A new resolution in stereo microscopy
By Dr. Winfried Busch Olympus Life & Materials Science Europa GmbH
We see in stereo to generate three-dimensional (3D) images because our world has depth.
However, what do we see when we observe a sample through a light microscope? What
information do we perceive? Most light microscopes with a single light path just present us
with a two-dimensional view of physical reality. This means one dimension is lost which
significantly restricts our perception of the physical reality viewed. Consequently, some 2D
microscope images will lack key information which may be essential to the interpretation of
certain samples.
When looking at two-dimensional images, based on experience, we can more or less
unconsciously extend the 2D view into the third dimension. But how can we be sure that
this interpretation is accurate? For example, some amazing 2D illusions can confuse our
brains into seeing things that are not actually correct (visit
http://www.michaelbach.de/ot/index.html for more information on optical illusions).
Furthermore, when looking at the night sky, we cannot tell whether two stars are close to
each other or are in fact hundreds of light years apart in space. Painting techniques,
however, offer numerous examples of how to create a distinct impression of a third
dimension using different lighting and shading for contrast, as well as through well-placed
usage of objects with familiar proportions. We are also accustomed to recognising different
lighting on a structure in a 3D way, this is how relief is shown – for example in contrast
methods used in microscopy (e.g. darkfield and oblique illumination).
The stereo view
Stereo microscopes, often called “binos”, provide views of a specimen that look natural,
with large depth of field and working distances. The ability to ‘see’ depth is based on
binocular disparity, as each of our eyes view an object from a slightly different angle due to
the distance between them. This, together with the interpretation by our brain, creates a 3D
view. To see objects down a microscope in a similar fashion, both eyes require a separate
light path to enable, observation of the specimen at an angle (called the convergence angle)
similar to the one they see naturally. Stereo microscopy provides each eye with views of the
same object from a slightly different perspective (Figure 1). This angle of convergence
determines the magnitude of the 3D effect, which will vary depending on the application.
For example, for delicate manipulation work such as microinjection, an enhanced 3D view is
ideal. However, for applications such as specimen identification, as natural a 3D view as
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possible is necessary. This is where stereo microscopes utilising different optics for differing
applications are highly useful.
Choosing the correct stereo microscope is now more important than ever. Different tasks
require different tools, and Olympus can provide a solution for each one of them.
Furthermore, techniques such as fluorescence, which revolutionised compound microscopy
to enable the clear identification of many parameters, are now applied on a whole organism
and tissue level. This not only requires a wider field of view, but also the addition of
different illumination technologies. Consequently, a fluorescence stereo system suitable for
even the most demanding tasks would prove to be an extremely useful tool for any research
laboratory. Through its new SZX2 series of stereo microscopes, Olympus has sought to
address the differing needs of stereo microscopy users. The SZX10 stereo microscope has
been introduced for standard as well as advanced routine and identification requirements.
Whereas the SZX16 has been developed for detailed research applications, including
fluorescence and accurate micromanipulation work.
As discussed above, with the increasing range of microscopy techniques for research, the
instruments required have become increasingly sophisticated to give clear views of ultrafine details. Stereo microscopes are designed for low magnifications compared to compound
systems. This is because as magnification and numerical aperture increase the depth of field
(the depth of the image in sharp focus) decreases (and vice versa). Therefore, there is a
limit to how much an object can be effectively magnified on a stereo microscope since past
a certain point there is not enough depth of field to maintain two separate light paths. The
Olympus SZX16 (Figure 2) though, is a completely new microscope designed with enhanced
technological features to extend the stereo effect and meet ever evolving modern research
needs.
A new resolution
The SZX2 series is designed using the parallel light path principle of a Galilean (or
telescope) optical system. This provides excellent flexibility since more optical features
(such as fluorescent illumination) can be placed into the parallel light paths. Using specially
adapted optics, which provide a greater distance between light paths, the SZX16 research
microscope can support larger lenses than conventional stereo microscopes. This enables
significantly increased numerical apertures (NA) to provide excellent light signal collection.
As a result, the SZX16 has a maximum numerical aperture of 0.3, giving an unprecedented
resolution of 900 line-pairs per millimetre. (Figure 3).
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When using stereo microscopes, there is a fine balance between higher NAs, which offering
increased resolution and brighter fluorescence, and a lower NA providing an increased depth
of field. It is often important to use a stereo microscope in tandem with compound
microscopes where a procedure requires the highest magnification and resolution possible.
In these situations, e.g. pre-screening, it is advantageous to have an advanced stereo
microscope to provide increased efficiency and clarity. It therefore needs to offer the best
resolution and brightest, clearest images possible within its usual working zoom range.
Consequently, through its specially adapted optics, the SZX16 provides the highest possible
NA, hence resolution, throughout its magnification range (Figure 4).
From whole organism to cellular detail
In addition to enabling improved image clarity, the specially adapted optics of the SZX16
have also extended the zoom range to provide far greater flexibility. Its world leading zoom
ratio of 16.4:1 means that its comprehensive range of parfocal objectives can zoom from
3.5x to 230x in one smooth movement, with the two position nosepiece attached. Such a
large magnification ratio is ideal for research areas such as developmental biology. For
example, an overview of the whole organism enables the identification of regions of interest
and with the extensive zoom range these can be focused in on very easily and quickly.
Furthermore, by using parfocal objectives there is no need to refocus when switching
objectives.
Enhanced 3D
As previously discussed, as a sample is magnified, the depth of field generally decreases.
The optical system used in the SZX16, provides an increased angle of convergence, which
gives an enhanced 3D effect enabling a clearer view of the available field. This not only
improves the depth information gained from the sample, but also allows much more
accurate sample manipulation. This is essential during manipulation procedures requiring
increased accuracy, such as those associated with embryos, e.g. micro-manipulation and
micro-injection. This enhanced 3D effect can also be of use in microscopic surgery
procedures.
Fluorescence
Fluorescence detection techniques now play a major role in the functional analysis of
organisms, so stereo microscopes are becoming powerful tools to visualise fluorescence at
low magnifications. Consequently, another benefit of its advanced optics and high NA, is
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that the SZX16 can be used as the perfect fluorescence microscope for whole organism to
cellular detail imaging.
Due to the unprecedented resolution and sensitivity achieved by the SZX16, even faint
fluorescence signals can be collected and viewed. Therefore all signals can be detected
easily from anywhere within the sample. This is also possible due to a series of specific
design enhancements, such as a unique fluorescence illuminator and whole system
optimisation to minimise intrinsic fluorescence. For any advanced fluorescence instrument,
the illumination source needs to be as close to vertical as possible and completely
integrated to provide even and controllable fluorescence. The SZX16 utilises a perpendicular
fluorescence illumination pathway and, as a result, avoids the artefacts generated when
using alternative pathways. Using high quality light sources and filter components means
that they will combine to produce the perfect fluorescence system for the user and
application.(Figure 5)
Easy viewing and documentation
To enable a significantly larger range of eye movement for viewing the 3D image and
making both short and long term use more comfortable, the SZX2 uses advanced Olympus
ComfortView eyepieces. These are designed to provide a larger focused area of view,
making it easier to form the stereo image and enabling greater head movements. This
results in a reduced occurrence of eyestrain. Tilting trinocular heads also help to minimise
user fatigue. For documentation, a small twist of the nosepiece moves the objective
producing an axial monoscopic image of exactly the same frame as viewed in stereo. By
combining this with the advanced Olympus DP71 camera, produces perfect colour
representation for brightfield images as well as highly sensitive fluorescence detection. The
camera uses the same colour reproduction technology as high definition television (HDTV)
and therefore, full-frame (1360 x 1024) live images can also be displayed at 15 frames per
second. Further to this the camera produces a maximum digital image resolution of 12.5
mega pixels for storage. Consequently, the SZX16 is designed for both optical and digital
use to create an extremely versatile macro-to-micro imaging system.
Also useful for user comfort and micromanipulation applications, the SZX2 range can be
used with an ultra-slim 40 mm LED illumination stand, which is the most ergonomic stage
height available. The long-life LEDs provide uniform illumination and constant temperature
at all intensity settings. A novel carousel offers several illumination modes including oblique
and darkfield illumination. Contrast inserts for brightfield and oblique illumination feature a
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unique system of fine lamellae, producing precise and completely even oblique illumination.
With this unique system, even transparent samples can be seen with the same contrast
across the entire field of view.
The Bigger Picture
With life being such a complex blend of known and unknown interactions, it is important to
take a step back and look at the bigger picture. To do this properly though, requires the
correct tools. Advances in the technology behind stereo microscopes, such as with the
Olympus SZX16, are certainly addressing these needs. Such technological developments
have now made it possible to apply advanced techniques when using stereo microscopes,
such as fluorescence, and gain completely clear 3D views from whole organism right down
to fine details.
Figures:
Figure 1: The stereomicroscopic (Galileo type) light path – two microscopes in one – offers
observation of specimens at the natural conversion angle of our eyes. This makes the 3D
topography visible.
Figure 2: The SZX16 stereo microscope designed for advanced research applications
Figure 3: The SZX16 achieves a maximum resolution of 900 line pairs/mm or 1.1 µm
Figure 4: The optimised NA / magnification ratio of the SZX16 research stereo microscope
(blue line) compared to a conventional stereo microscope (red line)
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Figure 5: A plant stamen illuminated using UV light, with pollen showing autofluorescence.
Taken using an SZX16 with a DP71 Camera.
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