, and fluorescence characteristics that will disqualify the lenses for certain applications. For many years, natural fluorite was commonly used in the manufacture of fluorite (semiapochromat) and apochromat objectives. Unfortunately, many newly developed fluorescence techniques often rely on ultraviolet excitation at wavelengths significantly

below 400 nanometers, which is severely compromised by autofluorescence that occurs from natural organic constituents present in this mineral. Also, the tendency of natural fluorite to exhibit widespread localized regions of crystallinity can seriously degrade performance in polarized light microscopy. Many of these problems are circumvented with new, more advanced materials, such as fluorocrown glass. Annealing of optical glass for the manufacture of objectives is critical in order to remove stress, improve transmission, and reduce the level of other internal imperfections. Some of the glass formulations intended for apochromat lens construction are slow-cooled and annealed for extended periods, often exceeding six months. True apochromat objectives are manufactured with a combination of natural fluorite and other glasses that have reduced transmission in the near-ultraviolet region. Extra Low Dispersion (ED) glass was introduced as a major advancement in lens design with optical qualities similar to the mineral fluorite but without its mechanical and optical demerits. This glass has allowed manufacturers to create higher quality objectives with lens elements that have superior optical corrections and performance. Because the chemical and optical properties of many glasses are of a proprietary nature, documentation is difficult or impossible to obtain. For this reason the literature is often vague about the specific properties of glasses utilized in the construction of microscope objectives.

Multilayer Antireflection Coatings - One of the most significant advances in objective design during recent years is the improvement in antireflection coating technology, which helps to reduce unwanted reflections (flare and ghosts) that occur when light passes through a lens system, and ensure high-contrast images. Each uncoated air-glass interface can reflect between four and five percent of an incident light beam normal to the surface, resulting in a transmission value of 95-96 percent at normal incidence. Application of a quarter-wavelength thick antireflection coating having the appropriate refractive index can increase this value by three to four percent. As objectives become more sophisticated with an ever-increasing number of lens elements, the need to eliminate internal

reflections grows correspondingly. Some modern objective lenses with a high degree of correction can contain as many as 15 lens elements having many air-glass interfaces. If the lenses were uncoated, the reflection losses of axial rays alone would drop transmittance values to around 50 percent. The single-layer lens coatings once utilized to reduce glare and improve transmission have now been supplanted by multilayer coatings that produce transmission values exceeding 99.9 percent in the visible spectral range. These specialized coatings are also used on the phase plates in phase contrast objectives to maximize contrast. Illustrated in Figure 3 is a schematic drawing of light waves reflecting and/or passing through a lens element coated with two antireflection layers. The incident wave strikes the first layer (Layer A in Figure 3) at an angle, resulting in part of the light being reflected (R(o)) and part being transmitted through the first layer. Upon encountering the second antireflection layer (Layer B), another portion of the light is reflected at the same angle and interferes with light reflected from the first layer. Some of the remaining light waves continue on to the glass surface where they are again both reflected and transmitted. Light reflected from the glass surface interferes (both constructively and destructively) with light reflected from the antireflection layers. The refractive indices of the antireflection layers vary from that of the glass and the surrounding medium (air). As the light waves pass through the antireflection layers and glass surface, a majority of the light (depending upon the incident angle--usual normal to the lens in optical microscopy) is ultimately transmitted through the glass and focused to form an image. Magnesium fluoride is one of many materials utilized in thin-layer optical antireflection coatings, but most microscope manufacturers now produce their own proprietary formulations. The general result is a dramatic improvement in contrast and transmission of visible wavelengths with a concurrent destructive interference in harmonically-related frequencies lying outside the transmission band. These specialized coatings can be easily damaged by mis-handling and the microscopist should be aware of this vulnerability. Multilayer antireflection coatings have a slightly greenish tint, as opposed to the purplish tint of single-layer coatings, an observation that can be employed to distinguish between coatings. The surface layer of antireflection coatings used on internal lenses is often much softer than corresponding coatings designed to protect external lens surfaces. Great care should be taken when cleaning optical surfaces that have been coated with thin films, especially if the microscope has been disassembled and the internal lens elements are subject to scrutiny. From the discussion above it is apparent that objectives are the most important optical element of a compound microscope. It is for this reason that so much effort is invested in making sure that they are well-labeled and suited for the task at hand. We will explore other properties and aspects of microscope objectives in other sections of this tutorial.

Objective Focal Length

Explore how changes in tube length affect the objective focal length. Start Tutorial As we have seen from the discussion above, the body tubes in modern microscopes contain a complex assembly of lenses, mirrors, and beamsplitters that transmit light from the objective into the eyepieces. Almost all microscope manufacturers are now designing their microscopes to support infinity-corrected objectives. Such objectives project an image of the specimen to infinity (the common description is not quite accurately stated as emerging parallel rays). To make viewing of the image possible, the body tube of the microscope, or in reflected light microscopy the vertical illuminator itself, must contain a tube lens. This lens has, as its main function, the formation of the image at the plane of the eyepiece diaphragm, the so-called intermediate image plane. The eyelens of the eyepiece "looks at" this real, inverted, magnified image and magnifies that image in the usual second stage magnification of the compound microscope. Infinity-corrected systems are especially valuable because they eliminate "ghost images" (caused by converging light passing through inclined plane glass surfaces) that often accompanied the older forms of instrumentation. Such systems have the advantage of being easier to design and also make possible the insertion of less costly accessories in the "parallel" light path. This advanced new optical system allows microscopes to support complex optical component clusters in the optical pathway between the objective and the lens tube. This is especially useful for techniques such as confocal, polarized, DIC, and epifluorescence microscopy where specialized lens systems must be employed for optimum results.

In modern infinity-corrected systems, the tube lens is a multi-element optic (to prevent introduction of coma or astigmatism even with increased "infinity light path space") built into and sealed in the observation tube. In this design, as many as two intermediate accessories can be accommodated, with no additional optics to correct the image, in the "infinity space" (see Figure 3) between the objective and the tube lens. Ghost images are eliminated as discussed above. Accessories are much easier to design; and unwanted extra magnification factors are avoided. The light paths illustrated in Figure 3 are diagrammatic representations of an infinity-corrected microscope system. The left-hand side of Figure 3 shows the focal points at the front focal plane of the objective and the eyepiece diaphragm plane. The right-hand side illustrates the "infinity space" between the objective and tube lens where intermediate attachments are placed into the light path. The schematic infinity-corrected systems illustrated in Figure 4 indicate how additional optical components can be inserted into the light path. Figure 4(a) is a diagrammatic representation of an infinity-corrected system showing a specimen on a microslide being illuminated by a substage condenser. The image-forming light rays pass through the objective and form a parallel light beam that is focused by the tube lens into the eyepiece. Accessories can be inserted into the parallel light beam without further optical correction as illustrated in Figure 4(b), which shows a Wollaston prism and several polarizers inserted into the pathway. Figure 4(c) illustrates the insertion of a beamsplitter into the

parallel light beam. This beamsplitter diverts light to the external accessory positioned on the right of the parallel beam.

Infinity-corrected objectives come in a wide range of magnifications, from 1.5X to 200X, and in various qualities of chromatic and spherical correctionfrom simple achromats to planachromats and precision planapochromats. Most, but not all, are designed to be used dry, that is with air in the space between the objective and the specimen. The brightfield series have the customary microscope thread for screwing into the nosepiece (see Figure 5). The objectives which are used for brightfield/darkfield observation usually have wider diameter threads and require a nosepiece with wider openings for attaching such objectives (these objectives are called Neo, BF/DF, or B/D objectives). Some reflected light objectives are designed to focus at a longer working distance from the specimen than is usual; such objectives are labeled on the barrel of the objective as LWD (long working distance) or ULWD (ultra-long working distance). The manufacturer usually designates the objective series to be used for reflected light Nomarski differential interference contrast studies; e.g., in the case of Olympus, the appropriate series is the MS Plan series for brightfield objectives and the Neo S Plan in

the brightfield/darkfield series. Such objectives are sometimes labeled NIC on the objective barrel or designated as strain-reduced.

Infinity-corrected objectives are inscribed with an infinity mark (). The magnification yielded by the objective is the quotient of the focal length of the tube lens divided by the focal length of the objective. For example, in the Olympus microscope system with the tube lens having a focal length of 180 millimeters, a 9 millimeter focal length objective will project a 20X magnified image onto the plane of the eyepiece diaphragm. With a tube lens of 180 millimeters, it is possible to design objectives with a magnification as low as 1.25X while still maintaining the parfocalizing distance of 45 millimeters. ntroduction to Stereomicroscopy

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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. Stereomicroscope Designs 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 threedimensional 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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Nikon SMZ1500 Stereomicroscope

Explore zoom magnification, focus, and changes in illumination intensity on a variety of specimens with a virtual Nikon SMZ1500 stereomicroscope. 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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Chromatic Aberration

Chromatic aberrations are important wavelength-dependent artifacts that occur because the refractive index of every optical glass formulation varies as a function of wavelength. 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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Distortion Optical Aberrations

Distortion is an aberration commonly seen in stereoscopic microscopy, which is manifested by changes in the shape of an image rather than the sharpness or color spectrum. 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 dtentes 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 blankout 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 / (n sin())

where d is the smallest resolvable distance, 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 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 Objective Magnification Color Code ED Plan 0.5x ED Plan 0.75x ED Plan 1x ED Plan 1.5x ED Plan 2x Plan Apo 0.5x Plan Apo 1x Plan Apo 1.6x Numerical Aperture Red Yellow White Green Blue N/A N/A N/A Working Distance (Millimeters) 0.045 0.68 0.09 0.14 0.18 0.066 0.13 0.21

155 117 84 50.5 40 136 54 24

Table 1

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 Numerical Aperture f-Number 0.023 0.029 0.052 0.085 0.104 0.118 0.128 0.131
Table 2

21.7 17.2 9.6 5.9 4.8 4.2 3.9 3.8

Lenses designed for general photography are rated with a system that is based on fnumbers (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 50millimeter 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 Objective Zoom Factor HR Plan Numerical Aperture 0.75 Depth of Field (Micrometer 10x 15x 20x 30x s) 0.023 1,348 1,072 934 796

Apo 1x

1 2 4 6 8 10 11.25

0.029 0.052 0.085 0.104 0.118 0.128 0.131

Table 3

820 239 80 48 35 28 26

655 193 66 41 30 24 21

573 170 59 37 27 22 21

491 147 52 33 25 21 19

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.
Interactive Flash Tutorial

Nikon SMZ1500 Light Pathways

Examine the optical system, lightpath, and aperture diaphragm operation in Nikon's SMZ1500 stereoscopic microscope with this interactive Flash tutorial. 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 versus Iris Diaphragm Opening Size Numerical Aperture Depth of Field (Micrometers) 0.131 0.095 0.063

10x 26 44 89
Table 4

15x 22 39 83

20x 21 37 79

30x 19 35 76

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.
Interactive Java Tutorial

Photomask Reticle Operation

Practice adjustment of the photomask reticle mounted in a focusing eyepiece using this interactive tutorial. 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 threedimensional 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. Conclusion

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 threedimensional 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.

The Making Of Voyager

The design and construction of Wayne Schmidt's

motorized binocular chair for a pair of 8-inch diameter aperture binoculars.

Telescopes are great... as long as you don't have to use them. Flat, one-dimensional views, straining to reach the eyepiece and fighting the cold all conspire to challenge even the most stalwart astronomy enthusiast. My solution to these problems was to mount a pair of large binoculars on a motorized chair complete with electrical heaters. I named it Voyager.

I used large binoculars because they reduce eyestrain, increase light sensitivity 40percent, color sensitivity 25-percent and most importantly provide beautiful threedimensional appearing images. The following drawing is an exploded view of Voyager to illustrate how it's put together:

I used F8.0 mirrors because they provided the optimum compromise between light capturing capability, ease of fabrication and image quality. By using a longer focal length than the usual 4.0 to 6.0 seen on most reflective binoculars, I was able to use smaller secondary mirrors and thinner spider vanes to reduce diffraction effects. Here's what the optical path looks like:

Unfortunately, one of the negatives of this design is that the image rotates as the tubes rotate. This means that the simple method of tube rotation for adjusting the interocular distance can't be used. Instead, right-hand tube is mounted on a cradle equipped with sideways push-pull screw pairs front and back to adjust eyepiece separation. For merging the images in both scopes, the left-hand tube was mounted on a cradle whose rear end could be moved left-and-right and the right-hand tube with a cradle that moves its end up-and-down by turning adjustment knobs located in the front of the chair. This way the observer can set the alignment while using the binoculars in the actual viewing position. This is important because unavoidable flexure resulting from tilting the 350-pound chair with a 170-pound rider can throw the alignment off. The ability of adjusting on-the fly makes tuning up the alignment easy at any time. The pivot points for the tubes are set at the eyepiece positions so alignment doesn't effect the interocular distance. By removing the opaque overhead light cover you can look don into the observing area of the chair:

The top set of eyepieces are 50mm Plossls that provide 32.5-power with a 1.4degree field of view. They are mounted on sliding carriages that allow the focus to be quickly adjusted for higher powers using a pair of 2.0X Barlow lenses stretched to 2.8X to provide 90X. The eyepieces themselves lock into focusers made from flush valves. The second. The smaller eyepieces are to a pair of very fine Adlerblick 7x50 binoculars. Their large field of view and erect image make them ideal finder scopes. Below that is a circular metal disc, which uses magnets to hold star charts in any desired position. In the middle of the star chart is a finder gauge consisting of two wire rings. The outer one corresponds to the field of view of the Adlerblicks while the smaller inner circle matches the field of view of the 8-inch, F8.0 binoculars. The wire hanging down on the left is a low-power resistive heater for both pairs of eyepieces to prevent dew formation. (Speaking of heaters, the chair has an electrically heated vest and boots to make sure the rider stays comfortable on even the coldest nights.) To move the binoculars forward and backward or up and down to use different eyepieces or reach the finder binoculars, the frame that holds the binoculars is equipped with two motors. One, mounted on the back of the rider's chair, moves the entire binocular assembly up and down using an electric motor driving a treaded rod through a captured nut. The 100+ pound weight of this assembly tended to grind down the nut and rod so it's important to keep both of them heavily greased and protected from dust. For moving the binoculars back and forth they were mounted on a pair of linear bearings and a second motor/captured nut drive pushed or pulled them as needed.

The motor drive that moves the binocular carriage forward and backward. The motor is a cheap, rechargeable drill with its handle cut off. Three similar motors drive the other chair motions. At this point it's necessary to make a comment about safety. When the chair is tilted back so that the observer is looking straight up, if the binoculars broke loose all 100+ pounds of them would come crushing down into the observer's eyes. For this reason it's necessary to have multiple safety measures in place to insure that if this ever happens, fail safe mechanical locks prevent the observer from being injured. The same thing applies to all aspects of such chairs. When being ridden, the observer is essentially imprisoned and every aspect of safety must be carefully considered and addressed. The chair's frame was made of 3/4-inch plywood strengthened where needed with 2x4-inch lumber. Straight-grained, kiln-dried wood was used through to avoid warping. The altitude bearings are 2-inch diameter metal casters. I found they produced less drag and ran smoother than traditional Teflon bearing. The altitude drive consists of a rubber roller pressing against a sector arc covered with 30-grit sand paper. The rubber wheel is driven by an electric motor. (Note: because this was a friction drive it is essential to keep the center of weight the same or the drive could slip. I found this to be the chair's biggest weakness because if a 170-pound rider shifts his position by only 2-inches, he's creating 340-inch pounds of additional torque, enough to cause the altitude drive to slip. (I tried a bent-bolt drive but it slipped even easier.) For this reason it's difficult for other people much different in size to myself to use the chair. I have counter-balancing weights that can be used to compensate for this problem but they are awkward to use.) The seating area of the chair is padded with a 5-inch thick layer of contoured foam to provide uncompromised comfort. The rotating part of the chair rests on a 12-inch diameter lazy Susan bearing stabilized with four casters mounted near the edge of the circular base. A similar drive system rotates the chair except the entire disk of the base is used so the chair can turn 360 degrees. Ordinary toggle switches on the right-hand armrest turn the motors on, off, and control the direction of slewing. Additionally, there's a switch that puts a one-ohm, 20-watt resistor into the power circuit to reduce the slew rate from 3-degrees per second to 0.5-degrees per second. Power is provided by a six-volt, 140-amp-hour deep discharge battery that has enough stored energy to run the chair for five nights, even in sub-zero weather.

I made the mirrors myself. They both pass 6-point Millies-Lacroix diffraction limit test, have smooth surfaces and good edges. The final focal lengths were 64-inches and 64.37-inches, well within the 1-percent agreement needed for good binocular image merging. It took an extra 5 hours of work to get the second mirror to match the first but then these were the first mirrors I'd ever made. A more experienced mirror making could certainly do it in less. I used 3, two-square-inch pads of foam mounting tape to secure each mirror to its cell. These mounts have held for years and never shown any sign of straining the mirrors or coming loose. The mirror cells themselves were simply two disks of 3/4inch plywood with push-pull adjusting screws set at 90-degrees, rather than the usual 60-degrees, for easier collimation. The secondary mirrors are 1.5-inches across and provide a 100-percent illumination field 20-minutes in diameter. Surveying 1,866 astronomical objects indicated that this large a field would be large enough for 100-percent illumination of 95-percent of them. The illumination drop off near the edge is unnoticeable. By using such a tight optical path I was able to employ a better baffling system that increased the scope's contrast. In the end, using the smaller diagonals actually provided brighter, sharper images. All three mirrors in each tube were coated with enhanced aluminum. This increased the overall reflection efficiency from 68-percent of normal coatings to 86-percent. The tubes for each telescope were cut from 11-inch diameter concrete forming tubes purchased from Home Depot. Mounted on front of them are 15-inch diameter, 40inch long baffling tubes used to reduce stray light from reaching the focal plain and reducing contrast.

To decide how to coat the insides of the tubes I conducted an experiment to see which surface treatment absorbed the most light. Above, from left to right, is flat black paint, black flocking paper, sawdust sprayed with flat black paint, black cloth and finally black velvet. The velvet was clearly the best so that's what I used. The four, 0.02-inch thick spider vanes used in most telescopes produce very large diffraction spikes. To reduce this I used a set of three vanes, which avoids spike reinforcement from in-line vanes, made of 0.008-inch music wire. The combined effect was to reduce the brightness of the diffraction spikes by 65-percent. While these spikes are easily seen in normal scopes when looking at 2nd magnitude stars, Voyager's wire spiders don't show them until you're looking at 1st magnitude stars, and even then they are barely discernable. Here's a cross section of what they look like:

Here's what the actual secondary holder looks like:

Below is a picture of the jig used to make each wire loop (necessary so that they are all the same length), a close-up of the nylon nut and nut cap that holds the wire firmly to the side of the telescope tube yet with enough slip so it can be adjusted and a close-up of the end of the central diagonal bar showing how two nuts tighten on the wire's end loops. The secondary's position is adjusted closer or further from the primary mirror by sliding the wires through the nylon capture nuts mounted on the telescope's tube. The secondary itself is foam-tape mounted on a wooden block and can be rotated by turning it on the threaded rod screwed into it that forms the central spar of the diagonal. Finally, the entire wire assembly can be angled by sliding one wire through its nylon nut further than the other two. Collimation using this system isn't easy but the reduced diffraction losses are worth it.

A word of warning: I found collimating a three-mirror optical path very difficult. There are so many rings within rings that have to all be concentric that I had a hard time keeping them straight and remembering what adjustment effected which ring. Fortunately, once the scopes were collimated they tended to remain that way even after transporting them hundreds of miles in a car.

Originally the chair broke down into 10 manageable pieces. Assembly on-site took half an hour. However, it was a tight fit to pack it into my station wagon so after a year I rebuilt it onto a small trailer. Everything except the telescope cradle and telescope tubes were permanently fixed to it. This reduced set-up time to less than 5 minutes. During transport I used wood wedges to raise the main chair off the lazy Susan bearing so heavy bounces wouldn't flatten it. A white vinyl cover sewed to fit the chair protected it from weather. The only drawback to this was that it made the chair look like a construction site port-a-potty.

So, how does it work? Outstanding! Besides the already mentioned advantages of binocular viewing, there is a very real psychological pleasure from physically looking in the same direction the telescopes are looking. One additional benefit I hadn't expected was the fact that when seated in the chair, the observer's head lays on a head rest. This immobilizes his head and therefore his eyes. By eliminating the small eye movements unavoidable when looking through a normal telescope I found that images looked brighter, sharper and much more concrete. I estimate that this increased the scope's limiting magnitude by at least half a magnitude. Large binoculars breathe life into astronomical views that must be seen to be appreciated and riding a motorized chair in warmth and comfort enormously increases the joy of observing, creating a sense of identity with the objects inhabiting the night sky unattainable any other way. The Orion nebula becomes a huge glowing cloud ripped by valleys of stygian blackness. The Triffid's color can be clearly seen and the Swan looks so solid and 3-dimensional it's hard to take your eyes off it. When riding Voyager the universe comes alive, inviting exploration.

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