Advances in Stereomicroscopy

Transcription

1 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: X/08/$18 doi: / Proc. of SPIE Vol P-1

2 INTEROCULAB 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 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 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. Proc. of SPIE Vol P-2

3 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 Workirnz V I I i I J I na Distance 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 D EP 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 f O of the common main objective one finds the numerical aperture na of the stereomicroscope as D na =. (1) 2 f EP O Proc. of SPIE Vol P-3

4 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 lp R = 3000 na. (2) mm 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 f E M TOT VIS ftl = Γ M E. (3) f O 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 VIS na < M TOT < 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 λ 0.34 mm δ = +. (5) 2 2 na M TOT VIS na 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 3- dimensional 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. Proc. of SPIE Vol P-4

5 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. Stereo Zoom..., / /, -, I, 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) = 2 f is kept the same as in the set up depicted in Fig. 2. The dashed line indicates O 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 Proc. of SPIE Vol P-5

6 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 f O = 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 ( f TL = 160 mm), and standard 10x eyepieces is considered. Proc. of SPIE Vol P-6

7 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 Spatial resolution 375 lp/mm 525 lp/mm Depth of field mm 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. Proc. of SPIE Vol P-7

8 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 f O = 80 mm attached a numerical aperture of is achieved, which allows a so far unachieved magnification range of x. This gives a ratio of M TOT 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. Proc. of SPIE Vol P-8

9 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. Proc. of SPIE Vol P-9

10 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. Proc. of SPIE Vol P-10

11 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 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. Proc. of SPIE Vol P-11

12 REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] Spektrum der Wissenschaft 41 05/08 Ian P. Howard, Brian J. Rogers, Binocular Vision and Stereopsis, Oxford Psychology Series No. 29, Born & Wolf, Principles of Optics, 6th Ed., Cambridge University Press, 694 (1980) Max Berek, Grundlagen der Tiefenwahrnehmung im Mikroskop, Marburger Sitzungsberichte 61, (1927) Collins, M.J. and A. Goode, Interocular blur suppression and monovision. Acta Ophthalmol (Copenh), 72(3), (1994) Johannsdottir, K.R. and L.B. Stelmach, Monovision: a review of the scientific literature. Optom Vis Sci, 78(9), (2001) Schor, C., L. Landsman, and P. Erickson, Ocular dominance and the interocular suppression of blur in monovision, Am J Optom Physiol Opt, 64(10), (1987) C. Schulthess, H. Schnitzler, D.C. Kiper, Spatial visual acuity at different depth planes: The role of eye of origin, Institute of neuroinformatics, University and ETH Zurich, to be published (2008) Proc. of SPIE Vol P-12