Abstract:
A microscope device having dual emission capability, wherein detrimental effects of image-aberrations and -distortions are reduced. By providing the means for reflecting the one beam in a manner so as to invert its handedness and the means for reflecting the second beam in a manner so as to preserve its handedness, a fully symmetrical configuration is obtained, where corresponding image points in both color/polarisation channels all experience the same field-dependent aberrations.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a microscope device, which is capable of producing images of a sample in different spectral ranges (“colors”) or polarisations on a single detector. 
     2. Description of Related Art 
     In 1991 a method for separating a microscope image into two images of different color placed next to each other on a single camera chip was proposed (K. Kinosita et al., “Dual-View Microscopy with a Single Camera: Real-Time Imaging of Molecular Orientations and Calcium”, J. Cell. Biol., 1991, 115(1), pages 67-73); see also U.S. Pat. No. 5,337,081). The commercial version of this concept was termed “w-View” because it not only provides a “double view” of the sample, but also because of the w-shape of the beam path it employs. (http://jp.hamamatsu.com/resources/products/sys/pdf/eng/e_aqfret.pdf). The design is used in all fields of microscopy where dual-emission images need to be recorded. Given that it allows recording two color images simultaneously rather than sequentially, the device is particularly suited for time-lapse studies, where switching of an emission filter would reduce time resolution. 
     A schematic of the W-view design as used in the prior art is shown in  FIG. 1 . A collimated image beam  10 , originating from an intermediate image (which is not shown in  FIG. 1 ) impinges onto a first dichroic beam splitter  12  and is separated into a first beam  14  and a second beam  16  of different color, i.e. the first beam  14  essentially consists of light of a first spectral range and the second beam  16  essentially consists of light of a second spectral range. The first beam  14 , which is reflected by the first beam splitter  12 , passes to a mirror  18  from where it is directed onto a second dichroic beam splitter  20 , which has the same spectral characteristics as the first beam splitter  12 . The second beam  16 , which is transmitted by the first beam splitter  12 , is reflected by a second mirror  22  and directed onto the second beam splitter  20 , where the first beam  14  and the second beam  16  are “reunited” (or “combined”) in their general direction. However, by adjusting the mirrors  18  and  22  in an appropriate fashion, the two beams  21 , corresponding to the first beam  14 , and  27 , corresponding to the second beam  16 , exhibit a slight angular offset relative to each other, i.e. they diverge relative to each other to a certain degree, so as to yield the desired spatial separation on the detector chip (not shown in  FIG. 1 ). 
     According to this prior art concept, color separation takes place in an infinity space of the optical beam path, which could be the space between the objective lens and the tube lens. However, in order to avoid image overlap from adjacent areas, it is advantageous to create an intermediate image and to confine it within boundaries defined by a suitable field-stop. Such field-stop has to reduce the field of view seen by the camera of the detector to one half of its original size, in order to accommodate the two semi-images projected side by side. Having an intermediate image requires a second set of optics (relay-lenses), which create another infinity space where beam separation takes place. One major advantage of this design is that using a beam splitter not only for separating beams, but also for reuniting them, allows maintaining telecentric optics throughout, thus avoiding vignetting and asymmetrical light-cones which are different for different areas on the detector chip. Moreover, the fact that both beam paths are transmitted by the same optics warrants that both color channels experience identical magnification and need no resealing before being compared. This is particularly important in co-localization studies. 
     However, since no optical system is perfect, there are always aberrations and distortions, and their extent depends on the position of a given point within the field. Usually, aberrations are less pronounced in the center and increase towards the edges of the field. However, due to the spherical symmetry of the imaging optics, aberrations and distortions are generally symmetrical with respect to the central axis of the optics. 
     According to U.S. Pat. No. 5,982,479 a single dichroic beam splitter may be used for separating a collimated image beam originating from an intermediate image into two different color channels, which are imaged by a common lens onto a common detector chip in order to obtain spatially separated semi-images on the detector. Each of the color channels is reflected twice prior to being projected onto the detector, whereas according to the prior art system shown in  FIG. 1 , both color channels are reflected 3 times or, in the original Kinosita paper, one color channel is not reflected at all whereas the other one is reflected 4 times A similar system is known from JP 2004361391 A, wherein splitting of the two color channels and double-reflection in each channel occurs in the space between the projection lens and the detector. All prior art has in common that the number of reflections for the two beam-paths are such that both color channels have the same handedness on the chip. 
     It is an object of the invention to provide for a microscope device having dual emission capability, wherein detrimental effects of image-aberrations and -distortions are reduced. 
     SUMMARY OF THE INVENTION 
     According to the invention, this object is achieved by a microscope device as defined in claims  1  and  16 , respectively, which enables separate “color channels” and by a microscope device as defined in claims  25  and  26 , respectively, which enables separate “polarisation channels”. 
     The invention is beneficial in that, by providing the means for reflecting the one beam in a manner so as to invert its handedness and the means for reflecting the second beam in a manner so as to preserve its handedness, a fully symmetrical configuration is obtained, where corresponding image points in both color/polarisation channels all experience the same field-dependent aberrations, whereas in the prior art systems, which either maintain the handedness of both color/polarisation channels or invert handedness of both color/polarisation channels, image points, which are close to the center line in one image are close to the edge of the image in the other image, so that their aberrations differ. 
     This is schematically illustrated in  FIGS. 2   a  and  2   b , where the symmetry of the images resulting from the two color channels on the detector with regard to the symmetry of the multi-color intermediate image is shown for the prior art concepts ( FIG. 2   a ) and for the present invention ( FIG. 2   b ). 
     According to the microscope device defined in claim  1  or  25 , each color/polarisation channel undergoes at least one additional reflection after having been separated from the other color/polarisation channel. This concept enables comfortable adjustment of the position of each of the color/polarisation channels on the camera-chip. 
     According to the microscope device as defined in claim  16  or  26 , only one of the color/polarisation channels undergoes additional reflections after having been separated from the other color/polarisation channel, while for the other color/polarisation channel the reflection needed for separating and reuniting the two beams remains the only reflection. Thereby a particularly simple and compact design can be achieved. According to a preferred embodiment, the means for reflecting the second beam and the separating means are integrated within a single member. 
     These and further objects, features and advantages of the present invention will become apparent from the following description when taken in connection with the accompanying drawings which, for purposes of illustration only, show several embodiments in accordance with the present invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a schematic view of a color splitting arrangement for a microscope device having the “W-view design” according to the prior art; 
         FIGS. 2   a  and  2   b  are schematic views of the image symmetry obtained by the color splitting arrangement of  FIG. 1  of the prior art and a color splitting arrangement according to the present invention; 
         FIG. 3   a  is a schematic view of a microscope device according to the invention comprising a first embodiment of a color splitting arrangement; 
         FIG. 3   b  is a schematic view of the arrangement of  FIG. 3   a  seen in the direction of the arrow A of  FIG. 3   a;    
         FIG. 4   a  is a schematic view of a microscope device according to the invention comprising a second embodiment of a color splitting arrangement; 
         FIG. 4   b  is a schematic view of the arrangement of  FIG. 4   a  seen in the direction of the arrow A; 
         FIG. 4   c  is a schematic view of the arrangement of  FIG. 4   a  seen in the direction of the arrow B; 
         FIG. 5   a  is a view like  FIG. 4   a , with a third embodiment of a color splitting arrangement of a microscope device according to the invention being shown; and 
         FIGS. 5   b  to  5   d  are a view of the arrangement of  FIG. 5   a  seen in the direction of the arrow A, wherein the incident multi-color image beam, the outgoing first beam/color channel and the outgoing second beam/color channel, respectively, are shown. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       FIGS. 3   a  and  3   b  show a first embodiment of a microscope device according to the invention, wherein a collimated multi-color beam  10  is generated by collecting light from a sample  11  by a compound microscope  13  (in the drawing consisting of objective  13   a  and tube lens  13   b ). The microscope creates an intermediate image  15  located in the focal plane of a projection lens  17 . Typically, the light collected from the sample  11  will be emission light, in particular fluorescence emission light, such as emission light obtained from Fluorescence Resonance Energy Transfer (FRET). A first dichroic beamsplitter  12  serves to reflect one wavelength component of the beam  10  (if the dichroic is a long-pass, beam  14  is the short-wavelength-part of beam  10 ) toward a mirror  18 , thereby generating a first beam  14 , whereas the component of the beam  10 , which is transmitted by dichroic  12 , constitutes a second beam  16 , which is directed towards a roof prism  23 . While the first beam  14  is reflected by the mirror  18  towards a second dichroic beamsplitter  20 , having the same spectral characteristics as the first beam splitter  12 , the second beam  16  is deflected by the roof prism  23  in such way that it meets the first beam  14  at the second beam splitter  20 . There the former is transmitted and the latter is reflected, thus reuniting beams  14  and  16  into a “combined” beam-bundle, consisting of beam  21  (formerly beam  14 ) and beam  27 , formerly beam  16  (the reunited beams  21  and  27  are “combined” in the sense that they later pass through the same optical elements). For the arrangement to serve its purpose the roof-prism  23  must be oriented in such a manner that its ridge  25  is located within the plane defined by the first beam  14  and the second beam  16 . 
     Beams  21  and  27  are projected by a projection lens  26  onto a detector  28  located in the focal plane of the lens  26 , so that an image of the sample  11  is generated on the active area of the detector  28 . In order to enable two images to be projected side by side onto the detector  28 , the intermediate image  15  is confined to the boundaries of about half of the size of the active area of the detector  28 . 
     Whereas in the view of  FIG. 3   a  the outgoing portion  21  of the first beam  14  and the outgoing portion  27  of the second beam  16  are superimposed,  FIG. 3   a , showing the beam-splitting part of the microscope device in a view in the direction of the arrow A of  FIG. 3   a , discloses that in the plane perpendicular to the paper plane of  FIG. 3   a  there is a slight angular offset between the beam portion  21  and the beam portion  27 . This angular offset is turned into a spatial offset of the images on the detector  28  (not shown) by means of the projecting lens. The exact angular offset—and hence the corresponding spatial offset—is controlled by appropriate relative adjustment of the elements  12 ,  18 ,  20  and  23 . Thereby two images of the sample  11  in two different spectral ranges which are determined by the beam splitters  12 ,  20  can be obtained side by side on a single detector  28 . 
     The first beam  14  undergoes an odd number of reflections, namely three, so that the handedness of the first beam  14  is inverted with regard to the handedness of the incident multi-color beam  10 , when being projected onto the detector  28 . By contrast, the roof prism  23  acts as a retroreflector, with the second beam  16  undergoing an even number, namely two, reflections, so that the handedness of the second beam  16  is maintained with regard to handedness of the incident multi-color beam  10 , when being projected on the detector  28 . It can be seen from  FIGS. 3   a  and  3   b  that the roof prism  23  acts as a retro-reflector only in one dimension, namely with regard to the direction perpendicular to the paper plane of  FIG. 3   a , whereas within the paper plane of  FIG. 3   a  it acts as a “normal” reflector in that the incident angle equals the outgoing angle of the beam. 
     The image symmetry obtained by the arrangement of  FIGS. 3   a  and  3   b  is schematically illustrated in  FIG. 2   b  which shows the handedness of the intermediate image  15  and the handedness of the resulting images  30  and  32  obtained on the detector  28  by projection of the outgoing portion  21  of the first beam  14  and the outgoing portion  27  of the second beam  16 , respectively. It can be seen that portions of the intermediate image  15  located close to the axial center line will be also located close to the axial center line for both final images, so that a fully symmetrical configuration is achieved wherein corresponding image points all experience the same field dependent aberrations when being projected by the projection lens  26 . 
     By contrast, with the arrangement of  FIG. 1  only for one of the final images  32 ′ an image point close to the center line in the intermediate image  15 ′ will remain close to the center line, whereas in the other final image  30 ′ such an image point will be located close to the edge of the field (see  FIG. 2   a ), so that the field dependent aberrations will be different for the two images  30 ′ and  32 ′. 
     It is to be understood that in the arrangement of  FIGS. 3   a ,  3   b  the beams are collimated between the projection lenses  17  and  26 , i.e. there is an infinity space between the lenses  17  and  26 . It is to be noted that in principle, rather than creating an angular offset of the beams  21  and  27  in a direction perpendicular to the paper plane of  FIG. 3   a , such angular offset could be alternatively achieved in the paper plane of  FIG. 3   a , so that in this case the beams  21  and  27  would coincide in the combined beam  24  in the view of  FIG. 3   b.    
       FIGS. 4   a  through  4   c  show a modified embodiment wherein only a single dichroic beamsplitter  112  is used, instead of two dichroic beamsplitters  12  and  20  as in  FIGS. 3A ,  3 B. In addition, rather than using two projection lenses  17  and  26 , only a single projection lens  126  is used. Its purpose is not only to collimate beam  110 , which originates from the intermediate image  115 , but also to project the outgoing beams  37  and  38  next to each other onto the detector  128 . 
     The intermediate image  115  of the sample  111  is located in the focal plane of the projection lens  126  and is confined to the boundaries of about half of the active area of the detector  128 . It is off-center relative to the optical axis  140  of the projection lens  126  in both the dimension displayed in  FIG. 4   a  (this off-center position allows separating the intermediate image  115  from the image on the detector  128 ) and in the dimension shown in  FIG. 4   b , which is perpendicular to the plane displayed in  FIG. 4   a.    
     Before the beam  110  intersects the optical axis  140  in the focal plane of the lens  126 , it reaches a dichroic beamsplitter  112  which serves to separate the incident beam  110  into a first beam  114  which is transmitted by the beamsplitter  112  onto a mirror  118  and a second beam  116 , which is reflected by the beam splitter onto a roof prism  23 . The mirror  18  is located in the focal plane of the lens  126  and serves to reflect the first beam  114  back to the beamsplitter  12 , where it is transmitted again. The mirror  118  is adjusted in such a manner that the outgoing portion  121  of the first beam  114  has an angular offset with regard to the incident beam  110  in the paper plane of  FIG. 4   a . The roof prism  123  is arranged in such a manner that the ridge  125  of the prism  123  is located in the paper plane of  FIG. 4   a  (in the shown example, the ridge  125  is parallel to the central optical axis  140 ) and that the second beam  116  received from the beamsplitter  12  is reflected back to the beamsplitter  12 , where it is reflected again in such a manner that it forms an outgoing portion  127  which coincides in the view of  FIG. 4   a  with the outgoing portion  121  of the first beam  114  reflected by the mirror  118  and transmitted by the beam splitter  112  in order to form a combined beam  124 . With regard to the dimension perpendicular to the paper plane of  FIG. 4   a , the prism  123  acts as a retro-reflector, whereas it acts as a “normal” reflector in the dimension in the paper plane of  FIG. 4   a . 
     While in  FIG. 4   a  the beamsplitter  112  is inclined at an angle of 45° with regard to the optical axis  140 , smaller angles also are conceivable. 
     The reflecting elements  112 ,  118  and  123  are adjusted in such a manner that the outgoing portions  121  and  127  of the first beam  114  and the second beam  116  have a slight angular offset relative to each other in the direction perpendicular to the paper plane of  FIG. 4   a , so that the image of the first beam  114  and the image of the second beam  116  on the detector  128  have a spatial offset. In order to create these images, the outgoing portions  121  and  127  of the first beam  114  and the second beam  116  pass through the projection lens  126  and, now called beam  37  and  38 , form separate images in the focal plane of lens  126 . 
     As shown in  FIG. 4   a , a prism  142  may be provided in the outgoing beams  37  and  38  for deflecting these beams onto the detector  128 , thus facilitating their separation from the incoming beam  110 . Alternatively, the prism  142  may also be placed in the incoming beam  110 . 
     Here again, as in the embodiment of  FIG. 3   a ,  3   b , in one of the two color channels (here that one formed by the first beam  114 ) the handedness is inverted (there is a single reflection at the mirror  118 ), whereas for the other color channel (here that one formed by the second beam  116 ) the handedness of the beam is maintained due to an even number of reflections (here: two reflections at the roof prism  123 ). 
       FIGS. 5   a  to  5   d  show an embodiment which is a simplified version of that of  FIGS. 4   a  to  4   c  in that the functions of the beam splitter  112 , the mirror  118  and the roof prism  123  are integrated into a single element  223 . As in the embodiment of  FIGS. 4   a  through  4   c , the intermediate microscope image  215  is radially shifted with regard to the optical axis  240  of the projection lens  226  not only in the dimension extending in the paper plane of  FIG. 5   a , but also in the dimension extending perpendicular to the paper plane of  FIG. 5   a , see  FIG. 5   b . The intermediate image  215  is located in the focal plane of the projection lens  226 . The collimated multi-color beam  210  is angled towards the optical axis  240  and impinges onto the element  223 , which is a roof prism having a dichroic coating  218  on the front surface. The ridge  225  of the roof prism  223  is arranged in the paper plane of  FIG. 5   a , and the coated front surface  218  is essentially perpendicular to the optical axis  240 . The dichroic surface  218  serves to split the incoming beam  210  into an outgoing first beam  221 , which is reflected at the surface  218  towards the projection lens  226 , and a second beam  216 , which is transmitted by the surface  218  into the interior of the roof prism  223 , where it is reflected back to the surface  218 . Then the second beam  216  is transmitted through the dichroic surface  218  towards the projection lens  226 , thus forming an outgoing beam portion  227 . The roof prism  223  acts as a retro-reflector in the dimension perpendicular to the paper plane of  FIG. 5   a , whereas it acts as a “normal” reflector in the dimension extending in the paper plane of  FIG. 5   a    
     Since the intermediate image  215  is radially shifted with regard to the optical axis  240  also in the dimension perpendicular to the paper plane of  FIG. 5   a , the collimated incident beam  210  is angled towards the optical axis  240  also with regard to that dimension (see  FIG. 5   b ). Since with regard to that dimension the roof prism  223  acts as a retro-reflector, the second beam  216  transmitted by the surface  218  is reflected back in the direction of the incoming multi-color beam  210 , so that—apart from the displacement of the combined beam  224  with regard to the incident multi-color beam  210  in the paper plane of  FIG. 5   a —beams  221  and  227  exhibit the same angle relative to the optical axis  240 , but having opposite signs. This warrants that the images created from the two beams  221  and  227  by the projecting lens  226  are next to each other in the plane of the detector  228 , in  FIGS. 5   b  and  5   c  above and below the optical axis  240  but both touching it. Given that the first outgoing beam  221  has experienced one reflection in both dimensions whereas the outgoing portion  227  of the second beam  216  undergoes one reflection in one and two reflections in the other dimension, their respective images created by the projection lens  226  differ in their handedness. 
     It is to be understood that in all embodiments the dichroic beamsplitter could be either a short pass or a long pass. 
     Due to the finite spectral selectivity of the beamsplitters the spectral separation of the two color channels (first beam and second beam, respectively) in practice never will perfect, so that here a certain color channel is to be understood as consisting of light containing the associated spectral range in higher relative amount than the spectral range associated to the other color channel. 
     The above embodiments, which serve to provide for two separate color channels, also could be used to realize two separate polarisation channels. In this case the light collected from the sample would include two different polarisations and the dichroic beamsplitters would be replaced by beamsplitters which split the incoming mixed polarisation beam into two beams having different polarisation. 
     While various embodiments in accordance with the present invention have been shown and described, it is understood that the invention is not limited thereto, and is susceptible to numerous changes and modifications as known to those skilled in the art. Therefore, this invention is not limited to the details shown and described herein, and includes all such changes and modifications as encompassed by the scope of the appended claims.