Advances in stereomicroscopy [7100-26]

Advances in Stereomicroscopy
H. Schnitzler*, Klaus-Peter Zimmer
Leica Microsystems (Schweiz) AG, 9435 Heerbrugg, Switzerland
ABSTRACT
Similar to human's binocular vision, stereomicroscopes are comprised of two optical paths under a convergence angle
providing a full perspective insight into the world's microstructure. The numerical aperture of stereomicroscopes has
continuously increased over the years, reaching the point where the lenses of left and right perspective paths touched
each other. This constraint appeared as an upper limit for the resolution of stereomicroscopes, as the resolution of a
stereomicroscope was deduced from the numerical apertures of the two equally sized perspective channels. We present
the optical design and advances in resolution of the world's first asymmetrical stereomicroscope, which is a technological
breakthrough in many aspects of stereomicroscopes. This unique approach uses a large numerical aperture and thus an,
so far, unachievable high lateral resolution in the one path, and a small aperture in the other path, which provides a high
depth of field ("Fusion Optics"). This new concept is a technical challenge for the optical design of the zoom system as
well as for the common main objectives. Furthermore, the new concept makes use of the particular way in which
perspective information by binocular vision is formed in the human's brain. In conjunction with a research project at the
University of Zurich, Leica Microsystems consolidated the functionality of this concept in to a new generation of
stereomicroscopes.
Keywords: Microscopy, Stereomicroscope, Binocular Vision, Zoom, Common Main Objective, Stereopsis
1. INTRODUCTION
Binocular vision and stereoscopic perception are one of our primary senses. The input from our eyes is processed into
three-dimensional picture of the space surrounding us. This seems to happen inconspicuously, continuously, and on a
fast time scale without significant delay.
As naturally and fundamental this physiological function appears in daily life, as complex are the processes involved.
Recent research shows, that the retina is not just a receptor. It rather works like a piece of the brain interpreting the image
information, like finding contours, motion, etc. About a dozen different kinds of interpreted information is send to the
brain via the optical nerve.
This and many more aspects of image processing, parallel and serial computing can be found in the physiology of human
vision. In the past as well as today understanding the physiology of human’s vision is of high interest in popular sciences
[1] as well as for regular research [2], and it seems there is still a lot to be discovered.
Insight into the small details in the microstructure of matter is another basic interest in research. The classical optical
compound microscope got many partners like the electron microscope or scanning techniques like confocal microscopes.
For three-dimensional observation, stereomicroscopes provide a perspective view into the world above micrometer scale,
and diverse new techniques come into the field in the last decades like tomographic techniques, holography, and
interferometric techniques.
As different as all these techniques might appear they all have the common goal to provide better views into our nano- to
micro-structures, at higher resolution, higher speed, and higher ease of use.
The presentation reports about the latest advances in stereomicroscopes. Stereomicroscopes might be seen as the
traditional approach to look at the micro world in three dimensions. They cannot compare with the highly dedicated
systems of later developments. However, their purely optical functionality, no principal requirement of electronics or
computers, and observation with bare eyes in real time make them rather universatile tabletop instruments well suitable
for a large variety of applications.
Two convergent optical beam path, zoom optics, long working distances, and the request of high resolution make
stereomicroscopes a challenge for the optical designers.
Optical Design and Engineering III, edited by Laurent Mazuray, Rolf Wartmann, Andrew Wood,
Jean-Luc Tissot, Jeffrey M. Raynor, Proc. of SPIE Vol. 7100, 71000P · © 2008 SPIE
CCC code: 0277-786X/08/$18 · doi: 10.1117/12.797409
Proc. of SPIE Vol. 7100 71000P-1
The presentation introduces the optical layout of two types of stereomicroscopes, Greenough- and CMO-type.
Challenges and limitations for the optical design and performance are revealed. In face with request to step further in
terms of resolution as well of depth of field, it is shown how an alternative new approach was found and validated.
Conclusively, the achieved advances are explained and summarized.
2. STEREOMICROSCOPES
A stereomicroscope allows stereoscopic observation of micro- and macroscopic objects. The optical layout comprises
two beam paths emerging from the object under a convergence angle. The two beams pass a pair of magnification
changers and are finally brought to the observer’s eyes with a binocular tube.
2.1 Greenough type stereomicroscopes
The invention of stereomicroscopes goes back to Horatio S. Greenough in the year 1892. It was found by the
combination of two compound microscopes under a convergence angle (Fig. 1). Besides of the two optical paths the
image erecting is of essential importance to achieve proper depth perception.
P4T
INTEROCULAB
BEVEL GEARS
Fig. 1. Optical system of a Greenough type stereomicroscope.
The drawback of Greenough’s optical set up is found in the tilted focal planes of left and right channel. Proper overlap of
the focal planes within the depth of field is given near the object’s centre, only. Nevertheless, Greenough type
stereomicroscopes allow a compact design and are of good use for many applications till today.
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2.2 Common-main-objective type stereomicroscopes
One could reconcile the focal planes of left and right perspective channels with the common main objective (CMO)
optical layout. A typical optical layout of a present high performance CMO stereomicroscope is sketched in Fig. 2.
Observer
Eyepieces
Intermediate Images
Image Inversion
Tube Lenses
Stereo Zoom
_______
_______
______ ______
Entrance Pupils
stereo basis
Common Main Objective
IL
V
I I i I J1151
I
-
Workirnz
.
Distance
nA
Object
Fig. 2. Optical layout of a CMO type stereomicroscope
The object plane is in the focal plane of the common main objective. This creates an afocal beam path after the objective,
which is also the interface to a large set of accessories offered together with CMO stereomicroscopes.
A telescope type magnification changer, typically a zoom, is placed between objective and tube.
The diameter of the entrance pupil DEP of the zoom is physically defined by the optical components of the zoom, and
varies with the zoom position to finally achieve a good ratio between magnification and resolution.
Together with the focal length fO of the common main objective one finds the numerical aperture nA of the
stereomicroscope as
nA =
DEP
.
2 fO
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(1)
2.3 Basic optical considerations
The optical resolution R of a stereomicroscope is limited by diffraction of light [3]. The resolution (in units of line pairs
per millimetre) translates from the numerical aperture as
R = 3000 ⋅ nA
lp
.
mm
(2)
Let the telescopic magnification of the zoom be Γ , the tube lens have focal length f TL , and the eyepiece have a visual
250 mm
magnification M E =
, then the total visual magnification of the instrument is
fE
M TOT VIS =
f TL
⋅Γ ⋅ ME .
fO
(3)
Considering the resolution of human’s eye, one finds a relation for the useful magnification of the instrument, in which
the resolution of the instrument matches the receiver’s resolution
500 ⋅ nA < M TOT VIS < 1000 ⋅ nA .
(4)
Great lateral resolution as described in (2) is only the half win in stereomicroscopy because the object’s topographic
properties are of significant interest, too. The sharpness in vertical direction in object space is commonly described by
Max Berek’s formula [4] for the depth of field
δ=
λ
2 ⋅ nA
2
+
0.34 mm
.
M TOT VIS ⋅ nA
(5)
As known, both formulas resolution (2) and depth of field (5) depend on the numerical aperture but in physically
conflicting directions.
2.4 Limitations of the current design of stereomicroscopes
Over the past years the resolution (2) was pushed to higher and higher levels. According to Fig. 2 two identical
telescopes are arranged side by side, symmetrically in the device. The plane of symmetry divides the object
symmetrically into a right and a left half. The distance between the telescope axes is referred to as stereo basis. To
achieve higher resolutions the entrance pupil diameters (1) of the telescopes were expanded. A limitation is set when the
physical dimensions of the telescopes lenses come into touch of each other.
Other options to further increase the numerical aperture are to decrease the focal length of the common main objective or
to expand the stereo basis (compare Fig. 2).
Shortening the focal length of the objective would decrease the working distance of the instrument. Working distance is
a key figure for stereomicroscopes where accessibility and manipulation of the specimen under 3-dimensional
observation in real time is a key application.
Increase of the stereo basis leads to more bulky mechanical design of the stereo zoom module, as well as for the optical
and mechanical design of the objectives.
Beyond that, both approaches increase the convergence angle. The object side convergence angle is a figure in the
physiological perception chain how the depth impression is formed. The convergence angle on the observer’s side for
CMO type stereomicroscopes is conventionally close to zero, which allows relaxed stereoscopic viewing. The mismatch
between object-side and observer-side convergence angle causes an exaggerated depth impression for human’s 3dimensional perception. In the past, the for the sake of higher resolution, the focal length of the objectives were already
brought down to the edge of what is reasonable within proper stereoscopic perception.
The figures of stereo basis and/or focal length of the objectives have already experienced many generations of
optimization. The margin for significant improvements on them appears to be rather exhausted.
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3. IDEAS TO ADVANCE
New ideas had to be found to really achieve significant improvements.
When the entrance pupils become larger than the stereo basis and if the stereo basis shall be conserved then the pupils
overlap as sketched in Fig. 3. The overlapping beams could be split into two separated optical paths by use of a
semitransparent mirror.
//
............,
,
-,I,
Stereo
Zoom
Stereo Basis
fo
ye1we
\inA
I
Fig. 3. Sketch of an optical layout where the entrance pupils are wider than the stereo basis. The
Stereo Basis
sin(Convergence Angle/2) =
is kept the same as in the set up depicted in Fig. 2. The dashed line indicates
2 ⋅ fO
a semi transparent mirror, which splits the overlapping entrance pupils into two separated optical paths.
Going into more details, this optical design turns out to become even more bulky as drawn in the sketch above: as the
pupils of the stereomicroscope are defined by the zoom lenses, the entrance pupils lay somewhere in the zoom. Due to
the field angle the long optical path between zoom and common main objective leads to a huge effective diameter
required for the lenses of objective. The result would be very bulky and also very costly and highly demanding on the
optical design of the objective.
Other stereoscopic approaches are found where common optics is used for the objective and the zoom, having the
perspective split after the zoom. Still, entrance pupils wider than the stereo basis still mean overlapping optical beams.
Such an optical design is reasonable as long as the two perspective optical beams are not too wide and not too far out of
the physical symmetry axes of the lenses. The optical correction of an off-symmetry zoom can hardly achieve the optical
quality, apertures, and the zoom range as known for the classic optical design. In consequence, this set up can only
achieve small convergence angles with very poor stereoscopic depth perception. Even more, the convergence angle
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changes with the zoom if the perspective split is placed after a variable focal length, which leads to a very unfavourable
perception.
Of course, combination of the above set ups is possible. But with respect to ease of use, handiness, and cost efficiency, it
is not the primary choice, neither from the designer’s nor from the customer’s point of view.
Having a view into the nature, we are rarely shown pupil sizes as wide (or wider) than the interpupillary distance. The
pupils of owls and eagles are still in good distance to each other. It seems that Mother Nature uses the optical perception
rather for distant objects and not for microscopic objects in close distance. But still the observation of nature indicates
the great potential hidden in the physiology of image processing, e.g. the sensitivity on motion for the eagle.
Use of existing physiological potential in the human’s brain is finally the key contribution to the advances that we
achieved with the new optical design for stereomicroscopes, which we titled “fusion optics”.
4.
FUSION OPTICS
The idea behind “Fusion Optics” is to apply differently sized entrance pupils for the left and right perspective channel.
The wide entrance pupil of the one channel (might be wider than the stereo basis) provides high spatial resolution.
The narrow narrow entrance pupil of the other channel simultaneously provides increased depth of field.
The fused image perceived by the observer will locally extract the best resolution available from either side, leading to
an stereoscopic perception superior in spatial resolution and in depth of field at the same time.
An optical layout of a CMO type stereo zoom with differently sized entrance pupils is sketched in Fig. 4.
Entrance Pupils
Stereo Basis
T
fo
Fig. 4. Optical layout of a CMO type stereomicroscope with differently sized entrance pupils.
While the idea is explained very quickly, still many aspects need to be evaluated, validated, and confirmed. The
specification of the optical design allows wide variations.
4.1 Specification
Without loss of generality, but for better quantitave discussion we now assume the stereo basis to be 24 mm and the
reference focal length to be fO = 80 mm, which is the same as established in current high performance stereomicroscopes.
A standard outfit comprising an objective of reference focal length, a standard tube ( fTL = 160 mm), and standard 10x
eyepieces is considered.
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The zoom shall be set to a maximum magnification position, resulting in a total visual magnification of 160.
The diameters of the entrance pupils are assumed to be 28 mm for the right channel and 20 mm for the left channel. This
describes the optical layout as it is chosen in our newly developed high performance stereomicroscope Leica M205.
Tab. 1 collects the optical key performance figures for a stereomicroscope of this kind, i.e. with differently sized
entrance pupils.
Left perspective channel
Right perspective channel
Entrance pupil diameter
20 mm
28 mm
Numerical aperture
0.125
0.175
Spatial resolution
375 lp/mm
525 lp/mm
Depth of field
0.035 mm
0.021 mm
Luminosity
51% of right channel
100%
Contribution to the
convergence angle (17°)
10°
7°
Diameter of exit pupil
0.39 mm
0.55 mm
Tab. 1: Optical key figures for a stereo zoom with differently sized entrance pupils. The figures are evaluated for a standard
outfit and the zoom set to maximum magnification. Detailed discussion in the text.
The consequences of the listed values will be discussed in the following; needless to say that magnification and object
field are always the same for both perspective channels throughout all configurations.
The exciting feature of this optical design is how the observer merges the two images, which locally show slightly
different details due to different resolution and depth of field, respectively. Extensive scientific studies were done at the
University and ETH Zurich which are presented in section 4.2.
The differences in luminosity between left and right channel principally could be compensated using a filter, but
fortunately the human eyes provide great flexibility in terms of individual adaptation.
There is no physiological coupling for the adaptation between left and right eye (different to humans’ eyes
accommodation process). Unequal adaption between left and right eye is well possible and even rather normal. Human
eyes adapt to different light situations quickly and individually, as we can easily observe in daily life, when exposed to
and moving in direct sunlight and when covering one eye. Beyond that, it is even found, that the human eye is not very
sensitive to differences in luminosity in the same image, e.g. vignetting of 50% to the sides of a images is hardly
recognized.
The overall convergence angle of the suggested stereomicroscope remains the same as what is was in a symmetric set up,
because stereo basis and focal length are kept the same.
If the objective were centred to the centre of the stereo channels the perspective impression would be accurately the
same. Since the effectively used area is laterally shifted to the right side, it is possible to avoid too bulky objective
designs if the objective is shifted the same way. Then, the viewing angles for left and right perspective in the centre of
the object field are not the same. This situation is the same as if the centre of the object would not be observed from
exactly above. Fortunately the human perception is well trained to look at extended objects over which the viewing angle
varies significantly. The particular point of the object field where the viewing angles of left and right side are equal not
perceived different from the other points. Slight displacements of the objective to the centre of the stereo basis therefore
do not mean a significant change for stereoscopic perception.
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4.2 Fusion of binocular images providing locally different resolutions
This section is to discuss the stereoscopic perception from a pair of perspective images with locally slightly different
resolutions, as received from a stereomicroscope like in Fig. 4.
The phenomenon of binocular rivalry is described in literature [2]. It is known that similar images presented to the two
eyes appear as one and are processed simultaneously rather than successively. Superimposed images from the two eyes
always rival by mutual inhibition (suppression theory). Only one eye’s input is seen at any one time, but the eye that
dominates varies from place to place and alternates over time, resulting in a mosaic of dominance and suppression.
Less is known what the rules of combination in the fusion process are like.
Several studies ([5], [6], [7]) showed that when one eye views a blurred version of the other eye’s image, an inter-ocular
blur suppression mechanism ensures a blur free percept. Moreover, Schor et al. [7] showed that inter-ocular blur
suppression acts locally: When two nearly identical monocular images each contain blurred regions at different locations,
the resulting binocular percept contains only non-blurred regions. In other words, the visual system is able to select, for
each spatial location, the monocular signal with the highest resolution. This remarkable ability explains why presbyopic
patients wearing one lens in the dominant eye for distant vision and a lens for near vision in the other (a situation known
as monovision) enjoy blur-free vision at all distances [7]. To date, it was not clear whether inter-ocular blur suppression
can occur for regions located at different depths. In other words, it was not known whether the human visual system can
use binocular disparities to derive depth information and simultaneously suppress blur in one of the two eyes.
A study at the University and ETH Zurich was performed to determine whether the human visual system is capable of
extracting the highest resolution available at each location, irrespective of the eye of origin. Visual acuity at different
depth planes created by binocular disparity was measured, and it was investigated whether the eye of origin of the signals
influence performance. The results show that the human visual system can use the highest resolution available between
the two monocular images, irrespective of eye of origin and depth plane [8].
4.3 Optical design
The specification of the first fusion optics type CMO stereomicroscope comprises a 20.5 x stereo zoom, with an entrance
pupil on one side as large as 28 mm. With the standard objective of fO = 80 mm attached a numerical aperture of 0.175 is
achieved, which allows a so far unachieved magnification range of 7.8...160 x. This gives a ratio of MTOT VIS/nA = 914 at
maximum magnification and so nicely fulfils the rules of useful magnification as given in (4). A field number of 23 mm
leads to an object field from 1.44 mm to 29 mm.
The optical layout is sketched in Fig. 5.
____J
____
rn
LH
Fig. 5. Optical design of the first stereo zoom with fusion optics. Upper half of the picture shows the lens position and
optical rays at minimum magnification position, lower half of the picture at maximum magnification position.
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Each zoom consists of 11 lenses in four groups with positive-negative-positive-negative refractive powers. Telescopic
magnification goes from .39x to 8x.
Lenses of both channels have the same optical design except for the free diameter of the first lens group. This results in
the same zoom curve for both sides. The outer lens groups stay at fixed positions while both of the inner lens groups
travel in opposite directions.
The zoom (and the complete microscope) is apochromatically corrected over the whole range.
An iris diaphragm is placed at a fixed position near the centre of the zoom.
The size of the entrance pupil depends on the zoom position. The discussed physical limitation of the entrance pupils due
to the spacing of the stereo basis is only a restriction near the maximum magnification position, where the high
resolution is required. For this reason the idea of fusion optics was actually implemented only for the high zoom
positions. In the middle and low zoom positions resolution and depth of field are sufficient, why the zoom design
implements equally sized beam paths for both channels. A detailed diagram of the behaviour of the numerical apertures
over the zoom range will be given in Fig. 6.
The physical size of the front lens group carefully needs to be taken into consideration also at the lower magnification
positions. Without further constraints the zoom might tend to pull the axial position of the entrance pupil to its interior.
The optical designer must make sure that the field angles have a proper path through the front lens group without
significant vignetting, also for the narrow channel.
The stereomicroscope is embedded in the system of Leica’s high performance stereomicroscopes offering a large variety
of common main objectives, among which a 2x objective is available. Using it, a numerical aperture of 0.35 is achieved,
so reaching a resolution of 1050 lp/mm. These resolutions until now were reserved for compound microscopes and have
now become available under full stereoscopic vision and at a working distance of 20 mm.
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4.4 Performance of fusion optics
The optical layout as described in 4.3 leads to a behaviour of the numerical apertures as shown in Fig. 6.
Fig. 6. Behaviour of the numerical aperture over the whole zoom range. Solid black line shows the numerical apertures of the
fusion optics designed stereo zoom, for left and right channel, respectively. Dashed gray line shows the numerical aperture
for a conventional stereo zoom with equally sized entrance pupils; since maximum achievable resolution is lower than with
the fusion optics layout, the magnification range ends at a lower position, too, to avoid empty magnification.
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The resolution achieved with a stereomicroscope having differently sized pupils is shown in Fig. 7. The right channel has
the highest peak resolution in the focal plane of the objective, while the left channel has a lower peak but higher depth of
field and thus a higher resolution out of the focal plane. For comparison, the dashed line shows the resolution for a
stereomicroscope with symmetrically sized entrance pupils.
Resolution
[lp/mm]
,/3OO
-0.04
-0.02
0
0.02
0.04
z [mm]
Fig. 7. Resolution in different levels z in object space at maximum magnification position. Solid lines show the right and left
channel, respectively. Thick solid line is the perceived resolution after fusion of both channels. Dashed gray line is the
achievable performance with a stereomicroscope of equally sized entrance pupils.
The perceived resolution with the fusion optics layout is superior in all z-planes.
5. CONCLUSION
In this presentation the limitation of conventional stereomicroscopes was shown, which prevents these microscopes to
achieve higher resolution.
An idea how to overcome this limitation was explained and validated. The “fusion optics” called optical layout supplies
the observer with slightly differently resolved stereoscopic images. The physiological fusion process finally lets the
observer perceive a 3-dimensional image with increased resolution and increased depth of field at the same time.
The optical design was presented and the performance described. This new approach combined with a 20.5x zoom and a
new series of CMO objectives now allows so far unachieved resolutions up to 1050 lp/mm under full stereoscopic
observation at a free working distance of 20 mm.
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[2]
[3]
[4]
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[6]
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[8]
Spektrum der Wissenschaft 41 05/08
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Max Berek, Grundlagen der Tiefenwahrnehmung im Mikroskop, Marburger Sitzungsberichte 61, 189-223 (1927)
Collins, M.J. and A. Goode, Interocular blur suppression and monovision. Acta Ophthalmol (Copenh), 72(3), 37680 (1994)
Johannsdottir, K.R. and L.B. Stelmach, Monovision: a review of the scientific literature. Optom Vis Sci, 78(9), 64651 (2001)
Schor, C., L. Landsman, and P. Erickson, Ocular dominance and the interocular suppression of blur in monovision,
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C. Schulthess, H. Schnitzler, D.C. Kiper, Spatial visual acuity at different depth planes: The role of eye of origin,
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