Around 1890, the American biologist and zoologist Horatio S. Greenough introduced a design principle for optical instruments which is still used by all major manufacturers today [1-3]. Stereo microscopes based on the “Greenough principle” deliver genuine stereoscopic images of a very high quality. In the late 1950s, Bausch & Lomb, presented its StereoZoom® Greenough design with a groundbreaking innovation: a stepless magnification (zoom) changer . Almost all modern stereo microscope designs are based on a zoom system. In 1957, the American Optical Company introduced a stereo microscope with optics based on the telescope or CMO (Common Main Objective) principle . This type of stereo microscope was soon offered, in addition to the Greenough type, by all manufacturers due to its modularity and high performance.
A stereo microscope can be a big investment, therefore, the selection process should be taken very seriously. To get the most out of a microscope, users should ask themselves the following questions:
- Does it involve screening and sorting?
- Is any sample manipulation needed?
- Is documentation necessary?
- Is high resolution more important than long working distance or the other way around?
- When it comes to using the microscope for many hours, it is important to consider ergonomic accessories as they can prevent repetitive strain injuries.
- Depending on the number of different users, it is advisable to have a microscope which can be adjusted to the preference of each user.
- Modular solutions may look like a higher investment, but in the long run they will save money thanks to their versatility, ability to accommodate different users, and large variety of add-ons and accessories.
- Users who tend to work at the same magnification don’t require a large zoom range.
- If the workflow requires search, find, and sample manipulation, then it may be useful to have a large zoom range to go from low to high magnification.
- At the same zoom magnification, a bigger or smaller object field can be seen, depending on the eyepieces. A larger object field allows users to maintain a better orientation on the sample.
- A larger working distance means a greater distance between the top of the sample and the objective’s front lens, allowing for easier handling of the sample during use.
- Higher NA results in higher resolution, but usually reduced depth of field.
- The FusionOptics technology combines high resolution with more depth of field.
- Plan optics: Correction for image flatness over the entire object field which is useful for all applications.
- Achromat (achromatic) optics: For applications where true color reproduction is not important and mainly geometrical features are assessed.
- Apochromat (APO; apochromatic) optics: For applications where color fringes can be disturbing, such as those which require fast color change and the colocalization of structures.
- Transmission: For applications requiring fine details on a sample to be visualized, then it is advantageous to use high-quality optics with better light transmission. For demanding applications, like research and development, optics with high light transmission can make a difference.
- Color reproduction: If seeing accurately the true colors of the sample is important, then high-quality optics and the appropriate illumination should be used.
- Ergonomic accessories can make working with the microscope easier and result in faster workflows. For example, can the zoom and focus knobs be easily adjusted while looking at the sample through the eyepieces?
- If the microscope is operated by different users, make sure that it can be adjusted to each user’s preferences.
- An optimal illumination should illuminate the entire field of view evenly, provide good contrast, and accurately reveal the true colors of the sample.
The total magnification of a stereo microscope is the combined magnifying power of the objective lens, zoom optics, and eyepieces .
The objective has a fixed magnification value. The zoom optics of the instrument allow the magnification to be changed over the zoom factor range. The eyepieces also have a constant magnification value.
To find out the magnification of the object observed via the eyepieces, the magnification factors of the objective, zoom optics, and eyepieces have to be multiplied.
The formula for total magnification is: MTOT VIS = MO x z x ME, where:
MTOT VIS is the total magnification (VIS stands for "visual");
MO is the magnification of the objective (1x for the case of a Greenough System with no supplementary lens);
z is the zoom factor; and
ME is the magnification of the eyepieces.
In general, values for MO are between 0.32x and 2x, for z between 0.63x and 16x, and for ME between 10x and 40x.
Influence of magnification on the object field
When looking into the eyepieces, a circular area called the object field becomes visible . The diameter of the object field depends upon the total magnification. For example, eyepieces with a 10x magnification have a field number of 23. The field number means that at a 1x combined magnification of the objective and zoom optics, the object field observed through the eyepieces is 23 mm in diameter.
The depth of field is determined by the correlation between numerical aperture, resolution, and magnification [5-7].
For the best possible visualization of an object, the proper adjustment of a modern microscope’s settings can produce an optimum balance between depth of field and resolution. Particularly at low magnifications, the depth of field can be significantly increased by stopping down, i.e., reducing the numerical aperture. It is therefore a matter of finding the optimum balance of resolution and depth of field depending on the size and shape of the object’s features.
High depth of field and high resolution with the FusionOptics technology
A sophisticated optical approach for stereo microscopes that allows both simultaneously high resolution and high depth of field is achieved with the FusionOptics technology from Leica Microsystems . With one light path, one eye of the observer sees an image of the object with higher resolution and lower depth of field. At the same time, via the other light path, the other eye sees an image of the same object with lower resolution and higher 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.
Chromatic aberration is a type of distortion in which there is a failure of a lens to focus all colors to the same convergence point [2,9]. It occurs because lenses have a different refractive index for different wavelengths of light (the dispersion of the lens). Spherical aberration occurs when light rays striking a spherical lens surface at a point away from its center axis are refracted to a greater or lesser degree than those that strike at points close to the center. The aim of a good optical design is to reduce or eliminate chromatic and spherical aberration completely. The following lenses can be used to limit the effect of these issues:
- Corrected for 2 wavelengths (red and green) which are brought into focus in the same plane.
- For standard applications in the visual spectral range.
- Corrected for 3 wavelengths (red, green, blue) which are brought into focus in the same plane.
- For applications with the highest specifications in the visual spectral range and beyond.
- A lens which is not plan corrected shows a non-uniform focus over the whole object (field of view).
- Advised for applications requiring observation over large object fields.
The working distance is the distance between the objective’s front lens and the top of the sample when it is in focus. Usually, the working distance of an objective decreases as magnification increases. The working distance has a direct impact on the usability of a stereo microscope, especially for inspection and quality control tasks.
Generally, the body size and working habits of people vary significantly. Therefore, the height (eyepieces) of a microscope equipped for a certain task with special accessories and a particular working distance may not be suitable for every 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 [10-12]. To compensate for these height differences, it is advisable to use a variable binocular tube . 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, therefore, are the preferred solution.
For stereo microscopy, the right illumination is a key factor . The most appropriate illumination will allow the sample features of interest to be visualized in an optimal manner and possibly new information to be revealed. It is important that the illumination works well for both the microscope used and intended application.
- Incident light
Used for opaque, non-transparent samples. Depending on the sample texture and application requirements, many different incident illumination solutions are available to give proper contrast of the sample details and features of interest. Refer to reference 13 below to see some examples of incident illumination for stereo microscopes.
- Transmitted light
Used for various kinds of transparent samples ranging from biological ones, such as model organisms, to polymers and glass.
- Standard transmitted brightfield illumination
Used for all types of transparent samples, it provides high contrast and sufficient color information.
- Oblique transmitted illumination
Used for samples that are nearly transparent and colorless; a greater contrast and visual clarity of the sample can be achieved.
- Darkfield illumination
Used for small features on flat areas of a sample which cannot be seen easily with brightfield, such as cracks, pores, fine protrusions, etc. on shiny or bright samples. It can also be used to reveal sample structures with a size below the resolution limit.
- Contrast method for clear, transparent specimens
Rottermann or relief contrast is an advanced oblique illumination technique that shows changes of the refractive index as differences in brightness. With positive relief contrast structures appear raised, while with inverted relief contrast they appear lowered. The positive and inverted relief contrast can make it easier to distinguish fine structures and extract the maximum amount of information from the sample.
- D. Goeggel, The History of Stereo Microscopy - Part I: 17th Century - The First Microscopes, Science Lab (2007) Leica Microsystems.
- D. Goeggel, The History of Stereo Microscopy - Part II: The 18th Century - Greater Demands are Placed on Optics, Science Lab (2007) Leica Microsystems.
- D. Goeggel, The History of Stereo Microscopy - Part III: The 19th Century - Breakthrough of Modern Microscope Manufacturing, Science Lab (2007) Leica Microsystems.
- J. DeRose, M. Doppler, What Does 30,000:1 Magnification Really Mean? Some Useful Guidelines for Understanding Magnification in Today’s New Digital Microscope Era, Science Lab (2018) Leica Microsystems.
- R. Rottermann, P. Bauer, How Sharp Images Are Formed: Depth of Field in Microscopy, Science Lab (2010) Leica Microsystems.
- M. Wilson, Collecting Light: The Importance of Numerical Aperture in Microscopy, Science Lab (2017) Leica Microsystems.
- M. Wilson, Microscope Resolution: Concepts, Factors and Calculation: Airy Discs, Abbe’s Diffraction Limit and the Rayleigh Criterion, Science Lab (2016) Leica Microsystems.
- D. Goeggel, A. Schué, D. Kiper, FusionOptics - Combines high resolution and depth of field for ideal 3d optical images, Science Lab (2008) Leica Microsystems.
- M. Wilson, Eyepieces, Objectives and Optical Aberrations, Science Lab (2017) Leica Microsystems.
- C. Müller, How to Turn Microscope Workplaces Ergonomic, Science Lab (2017) Leica Microsystems.
- M. Birlenbach, R. Holenstein, Higher Motivation, Longer Concentration - Ergonomics as a Competitive Advantage: Microscope Workplace Design in Quality Control, Science Lab (2013) Leica Microsystems.
- C. Müller, J. Ludescher, Investing in Ergonomically Designed Microscope Workplaces Pays Off, Science Lab (2013) Leica Microsystems.
- J. DeRose, M. Schacht, Illumination (Lighting) Systems for Stereo Microscopes: Obtaining the optimal results for industrial applications, Science Lab (2015) Leica Microsystems.
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