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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:
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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,
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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.
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A
B
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
100
10
1
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
NA
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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