Transcript
Imaging Systems
Factors to Consider When Selecting a Stereo Microscope By Daniel Goeggel at Leica Microsystems
The stereo microscope is the workhorse of the pharmaceutical research laboratory and production department, and decision-makers need to ensure that an instrument is chosen that is 100 per cent tailored to the needs of the user.
Stereo microscopes are often nicknamed the workhorse of the laboratory or the production department. Users spend many hours behind the ocular – inspecting, observing, documenting or dissecting samples. So what are the factors that need to be considered when selecting a stereo microscope? The answer is: “It depends”. Why is that? Because it depends on the application, on the task the user wants to accomplish. Basically, a stereo microscope is a tool for magnifying a three-dimensional object in three dimensions and – unlike a compound microscope – a stereo microscope is well able to cope with this task.
presented its StereoZoom® Greenough design with a ground-breaking innovation: a stepless magnification changer (zoom). Almost all of today’s designs are based on a zoom system.
Criteria for Selecting a Stereo Microscope Stereo microscopes are still based on the abovementioned technical approaches – the Greenough and CMO principles. But what other factors need to be considered? First, four things need to be carefully assessed: ●●
Greenough and Cycloptic® Principles The binocular microscopes of the old days featured a simple lens system, and the same design is used for traditional compound microscopes. These dissecting microscopes, as they were then known, were used primarily in biology for dissection purposes; there were no technical applications for them at the time. Around 1890, the American biologist and zoologist Horatio S Greenough introduced a design principle that is still used today by all major manufacturers of optical instruments. Stereo microscopes based on the ‘Greenough Principle’ deliver genuine stereoscopic images of a very high quality.
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What is the application? Which structures need to be observed, documented or visualised? How many people will be using the microscope? What is the available budget for the solution?
Once these factors are known, the decision boils down to the following criteria: Figure 1: Cycloptic®, the first modern stereo microscope based on the telescope principle
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Images: Leica Microsystems
In 1957, the American Optical Company introduced the modern stereo microscope design with a shared main objective and named it ‘Cycloptic®’ (see Figure 1). Its modern aluminium housing contained two parallel beam paths and the main objective, as well as a five-step magnification changer. Keywords This type of stereo microscope – which was based on the telescope or Common Stereo microscope Main Objective (CMO) principle – was adopted in addition to the Greenough Greenough principle type by all manufacturers and used for Magnification modular, high-performance instruments Achromatic/Apochromatic (see Figure 2). Two years later, another Illumination American company, Bausch & Lomb, Innovations in Pharmaceutical Technology Issue 39
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Figure 2: Two basic stereo microscope principles: a) the telescope or CMO principle; and b) the Greenough principle
Magnification, zoom range and object field Depth of field and numerical aperture Optical quality and working distance Ergonomics Illumination
Magnification, Zoom Range and Object Field The total magnification of a stereomicroscope is the combined magnification of the magnification changer, the objective and the eyepieces.
The Magnification Changer or Zoom Body Like a magnifying glass, the magnification changer consists of optical lenses that can be used to change the magnification of the instrument. Changing the position of the magnification changer alters the degree to which the image is magnified (the magnification factor). Modern stereo microscopes are able to provide up to 16x magnification (zoom body only) with a 20.5:1 zoom range, and feature motorisation or encoding to allow reliable measurements. Next, the image is magnified further by the eyepieces and main objective. To find out the magnification of the object being observed in the eyepieces, the user has to multiply the magnification factors of the magnification changer, main objective and the eyepieces. For the sake of completeness, however, the formula is as follows: MTOT VIS = z x ME x MO Where: MTOT VIS is the total magnification that we want to calculate (VIS stands for ‘visual’) z is the level of the magnification changer ME is the magnification of the eyepiece MO is the magnification of the main objective (1x in case no supplementary lens is used in a Greenough system)
Object Field When looking into the eyepieces from the proper distance and with the interpupillary distance set correctly, a circular area called the object field is visible. The diameter of the object field changes depending on the magnification. In other words, a mathematical relationship exists between the magnification and the diameter of the object field. Eyepieces with 10x magnification provide a field number of 23. That means that at a 1x magnification of the zoom body and the main objective, the object field is 23mm in size. At 3x magnification, the object field is reduced to one third – that is, the object field has a diameter of only 7.66mm. Innovations in Pharmaceutical Technology Issue 39
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Depth of Field and Numerical Aperture In microscopy, depth of field is often seen as an empirical parameter. In practice, it is determined by the correlation between numerical aperture, resolution and magnification. For the best possible visual impression, the adjustment facilities of modern microscopes produce an optimum balance between depth of field and resolution – two parameters that, in theory, are inversely correlated.
Practical Values for Visual Depth of Field In DIN/ISO standards, the depth of field on the side of the object is defined as the “axial depth of the space on both sides of the object plane within which the object can be moved without detectable loss of sharpness in the image, while the positions of the image plane and the objective are maintained.” The author of the first publication on the subject of visibly experienced depth of field was Max Berek, who published the results of his extensive experiments as early as 1927. Berek’s formula gives practical values for visual depth of field and is therefore still used today. In its simplified form, it is as follows: TVIS = n [ λ/(2 x NA2) + 340μm/(NA x MTOT VIS)] Where: TVIS is visually experienced depth of field n is refractive index of the medium in which the object is situated. If the object is moved, the refractive index of the medium that forms the changing working distance is entered in the equation λ is wavelength of the light used, for white light, λ = 0.55μm NA is numerical aperture on the side of the object MTOT VIS is total visual magnification of the microscope
If, in the above equation, the total visual magnification is replaced by the relationship of useful magnification
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10,000
Optical Quality The optical quality for stereo microscopes is usually listed as Achro or Achromat (achromatic), and as Apo (apochromatic) for the highest degree of correction for spherical and chromatic aberrations. Field curvature corrections are abbreviated Plan, while PlanApo designates a combination of chromatic aberration and field curvature correction (see Table 1).
Depth of field (µm)
1,000 M = 500 NA M = 1,000 NA
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Figure 3: Depth of field as a function of the numerical aperture for λ = 0.55µm and n = 1
(MTOT VIS = 500 to 1000 x NA), it can be seen that, to a first approximation, the depth of field is inversely proportional to the square of the numerical aperture (see Figure 3). Particularly at low magnifications, the depth of field can be significantly increased by stopping down – that is, reducing the numerical aperture. This is normally done with the aperture diaphragm or a diaphragm on a conjugated plane. However, the smaller the numerical aperture, the lower the lateral resolution. It is therefore a matter of finding the optimum balance of resolution and depth of field depending on the structure of the object. In the case of stereo microscopes, it is often necessary to make a certain compromise in favour of a higher depth of field, as the z dimension of threedimensional structures frequently demands it.
Even More Depth of Field – FusionOpticsTM A sophisticated optical approach that cancels the correlation between resolution and depth of field in stereo microscopes is FusionOptics™. Here, one of the light paths provides one eye of the observer with an image of high resolution and low depth of field. Via the second light path, the other eye sees an image of the same object with low resolution and high depth of field. The human brain combines the two separate images into one optimal overall image that features both high resolution and high depth of field. Figure 4: Modern stereo microscope featuring a 20.5:1 zoom range with apochromatic corrected optics and FusionOptics™
Table 1: Optical quality terms for stereo microscopes Achro, achromat
Achromatic aberration correction
Plan
Flat field optical correction
PlanApo
Apochromatic and flat field correction
In optical instruments such as stereo microscopes, achromatic aberration is a type of distortion in which there is a failure of the lens to focus all colours to the same convergence point. It occurs because lenses have a different refractive index for different wavelengths of light (the dispersion of the lens). The refractive index decreases with increasing wavelength. The aim of a good optical design is to reduce or eliminate this effect completely. An achromatic lens or achromat is a lens that is designed to limit the effects of chromatic and spherical aberration. Achromatic lenses are corrected to bring two wavelengths (typically red and blue) into focus in the same plane. These types of lenses or microscopes are used for tasks where colour reproduction is not imperative, and mainly geometrical characteristics are assessed. Apochromatic lenses, on the other hand, are designed to correct three wavelengths (red, green and blue) and bring them into focus in the same plane.
Working Distance This is the distance between the objective front lens and the top of the specimen when the specimen is in focus. In most instances, the working distance of an objective decreases as magnification increases. In stereo microscopy, working distance is one of the most important criteria, since it has a direct impact on the usability of the microscope as a tool.
Ergonomics – People are Very Different There are tall and short people, and this makes instrument requirements a personal matter. For example, the existing height of a microscope equipped for a certain task with accessories and with a particular working distance may be quite unsuitable for the specific user. If the viewing height is too low, the observer will be forced to bend forward while working, resulting in muscular tension in the neck region. Ideally, therefore, the viewing height and the viewing angle of
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the microscope should be adjustable to the build of the user. In addition, a variable viewing height is the best way to prevent an entirely sedentary posture. It permits the observer to adopt a personal sitting posture and to change it periodically in accordance with the natural urge to shift around from time to time. It is true that the height of a chair can be altered so that a relaxed, slightly bent posture is substituted for the previous rigidly upright one – but this is not the best approach. It is much simpler and more comfortable to use a variable binocular tube in order to compensate for the height difference. Figure 5 (top): Ergo tube – relaxed body and head, arms comfortably supported, adequate space for the legs, good use of the chair Figure 6 (bottom): Modern stereo microscope illumination systems are based on long lasting light emitting diodes (LEDs) and provide unique ways to integrate the solution into the overall microscope system. Highly integrated ring light, with applied polariser to reduce glare on the specimen
This is used for all kinds of transparent specimens with high contrast and sufficient colour information.
Oblique Transmitted Illumination This illumination technique is used for specimens that are nearly transparent and colourless. Due to the oblique position of the illumination, a greater contrast and visual clarity of the specimen can be achieved.
Darkfield Illumination Darkfield observation in stereomicroscopy requires a specialised stand containing a reflection mirror and light-shielding plate to direct an inverted hollow cone of illumination towards the specimen at oblique angles. The principle elements of darkfield illumination are the same for both stereo microscopes and more conventional compound microscopes, which are often equipped with complex multi-lens condenser systems or condensers having specialised internal mirrors containing reflecting surfaces oriented at specific geometries.
Contrast Method for Clear, Transparent Specimens
Thanks to the modular product approach, stereo microscopes with a CMO design offer many ways of tailoring the instrument to the user’s size or working habits, and are therefore the preferred solution.
Rottermann Contrast™ is a partial illumination technique that shows changes of the refractive index as differences in brightness. Phase structures then typically appear as spatial, relief-type images like hills in positive relief contrast and as indentations in inverted relief contrast. This technique offers many variable views for extracting the maximum possible amount of information.
Illumination
Conclusion
In stereo microscopy, illumination is the key that will bring all of the work to light. The correct illumination will allow the required structures to be visualised or perhaps new information about a sample to be discovered, just by changing the type of light. It is important that the illumination is matched correctly to the right microscope and the right application.
Careful assessment of the application requirements for a stereo microscope is the key element for lasting satisfaction of the user. Since it is the workhorse of the laboratory or the production department, decision makers need to ensure that they are able to tailor the instrument 100 per cent to the requirements of the user. This requires a microscopy solution provider that is able to cope with the increasingly demanding requirements of the biopharmaceutical industry.
Incident Light This is used with primarily non-transparent specimens. The method of delivering this light (ring light, spots and so on) will depend on the texture of the specimen and the application requirements. Incident light is needed for all kinds of non-transparent specimens. Depending on the texture of a specimen and the goal of the results, an eclectic selection of incident illumination solutions is available. Transmitted light is desirable for various kinds of transparent specimens ranging from biological samples such as model organisms to polymers.
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Standard Transmitted Brightfield Illumination
Daniel Goeggel heads the Product Management of the Industry Division of Leica Microsystems (Heerbrugg, Switzerland). He studied Electrical Engineering at the University for Applied Science in Winterthur (Switzerland), and holds an Executive MBA for Strategy and Leadership. He joined Leica Microsystems in 2001, and – with his team – oversees the global stereo microscopy, digital camera and digital microscopy business. Email:
[email protected]
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